A process for the manufacture of paper from pulp employs at least one graft copolymer of starch selected from the group consisting of starch graft-polymethacrylic acid, starch graft-polyacrylic acid, cationic starch graft-polymethacrylic acid and cationic starch graft-polyacrylic acid as a wet-end additive. The graft copolymer preferably has an add on amount of acid of from about 0.1 to about 50% based on the weight of the starch, and is typically added to cellulosic pulp in an amount of from about 0.1 to about 10% based on the weight of the pulp. Such wet-end additives are also useful in processes involving relatively high alum levels of up to about 15 or 25%, based on the weight of the pulp.

In paper making, cellulosic pulp is prepared by mechanical treatment in beaters which increases the surface area of fibers in the pulp by cutting, fibrillation and hydration. Subsequently, a dilute suspension of the pulp along with other materials (like alum, resin and fillers) is filtered on a wire screen. Water drains off through the wire screen and a wet fibrous mat retained on the wire screen is mechanically processed and dried. "Wet-end" additives are added as dispersions in water at a suitable stage to the pulp slurry prior to sheet formation.
In paper manufacture, starch derivatives may be used as wet-end additives to improve the processability (drainage and retention) and strength properties of paper. Cationic and amphoteric starches are, thus, widely used in the paper industry. Graft copolymers of starch with cationic monomers like dialklamino-alkyl(meth)acrylamide (JP 88,219,696, JP 87,104,998), dimethylaminopropyl acrylamide (JP 88,275,795) and 2-(methacryloyloxy) ethyltrimethylammonium Me sulfate (1986 Eur. Pat. Appl. EP 194,987) along with acrylamide (in most cases) have recently been reported as improving (when used as a wet-end additive) the drainage and/or retention during paper manufacture and/or dry strength properties of
In the process of paper manufacture, whilst we do not wish to be bound by theory, we believe, the "mileage" obtained by use of wet-end additives results from improving fiber to fiber or/and fiber to filler bonding by anchoring onto reactive sites on the fiber and/or filler.
Interfiber hydrogen bonds are formed as a result of a wet paper web drying in the absence of any additive. The cationic starch based wet-end additives are known to anchor through their cationic functional groups onto the anionic reactive sites on cellulosic fibers (cellulose fibers in water are negatively charged due to ionised groups and residual lignin present on the surface of the fibers). Thus, the combination of natural fiber-fiber interaction through hydrogen bonding and the interfiber and fiber-filling bonding through anchoring of cationic polymers helps to improve drainage, retention and strength properties in paper manufacture.
However, the improvement in processing and properties brought about by these additives is influenced by (i) presence of other cationic species in the paper furnish, and (ii) the reactivity of the fibers and fillers for the cationic additives. There exist chemicals like aluminium sulphate (alum) giving rise to cationic species in water which are used extensively in paper-making, their primary role being to set rosin size. Alum is also known to have an impact on retention, drainage, paper strength and in addition it effects the pH and total system ionic charges.
The amount of alum used in paper making depends on the conditions optimised by a particular paper manufacturing unit. It is based on various considerations, such as, quality of pulp, water and type of fillers, etc. Alum is known to effect the performance of cationic and/or amphoteric starch based wet-end additives. Aluminium sulphate in solution gives rise to Al.sup.+++, Al(OH).sub.2.sup.+, Al(OH).sup.++ and Al.sub.8 (OH).sub.20.sup.+4 species depending on the pH. These cationic species suppress the anionic charges on the fiber or filler and therefore reduce their reactivity to the cationic/amphoteric wet-end additives. Thus, although lower levels of alum are useful for rosin sizing and neutralising the anionic colloidal impurities in the furnish, higher levels of alum present in the furnish is detrimental to the performance of cationic and/or amphoteric wet-end additives.
A given paper making system can only tolerate a particular amount of combined cationic additives to deliver optimum performance as regards processing and properties of the finished product. The higher the concentration of alum used, the lower will be the performance of these polymers as wet-end additives and their use is restricted in such cases. Although alum as well as cationic polymers produce cationic species, their roles are different and many cationic polymers do not function fully in the presence of high amounts of alum. Thus, paper manufacturing processes are restricted to certain usage levels of alum/cationic polymers.
K. Tanaka, F. Masuda and K. Mita in a Japanese Patent JPB 55-42200 describes the synthesis of graft copolymers of starch and a carboxyl group - containing ethylenic unsaturated monomer, (sodium salt) and their use in strengthening of paper. The graft copolymers were synthesised from gelatinised starch. In more detail, 50 parts starch in gelatinised form is reacted with 50 parts of a carboxylated ethylenic monomer, with a synthetic monomer content of 10% to 1000% (by weight of starch) using hydrogen peroxide as catalyst. The product is neutralised with sodium hydroxide at the end of the reaction.
Treatment of paper pulp with 0.5% of this polymer is shown to improve the dry breaking length of paper at low alum levels of 0.5%, based on the weight of the pulp. The advantages described by the authors for the additive are (1) water solubility as compared to many starch derivatives used as paper additives, and (2) improved performance in improving some of the paper properties. The extent of property improvements described in this patent are also known to be achieved by commercially available cationic and amphoteric starch derivatives with the same levels of incorporation. However, the products exemplified can be expected to be costly owing to the fact that their synthesis involves the large amounts of synthetic monomers, to the difficult processing conditions associated with use of viscous starch dispersions, etc. and recovery of the product by energy intensive unit operations like drum drying.
There are no definite property attributes indicated in the aforementioned document for these additives vis-a-vis existing high performance wet-end additives under specific paper manufacturing conditions. Comparisons have been made with low performance dry strength improvement additives.
We have now surprisingly found an alternative process for making paper which may improve drainage and retention during processing and also improve the dry strength of paper. Process performance may improve with increasing amounts of alum up to a limit (about 10%) and further increases in the alum levels do not detrimentally affect the performance. Thus, the present process is also surprisingly effective for paper mills operating at high alum levels in addition to mills operating at moderate alum levels. This is in contrast to the case of some processes using cationic and/or amphoteric starch derivatives where performance is detrimentally affected due to presence of high amounts of alum.

National Starch and Chemical Investment Holding Corporation


A technique for boresighting an antenna mounted on a moving platform is described. The technique uses a concurrently moving calibration target. Target navigation data and platform navigation data are used to compensate for movement of the target and the platform. The antenna pointing direction is biased in a direction which provides a best signal quality, and the bias used to determine antenna boresight calibration factors. The boresight correction factors can be used for open loop pointing.

Communications systems often use directional antennas to focus transmitted and received power in a desired direction. The direction of maximum transmitted and received power is sometimes referred to as the "boresight" of the antenna. Highly directional or narrow beamwidth antennas must be pointed accurately to ensure the antenna boresight is oriented in the desired direction. For example, for communication between two moving platforms, narrow beamwidth antennas are constantly adjusted as the pointing angles change due to relative movement of the platforms. Additional stabilization may also be required to accommodate changes in platform orientation, for example, using an inertial reference that relates the platform orientation (roll, pitch, and yaw/heading) to the earth.
Antenna pointing can be performed "closed loop," where the antenna pointing angles are continuously adjusted to maximize received (or transmitted) signal power. Closed loop pointing systems typically require complex feed arrangements or complex receiver signal processing, which can increase cost. Hence, in many applications, closed loop pointing is undesirable.
Antenna pointing can also be performed "open loop," where the antenna pointing direction is determined algorithmically and the antenna is directed to point in a particular direction. Typically, open loop antenna pointing algorithms translate platform and target locations from inertial coordinates, determined by an inertial reference unit or attitude reference system, into relative ship's coordinates of azimuth and elevation which is provided to the antenna. In open loop pointing, the antenna is therefore calibrated to ensure that the boresight is accurately directed towards the specified azimuth and elevation.
Various sources of error can occur in an antenna system which results in boresight errors. For example, antennas are often stabilized with respect to an attitude reference system to compensate for attitude changes (roll, pitch and yaw) of the platform. For example, stabilization for platform orientation sometimes involves providing an attitude reference system at or near the antenna. The attitude reference system can be, for example, a set of accelerometers or a complete inertial navigation system. There can, however, be alignment errors between the axes of the antenna system and the axes of the attitude reference system. These alignment errors result in misalignment or boresight error between the desired and actual antenna pointing direction. Replacement of components in the antenna or attitude reference system can make recalibration of the boresight necessary. Other potential sources of boresight error include mechanical tolerances in the antenna positioning equipment, mechanical tolerances in the antenna beam forming elements, and electrical tolerances.
Traditionally, boresighting an antenna has been performed by holding the platform to which the antenna is mounted stationary (fixed to the earth) while the antenna is pointed at a fixed calibration target. This approach is undesirable in many circumstances. For example, a ship cannot be held in a stationary position while at sea due to sea swell, and thus is returned to port to boresight an antenna. Similarly, an aircraft is usually required to land in order to perform boresighting. This can prove inconvenient, for example in military applications, since either the mission must be interrupted or the antenna operated without the boresight calibrated. Furthermore, requiring the calibration target to remain in a fixed position can prove inconvenient. For example, this has typically meant that calibration targets are land based, again making it difficult to boresight an antenna on a ship at-sea.

Method and device for boresighting an antenna on a moving platform using a moving target


Subscription based content is decrypted for one or more trial periods without shortening the subscription period for the device. The subscription period for the content does not start until the expiration of the last trial period. The subscription logic included with the device monitors a packet value received in a broadcast to determine when the device is receiving a packet within a trial period. The packet value is time-based such that the subscription logic can determine when the device has exceeded the number of allowed trial periods as well as to determine when the subscription ends.

Encryption is used in many different subscription-based systems to protect data from being accessed without authorization. Some subscriptions impose a time-limit on the ability of a device to decrypt data. When the time-limit expires on the device, the subscription ends and the device no longer decrypts the data. Another way to protect subscription data is to use a "smart card" that is incorporated within a device. The smart card contains its own decryption mechanism along with a set of rules that determine the duration of the subscription. When the subscription ends these smart cards can be manually recharged or replaced by the user, or some other authorized entity. Additionally, the smart card must either be replaced or recharged if a user returns a device after only using a portion of the subscription during a trial period such that a user who obtains the device after its return receives the full subscription.

Trial usage for encrypted subscription-based data


An exhaust pipe of an internal-combustion engine in which self-regulating throttle element is arranged behind the catalyst. In the warm-up phase of the internal-combustion engine, the throttle element closes off the exhaust pipe almost completely. The resulting increased exhaust back pressure causes a higher mass flow rate of the engine, whereby the exhaust gas temperature rises and the catalyst is warmed up rapidly.

The present invention relates to an exhaust pipe of an internal-engine, having at least one catalyst in the exhaust pipe and at least one adjustable throttle element in the exhaust pipe.
From the German Patent Document DE-OS 23 21 578, a throttle element made of bimetal for the exhaust pipe of an internal-combustion engine is known which, when the exhaust gas temperature is low, opens up the full cross-section of an exhaust gas pipe and, when the temperatures are higher, swivels a damming element into the exhaust gas flow. By means of this arrangement, a faster heating of the core zone of the catalyst after the start of the internal-combustion engine is achieved as well as a uniform durability-increasing action on the catalyst after it has reached its working temperature.
An object of the invention is to provide an exhaust pipe of an internal-combustion engine comprising a catalyst in which the catalyst, after the starting of the internal-combustion engine, reaches its working temperature as fast as possible.
This and other objects are achieved by the present invention which provides an exhaust pipe of an internal-combustion engine. This exhaust pipe includes at least one catalyst arranged in the exhaust pipe and one adjustable throttle element arranged in this exhaust pipe. The throttle element has an upstream stationary end adjacent to a first side wall of the exhaust pipe, and a downstream, movable end. The throttle element is positioned such that when the internal-combustion engine is cold, the movable end forms a gap with a second side wall opposite to the first side wall.
By arranging a throttle element in the exhaust pipe which, when the internal-combustion engine is cold, reduces the cross-section of the exhaust pipe down to a residual gap, the exhaust back pressure is increased. For compensating this increased back pressure, the mass flow rate of the engine is increased, whereby the exhaust gas temperature rises and the catalyst reaches its working temperature faster. The throttle element may be constructed as a bimetal or a memory metal and, with its upstream stationary end, is arranged adjacent to a side wall of the exhaust pipe. In the cold condition, the downstream free end forms a gap with the opposite side wall. In the cold state, the throttle element thus blocks the exhaust pipe down to the gap required for the flowing-off of the exhaust gas quantity occurring in the cold start phase. As the internal-combustion engine warms up, the exhaust gas temperature increases, whereby the throttle element deforms and in this case progressively opens up the whole cross-section of the exhaust pipe. The elasticity of the throttle element is designed such that, in the case of a cold start and an immediate high load, the exhaust gas flow lifts the throttle element thus enlarging the gap.
Advantageously, the arrangement of the present invention is self-regulating so that no sensors or control elements are required. It has a simple maintenance-free construction and is preferably arranged downstream of the catalyst, whereby the catalyst is warmed up in an optimally short time.
Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.

Exhaust pipe of an internal-combustion engine


Kits and assays, incorporating antibodies specific for glutathione peroxidase, for determining the fertilizing ability of a male at home or in a clinical laboratory are disclosed.

According to recent studies, male infertility is responsible almost 40% of the time that a couple is unable to conceive a child. By convention, male infertility is diagnosed based on low sperm motility and/or count. When no sperm are observed to move in a sample obtained from a male, that male is diagnosed as being infertile. However, besides zero motility, there are really no motility thresholds for distinguishing fertile sperm from infertile sperm. With regard to sperm count, twenty million sperm per milliliter or greater is generally considered to be in the fertile range. Sperm count and motility can be assessed using commercially available instruments, such as computerized videoanalysis systems.
U.S. Pat. No. 5,068,089 describes a home kit for testing fertility of human sperm, based on ability of the sperm to reduce a dye. The extent of reduction (displayed calorimetrically), is said to be indicative of sperm fertilizing ability. However, this test is complicated to perform, requires incubation at a temperature above room temperature and does not distinguish between reduction due to sperm cells or other cells, which may be present in a semen sample.
U.S. Pat. No. 5,219,729 describes a laboratory assay for determining the fertilizing ability of sperm based on the affinity of binding between to a oocyte zona pellucida fragment. The tighter the binding, the greater the fertilizing ability of the sperm sample. However, this assay requires freshly prepared oocyte fragments and at least four hour's time during which the sperm must be kept in contact with the oocyte fragment.
A simple and rapid assay for determining the pregnancy potential of sperm in a laboratory or at home is needed.

Male fertility and contraception home test kits


Larrea tridentata and Ambrosia deltoidea are shown to produce compounds that inhibit replication of HIV. The compounds can be extracted by differential fractionation and by counter-current chromatographic methods described herein. Extracts prepared from L. tridentata and A. deltoidea are found to be effective in inhibiting viral replication. The extracts from L. tridentata have been characterized and found to contain tricyclic lignans. The extracts can be administered to subjects for treatment of viral infection.

1. Field of the Invention
The present invention is directed to extracts from the leaves and flowers of the creosote bush (Larrea tridentata, Zygophyllaceae) having anti-HIV activity. The extracts of at least L. tridentata contain a variety of tricyclic compounds, including cyclolignan compounds, which are also the subject matter of the present invention. The invention also embraces derivatives and isomers of the tricyclic compounds found in the extracts. Finally, the present invention also encompasses methods for treating retroviral infections, especially HIV infection, by administering the extract and/or purified tricyclics or cyclolignans to an HIV-infected subject.
2. Description of the Related Art
Articles of the scientific literature cited throughout this application are hereby incorporated by reference in their entirety by such citation.
It is becoming clear that control or cure of HIV infection in a subject will require a multifacetted approach, which partly relies upon inhibition of the HIV replication cycle. Furthermore, it is clear that a several aspects of viral replication will have to be attacked. For instance, it has recently been shown that a regimen of multiple drugs, such as inhibitors of reverse transcriptase (RT), AZT, ddI, and the protease inhibitor, 3TC, has proved more effective in controlling the virus than administration of any one of these drugs alone.
It has been shown that the creosote bush and related species contain active principles with antimicrobial and antiviral activity (Ayres et al., Ch. 3, p. 85 in Lignans: Chemical, Biological and Clinical Properties--(Chemistry and Pharmacology of Natural Products), Ayres and Loike, Eds., c. 1990 by Cambridge University Press, New York,).
Tat is a transactivator of human immunodeficiency virus (HIV) gene expression. Tat and Rev are indispensable viral regulatory factors for HIV gene expression. Tat acts by binding to the TAR RNA element and activating transcription from the long terminal repeat (LTR) promoter of the HIV-1 proviral DNA.
It has been shown that upon infection with AIDS virus, basal viral gene transcription is initiated. Activation of viral genes occurs upon antigenic stimulation and results in a burst of viral replication. Expression of the tat gene has been shown to result in several thousand-fold increased levels of HIV LTR-driven gene expression at both the transcriptional and postranscriptional levels.
The Tat protein stabilizes elongation of transcription complexes and has also been shown to be involved in transcription initiation. Previous studies have shown that Tat mediates reduction of antibody-dependent T cell proliferation, contributing substantially to the failure of the immune response. Tat also directly stimulates the growth of Kaposi's sarcoma cells. Tat is indirectly involved in HIV-related dementia in patients with advanced AIDS.
Since Tat has no apparent cellular homologs, this strong positive regulator has become an attractive target for the development of anti-AIDS drugs (see FIG. 1). Currently available AIDS drug such as HIV reverse transcriptase inhibitors (AZT, DDI) or the protease inhibitor (3TC) target viral gene products (enzymes and specific viral proteins). These drugs are capable of suppressing the replication of a wild-type AIDS virus. However, they are ineffective in destroying a small pool of coexisting mutant viruses which continue to replicate at a high rate in the presence of these drugs. An inhibitor which targets Tat transactivation and viral gene transcription at the level of integrated proviral DNA will arrest the virus at an early stage (Hsu et al., Science, 254:1799-1802, 1991). Because replication of the viral genome is suppressed, virtually no viral resistance to the drug should develop.
Efforts aimed at the elucidation of factors which control gene expression at the transcriptional and post-transcriptional levels in host eukaryotes have recently made possible quantitative assessment of Tat function (Sim, Ann. N.Y. Acad. Sci., 616:64-70, 1990).
The HIV-1 integrase (HIV-1 IN) mediates the integration of the transcribed viral DNA into the host chromosome, a crucial step in the HIV replication cycle. This enzyme has no known cellular homolog, and relatively simple in vitro assay systems have enabled functional characterization and the screening of specific inhibitors of the HIV-1 IN. Therefore HIV-1 IN has also become a major target for the development of specific anti-AIDS drugs.

Extracts of Larrea tridentata having antiviral activity and their use for treating viral infections


The invention relates to a system for the protection of neuroprosthesis operation against unauthorized access to the function or operating data. The system is assigned to neuroprostheses for treating functional disorders of the nervous system. This neuroimplant protection system (NIS) comprises at least one neuroprosthesis component which is in contact or operational connection with nerve tissue. Neuroprosthesis operation takes place only during the period of a specific authorization and/or comprises an authorized data transmission between external component and implanted component and/or an authorized communication for monitoring and/or defining the neuroprosthesis functional state. The communication between external and implanted component is in encrypted form.

The invention relates to a method for the operation of a neuroprosthesis and to a device therefor.
In the context of the present patent application, neuroprostheses are understood as meaning devices for use in contact or active connection (unidirectional or bidirectional influencing through release of active ingredients) with the central nervous system inside the skull, the spinal cord (or peripheral nerves connected to the spinal cord) and also optical prostheses or hearing prostheses with an implanted internal component and a non-implanted external component.
Several known neuroprostheses for the treatment of inter alia functional disorders of the vision system, the hearing system, the intracranial nerve system, the vegetative nerve system, the spinal cord or the peripheral nervous system have an implanted component in contact with nerve tissue in which data transmission is provided between an external and the internal implanted component for the purposes of operation, of functional monitoring or of function determination (see for example WO 98/36793, WO 98/36795 and U.S. Pat. No. 6,002,996).
Several systems have been disclosed for protected data transmission between mutually remote components by inter alia mobile radio, satellite communication or in local computer networks or the Internet in for example telebanking, private car theft protection or telesurgery (see for example DE 2618401, U.S. Pat. No. 5,646,456, U.S. Pat. No. 5,734,330, U.S. Pat. No. 5,940,515, U.S. Pat. No. 5,930,362, U.S. Pat. No. 5,963,621, U.S. Pat. No. 5,940,799, DE 19630920 and EP 0946018).
Known implants for inter alia personal identification or animal tracking location are in signal connection with an external component without contact to nerve tissue (see for example U.S. Pat. No. 4,399,821, U.S. Pat. No. 4,909,250, U.S. Pat. No. 5,855,609, WO 97/00708, EP 0896828 A2 and WO 98/29160).
Implants as heart pacemakers whose operating state can be checked and/or altered by means of an external component are known (see for example in U.S. Pat. No. 4,361,153, DE 2944542, U.S. Pat. No. 5,871,451 and U.S. Pat. No. 5,891,178).
There are known encrypting and decrypting devices and methods for avoiding unauthorized access to digital or analogue data transmissions inter alia in industrial or medical-engineering fields of application (see for example U.S. Pat. No. 4,766,516, U.S. Pat. No. 5,365,225, U.S. Pat. No. 5,696,825, U.S. Pat. No. 5,987,440 and WO 98/10836).
Available neuroprostheses or neuroimplants (eg a cochlear implant for deaf people or a retina implant with learning capability for blind people with retina degeneration) do not have an access control that makes access to data and operation dependent on a specific authorization. In principle, the external components specially adjusted for a given implant wearer can be exchanged or mixed up and thus result in appreciable and possibly harmful functional disturbances in the industrial and/or biomedical field.
Furthermore, personal data requiring protection and/or implant data can be retrieved and/or altered without authorization to the detriment of the implant wearer and/or of the implant producer/operator.
A communication connection between the implanted component permanently connected to the implant wearer and an essentially exchangeable external component is typically set up for neuroprosthesis operation without prior attention having been paid to protection against unauthorized data access, etc. Therefore the basic risk of misuse or of an unintenional incorrect operation is significant.
It is therefore an object of the present invention to eliminate these disadvantages and to disclose a neuroimplant protection system (NIS) that prevents unauthorized access.
Thus viewed from one aspect the present invention provides a method for operating a neuroprosthesis in the central nervous system within the skull, the spinal cord or peripheral nerves connected to the spinal cord, or a vision prosthesis or hearing prosthesis, the neuroprosthesis comprising an implanted internal component and a non-implanted external component, a wireless data transmission being provided between the internal component and the external component, wherein some or all of the data are transmitted only if an authorization signal transmitted by the external component to the internal component has been tested and accepted. Because data transfer or operation of the neuroprosthesis can take place only after authorization with the aid of the implanted component, numerous conceivable types of operational misuse may be prevented.
The preferred embodiments of the authorization procedure are based on essential functional features of the implanted component that cannot be discovered, copied or simulated in the implanted state.
Since the authorization procedure requires the interaction of the implanted component with the external component, data stored in protected form in the internal, the external and (optionally) a further internal component can include not only data for the purpose of the neuroprosthesis but also (for example) economically or legally important personal data.
The construction and authorization system of a neuroprosthesis designed in this way prevents the copying of the individual components.
The neuroimplant protection system (NIS) of the invention provides a neuroprostheses expanded or additionally equipped with a number of important advantages over known neuroprostheses without NIS. This substantially increases the operational security and the acceptance of neuroprostheses in relation to inter alia data protection and protection against operating faults. For the first time, the present invention provides a system that prevents operational misuse by coupling the implanted component to external components not specifically assigned. This ensures for the first time that the implant wearer is protected against functional injury owing to the use of wrong components.
The present invention makes it possible for the first time to prevent unauthorized access to data and function determination of the implanted components for neuroprostheses equipped with NIS.
Furthermore the present invention makes it possible for the first time to prevent unauthorized access to data transmission between external and implanted components. As a result, the personal data protection of the implant wearer (which in the case of conventional treatment methods is protected by inter alia the attendant doctor""s duty of confidentiality) is secured permanently for the first time in relation to the neuroprosthesis operation.
In addition, the present invention can prevent the unauthorized access to important functional properties of the neuroprosthesis. This prevents the unauthorized imitation (reverse engineering) of components of the neuroprostheses because (on the one hand) important functional properties are lacking for the unauthorized imitation and (on the other hand) such unauthorized imitation components cannot be operated with the remaining components because of lack of compatibility and lack of authorization of the component communication.

Device for the protected operation of neuroprostheses and method therefor


A control arrangement for an arrangement of hydraulically couplable machines has an interface for hydraulic coupling of the machines, and a hydraulic machine for supplying pressure medium to at least one hydraulic consumer of the machines in accordance with requirements. An electronic control device is configured, in accordance with a load of the consumer, to generate an electronic control signal, which can be converted by a transducer of the control arrangement into a hydraulic control signal, in accordance with which the hydraulic machine can be operated.

An arrangement of mobile machines has at least two mobile machines. In general, one of these is a towing machine, e.g. a tractor, and another is a towed machine, e.g. a harvest wagon. Both machines have at least one hydraulic consumer, e.g. a hydraulic motor or a hydraulic cylinder, which needs to be supplied with pressure medium. For this purpose, a hydraulic pump or hydraulic pump unit is arranged on the machine designed as a towing machine and supplies the consumers of the towing machine. Via a hydraulic interface, which is also known as a "power beyond", it is also possible to supply the consumers of the towed machine.
In order to provide the pressure medium in accordance with requirements, that is to say, in particular, in a manner tailored to the load pressure of the consumers, arrangements of this kind are generally fitted with load sensing systems for the working hydraulics. In such systems, the highest of the load pressures of the various consumers is determined by means of a hydraulic selection device, e.g. a shuttle valve cascade, and indicated to the hydraulic pump. A pump controller then adjusts the pump pressure provided by the hydraulic pump in accordance with the highest load pressure to a value which is equal to the highest load pressure plus a differential pressure. This makes available the required pressure difference that enables the consumers to be supplied via control valves with sufficient pressure medium and to overcome the flow losses of the hydraulic lines.
In conventional practice, the differential pressure at the pump controller is set to a fixed value of between 20 and 40 bar. In this case, the level of the value set is affected by the line cross sections, the length thereof and the pressure medium volume flows appropriate to the purpose of the consumers since it is on these that the flow losses depend.
A fundamental document on the load-sensing control of a hydraulic pump for a hydrostatic drive system is German Laid-Open Application DE 10 2005 059 240 A1. In this document, the load pressure is detected electronically and indicated to an electronic control device. In this way, it is possible to avoid long hydraulic load-indicating lines to the controller of the hydraulic pump. These have the problem, for example, that the hydraulic load pressure signal indicated via said lines may be prone to oscillate owing to a slight but nevertheless present compressibility of the pressure medium and an elasticity of the lines. This makes it more difficult to indicate a load to the hydraulic pump in a manner which is reliable in terms of the process involved, and it can have a disadvantageous effect on the control behavior of the pump controller. These problems can be counteracted by the use of electronic load indication and processing. Moreover, electronic processing of the indicated load pressure enables said pressure to be modified or falsified in order to control the pump as desired.
The use of electronic load indication, further processing and modification of the originally hydraulic load pressure signal is shown by document DE 10 2014 103 932 B3. In this case, the towed machine has arranged on it a control device which electronically processes for a difference of the pump pressure and the highest load pressure of the consumers of said machine and indicates this difference electronically to a hydraulic pump controller. Hydraulic operation of a hydraulic adjusting element of the adjustable hydraulic pump is then performed in accordance with the electronic signal by means of the pump controller. In order to keep the hydraulic lines for load indication relatively short and to limit the abovementioned effect, electronic detection of the load pressure of the consumers of the towed machine is performed in the region of said machine in this case.
The disadvantage with this solution is that, when using various or several towed machines, the advantage of electronic processing of the load pressure signal is only available if each of the towed machines is fitted with a control device in this way. This represents a considerable outlay in terms of equipment and therefore there is an obstacle to equipping towed machines for electronic detection of the load pressure.

Hydraulic control arrangement for an arrangement of mobile machines, and arrangement of mobile machines


The present invention provides mice which are deficient in the normal expression of one or more members of the RAR or RXR class of receptors, to mice heterozygous for such deficiency, and to cell lines, preferably pluripotent or totipotent cell lines, which are heterozygous or homozygous for such deficiency. The present invention further provides the use of any of the above mice and cell lines in situations where the absence of at least one RAR or RXR receptors, or the normal expression thereof, is desirable.

1. Field of the Invention
The present invention relates to the fields of retinoic acid receptor (RAR) biology and transgenic mice. Specifically, the present invention relates to mice which are deficient in the normal expression of one or more of the genes encoding members of the RAR or RXR class of receptors, to mice heterozygous for such deficiency, to cell lines, preferably pluripotent or totipotent cell lines, which are heterozygous or homozygous for such deficiency, and to methods of using said mice or said cell lines to identify agonists and antagonists of specific members of the RAR or RXR class of receptors.
2. Description of the Related Art
It has long been established that retinoids (vitamin A derivatives) are crucial for normal growth, vision, maintenance of numerous tissues, reproduction and overall survival (Wolbach, S. B., and Howe, P. R., J. Exp. Med. 42:753-777 (1925); for reviews and refs see Sporn et al., The retinoids, Vols. 1 and 2, Sporn et al., eds., Academic Press, Orlando, Fl. (1984); Livrea and Packer, in Retinoids, Livrea and Packer, eds., Marcel Dekker, New York (1993)). In addition offspring of vitamin A deficient (VAD) dams exhibit a number of developmental defects, indicating that retinoids are also important during embryogenesis (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953)). With the exceptions of vision (Wald, 1968) and possibly of spermatogenesis in mammals (Thompson et al., Proc. Royal Soc. 159:510-535 (1964); van Pelt, H. M. M., and De Rooij, D. G., Endocrinology 128:697-704 (1991); and refs therein), most of the effects generated by VAD in fetuses, young, and adult animals can be prevented and/or reversed by retinoic acid (RA) administration (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953); Thompson et al., Proc. Royal Soc. 159:510-535 (1964)). The dramatic teratogenic effects of maternal RA administration on mammalian embryos (Shenefelt, R. E., Teratology 5, 103-108 (1972); Lammer et al., N. Eng. J. Med. 313:837-841 (1985); Webster, W. S. et al., J. Cranofac. Genet. Dev. Biol. 6:211-222 (1986); Kessel and Gruss, Cell 67:89-104 (1991); Kessel, M., Development 115:487-501 (1992); Creech Kraft, J., xe2x80x9cPharmacokinetics, placental transfer, and teratogenicity of 13-cis-retinoic acid, its isomer and metabolites,xe2x80x9d In Retinoids in Normal Development and Teratogenesis, G. M. Morriss-Kay, ed., Oxford University Press, pp. 267-280 (1992)), and the spectacular effects of topical administration of retinoids on embryonic development of vertebrates and limb regeneration in amphibians (Mohanty-Hejmadi et al., Nature 355:352-353 (1992); for review and refs see Tabin, C. J., Cell 66:199-217 (1991)), has markedly contributed to the belief that RA could in fact be a morphogen (conferring positional information during development), and may also play a critical role during organogenesis.
With the exception of visual perception (Wald, G. et al., Science 162:230-239 (1968)), the molecular mechanisms underlying the highly diverse effects of retinoids has remained obscure until recently. The discovery of nuclear receptors for RA (Petkovich et al., Nature 330:444-450 (1987); Giguxc3xa8re et al., Nature 330:624-629 (1987)) has greatly advanced the understanding of how these simple molecules could exert their pleiotropic effects (for reviews see Leid et al., TIBS 17:427433 (1992); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992)). It is thought that the effects of the RA signal are mediated through two families of receptors which belong to the superfamily of ligand-inducible transcriptional regulatory factors that include steroid/thyroid hormone and vitamin D3 receptors (for reviews see Evans, R. M., Science 240:889-895 (1988); Green and Chambon, Trends Genet. 4:309-314 (1988); Beato, M., Cell 56:335-344 (1989); Gronemeyer, H., Ann. Rev. Genet. 25:89-123 (1991); de Luca, L. M., FASEB J. 5:2924-2933 (1991); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992); Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433 (1992)).
The RAR family (RARxcex1, xcex2 and xcex3 and their isoforms) are activated by both all-trans and 9-cis RA, whereas the retinoid X receptor family (RXRxcex1, xcex2 and xcex3) are activated exclusively by 9-cis RA (for review and refs see de Luca, L. M., FASEB J. 5:2924-2933 (1991); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992); Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427433 (1992); Kastner et al., xe2x80x9cThe role of nuclear retinoic acid receptors in the regulation of gene expression,xe2x80x9d in Vitamin A in health and disease, R. Blomhoff, ed., Marcel Dekker, New York (1993); Allenby et al., Proc. Natl. Acad. Sci. USA 90:30-34 (1993)). Within a given species, the DNA binding (region C) and the ligand binding (region E) domains of the three RAR types are highly similar, whereas the C-terminal region F and the middle region D exhibit no or little similarity. The amino acid sequences of the three RAR types are also notably different in their B regions, and their main isoforms (xcex11 and xcex12, xcex21 to xcex24, and xcex31 and xcex32) further differ in their N-terminal A regions (reviewed in Leid et al., TIBS 17:427-433 (1992)). Similarly, the RXRs characterized to date also markedly differ in their N-terminal A/B regions (Leid et al., TIBS 17:427-433 (1992); Leid et al., Cell 68:377-395 (1992); Mangelsdorf et al., Genes and Dev. 6:329-344 (1992)). Amino acid sequence comparisons revealed that the interspecies conservation of a given RAR or RXR type is greater than the similarity found between the three RAR or RXR types within a given species (reviewed in Leid et al., TIBS 17:427433 (1992)). This interspecies conservation is particularly striking in the N-terminal A regions of the various RARxcex1, xcex2 and xcex3 isoforms, whose A region amino acid sequences are very divergent from each other. Taken together with the distinct spatio-temporal expression patterns observed for the transcripts of each RAR and RXR type in the developing embryo and various adult mouse tissues (Zelent, A., et al., Nature 339:714-717 (1989); Dollxc3xa9 et al., Nature 342:702-705 (1989); Dollxc3xa9 et al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990); Ruberte et al., Development 111:45-60 (1991); Mangelsdorf et al., Genes and Dev. 6:329-344 (1992)) this interspecies conservation has suggested that each RAR and RXR type (and isoform) may perform unique functions. This hypothesis is further supported by the finding that the various RAR isoforms and RXR types contain two transcriptional activation functions (AFs) located in the N-terminal A/B region (AF-1) and in the C-terminal E region (AF-2), which can synergistically, and to some extent differentially, activate various RA-responsive promoters (Leid et al., TIBS 17:427-433 (1992); Nagpal et al., Cell 70:1007-1019 (1992); Nagpal et al., EMBO J., in press (1993)). Moreover, it has been shown that activation of RA-responsive promoters likely occurs through RAR:RXR heterodimers rather than through homodimers (Yu, V. C. et al., Cell 67:1251-1266 (1991); Leid et al., Cell 68:377-395 (1992b); Durand et al., Cell 71:73-85 (1992); Nagpal et al., Cell 70:1007-1019 (1992); Zhang, X. K., et al., Nature 355, 441-446 (1992); Kliewer et al., Nature 355:446-449 (1992); Bugge et al., EMBO J. 11:1409-1418 (1992); Marks et al., EMBO J. 11:1419-1435 (1992); for reviews see Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433 (1992); Laudet and Stehelin, Curr. Biol. 2:293-295 (1992); Green, S., Nature 361:590-591 (1993)). Thus, the basis for the highly pleiotropic effect of retinoids may reside, at least in part, through the control of different subsets of retinoid-responsive promoters by cell-specifically expressed heterodimeric combinations of RAR:RXR types (and isoforms), whose activity may be regulated by cell-specific levels of all-trans and 9-cis RA (Leid et al., TIBS 17:427-433 (1992).
The apparently ubiquitous distribution of RARxcex1 transcripts (mainly the RARxcex11 isoform; Zelent, A., et al., Nature 339:714-717 (1989); Leroy et al., EMBO J. 10:59-69 (1991); Leroy et al., Proc. Natl. Acad. Sci. USA 88:10138-10142 (1991); Dollxc3xa9 et al., Nature 342:702-705 (1989); Dollxc3xa9 et al., Development 110:1133-1151 (1990); Ruberte et al., Development 111:45-60 (1991); E. Ruberte, P. Dollxc3xa9, D. Decimo and P. C., unpublished results) during development and in adult tissues suggests that RARxcex11 may play some general housekeeping function (Brand et al., Nucl. Acid Res. 18:6799-6806 (1990); Leroy et al., EMBO J. 10:59-69 (1991)). RARxcex2 transcripts exhibit a more restricted pattern of distribution in developing embryos and adult tissues, suggesting that RARxcex2 isoforms could be involved in the differentiation of certain epithelia, as well as in the ontogenesis of the nervous system (Dollxc3xa9 et al., Development 110:1133-1151 (1990); Ruberte et al., Development 111:45-60 (1991); Mendelsohn et al., Development 113:723-734 (1991)). In situ hybridization studies indicate that RARxcex3 transcripts are apparently restricted to the presomitic caudal region of day 8.0 p.c. embryos, and to the frontonasal mesenchyme, pharyngeal arches, sclerotomies and limb bud mesenchyme at day 8.5 to 11.5 p.c. At later stages, RARxcex3 transcripts are found in precartilaginous condensations (day 12.5 p.c.), with subsequent restriction to cartilage and differentiating squamous keratinizing epithelia (day 13.5 p.c.), regardless of their embryonic origin (Dollxc3xa9 et al., Nature 342:702-705 (1989); Dollxc3xa9 et al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990)). These observations suggest a role for RARxcex3 in morphogenesis, chondrogenesis and differentiation of squamous epithelia (Dollxc3xa9 et al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990)). In addition, Northern blot analysis indicates that the RARxcex32 isoform is the predominant isoform in the early embryo (day 8.5 to 9.5 p.c.), whereas RARxcex31 is the predominant RARxcex3 isoform transcript found later in embryogenesis as well as in newborn and adult skin (Kastner et al., Proc. Natl. Acad. Sci. USA 87:2700-2704 (1990)).
The mouse is the model of preference in the study of the mammalian genetic system, and a great deal of research has been performed to map the murine genome.
It would be of great importance to be able to establish a living model wherein the role of the various members of the RAR class of receptors could be studied in a definitive manner.
Accordingly, it is an object of the present invention to generate strains of mice which do not express, or express at undetectable levels, one or more members of the RAR or RXR class of receptors. Such mice would be of great value for a better understanding of the role each of the members of the RAR or RXR class of receptors because such animals and cell lines would allow direct testing of the function of specific genes, either deleted or reintroduced by transgenesis, and would serve as an assay system to identify compounds which act as antagonists or agonists of specific members of the RAR or RXR class of receptors.
To establish the actual functional role of RAR and RXR isoforms in vivo during mouse development and post-natal life, the present invention describes the generation of transgenic mice, produced via homologous recombination in embryonic stem (ES) cells, in which RARxcex32, all RARxcex3 isoforms, RARxcex11, all RARxcex1 isoforms, RARxcex22, all RXRxcex1 isoforms, or all RXRxcex2 isoforms have been functionally inactivated.
Thus, the present invention provides mice and mouse cell lines which are deficient in the normal expression (either incapable of total or detectable functional expression) of one or more subtypes or specific isoforms of RAR or RXR receptors. Specifically, the present invention describes mice and cell lines which have been genetically altered such that the normal expression of one or more of the genes encoding a subtype or specific isoform of a RAR or RXR receptor has been disrupted such that it no longer encodes functional or detectable levels of the given receptor subtype or isoform.
The invention further provides mice and cell lines which are heterozygous for the above deficiency.
Utilizing one or more of the aforementioned mice or cell lines, the present invention further provides methods of identifying antagonists and agonists of specific members of the RAR or RXR class of receptors. Specifically, the isoform or subtype of RAR or RXR specific for a given agent, and the effects the agent has on inducing RA dependent expression, can be assayed by first incubating an agent with a cell line, a transgenic mouse, or cells or tissues derived therefrom, which is deficient in the normal expression of one or more isoforms or subtypes of RAR or RXR receptors, and then determining the amount of agent bound or determining the level of RA dependent gene expression which is induced in the cell lines, or specific tissues of the transgenic mice.
FIG. 1 (Panels a-d). Generation of RARxcex3 mutant mice.
RARxcex3 locus, RARxcex32 and RARxcex3 targeting constructs, targeted loci, heterozygote inbreeding analysis, and RNase protection analysis.
(Panel a) Diagram of the RARxcex3 locus, targeting constructs and targeted alleles. The RARxcex3 A to F regions, the DNA-binding domain (DBD) and ligand-binding domain (LBD) are shown at the top, as are the alternate promoters (P1 and P2) and alternate splicing of exons (E1-E8) which generate the major RARxcex3 isoforms, RARxcex31 and RARxcex32. The two targeting constructs are shown below the RARxcex3 locus. Plasmid vector sequences are not shown, nor are the constructs drawn to scale. The predicted RARxcex32 and RARxcex3 targeted alleles, the restriction enzyme digests and the DNA probes (Probes 1 and 2) used for Southern blotting are shown below. The locations of the neomycin resistance and HSV tk genes are denoted by GTI-II.NEO and GTI-II.tk respectively. B, BamHI; E, EcoRI; Ea, EagI; K, KpnI.
(Panel b) Southern blot analysis of offspring from intermatings of RARxcex32+/xe2x88x92 (top) or RARxcex3+/xe2x88x92 (bottom) mice. The positions of the wild type (WT,+) and mutant (xe2x88x92) alleles are shown to the right and the size of each allele is shown to the left of each blot. The genotype is indicated above each lane. +/+, WT; +/xe2x88x92, heterozygote; xe2x88x92/xe2x88x92, homozygote. The probes (Probe 1, RARxcex3 analysis; Probe 2, RARxcex32 analysis) and digests (KpnI, RARxcex32; BamHI, RARxcex3) correspond to the probes and restriction digests shown in panel a.
(Panel c) Representation of the strategy for RNase protection analysis of WT and mutated RARxcex3 transcripts. RARxcex31 and RARxcex32 WT transcripts are represented at the top of the figure, with the predicted RARxcex32 and RARxcex3 mutated transcripts shown immediately below. NEO indicates the position of the neomycin resistance gene in each transcript resulting from targeting of the respective cognate alleles. The riboprobe used to detect WT and mutated RARxcex3 transcripts is shown in the middle of the diagram, followed by the protected fragments for both WT and mutated RARxcex31 and RARxcex32 RNAs as indicated on the left. The identity and size in nucleotides (nt) for each protected fragment is shown on the right of the figure. mut, protected fragment derived from RNase protection of mutated RARxcex3 transcripts.
(Panel d) RNase protection analysis of RNA from either day 10.5 p.c. (RARxcex32) or day 13.5 p.c. (RARxcex3) WT, heterozygote and homozygote embryos for either the RARxcex32 (lanes 1-3) or RARxcex3 (lanes 4-6) disruption. The identities of the protected fragments are indicated to the right. The size of the RARxcex32 WT, RARxcex32 mutant or RARxcex31 WT fragments are indicated to the left of each gel. The source of the RNA used in the protection assays was as follows; lane 1, RARxcex32 WT; lane 2, RARxcex32+/xe2x88x92; lane 3, RARxcex32xe2x88x92/xe2x88x92; lane 4, RARxcex3 WT; lane 5, RARxcex3+/xe2x88x92; lane 6, RARxcex3xe2x88x92/xe2x88x92. The Histone H4 protection was included as an internal control for the quantitation and integrity of the RNA samples. The protected fragment for the RARxcex31 mutated transcript obtained from RARxcex3xe2x88x92/xe2x88x92 or RARxcex3+/xe2x88x92 samples was not seen on this autoradiogram due to the small size of the fragment (41 nt; see panel c).
FIG. 2 (Panels a-l). Axial skeletal and tracheal cartilage defects in RARxcex3 null fetuses.
(a to d), lateral view of the occipital, cervical and upper thoracic region of WT (a, c) and RARxcex3xe2x88x92/xe2x88x92 (b, d) skeletons. (a and b), the large arrow in panel b between the basioccipital bone (BO) and the anterior arch of the atlas (AAA) indicates an ossified fusion between these structures (see also FIG. 3a to d); the large arrow in panel b between xe2x80x9cC1xe2x80x9d and the third cervical vertebra indicates a fusion between the neural arches of these vertebrae; AAA*, ectopic anterior arch of atlas; xe2x80x9cC1xe2x80x9d, anterior transformation of the second cervical vertebra to a first cervical identity; xe2x80x9cC6xe2x80x9d, anterior transformation of the seventh cervical vertebra to a sixth cervical identity (see also panels i to l); C1 to C7, first to seventh cervical vertebrae; E, exoccipital bone; T1, first thoracic vertebra; TR, tympanic ring. (c and d), fusion of first and second ribs (large arrow in panel d), fusion and disruption of tracheal cartilaginous rings (T in panel d, compare to T in panel c; see also FIG. 3e and f), and ectopic anterior arch of the atlas (AAA* in panel d, compare to panels a and c). Numbering (1 and 2) indicates the first and second vertebrosternal ribs. (e, f and g), lateral (e and f) and ventral (g) views of WT (e) or RARxcex3xe2x88x92/xe2x88x92 (f and g) skeletons. *8 (panel f) or R8* (panel g) indicates anterior transformation of the eighth thoracic vertebra to a seventh thoracic identity; arrows indicate bilateral fusions between the first and second ribs (panel g, see also panel d). Numbering (1-7 in panels e and f), normal vertebrosternal ribs; R1-R13 (panel g), ribs. (h), the large arrow indicates fusion between the first and second cervical vertebrae (compare to panel a). (i to l), fifth to seventh cervical vertebrae from either a WT (i) or RARxcex3xe2x88x92/xe2x88x92 (j to l) skeletons. (i), normal aspect of fifth, sixth and seventh cervical vertebrae showing normal foramina transversaria (FT) on the fifth and sixth vertebrae, and the normal (bilateral) positioning of the tuberculi anterior (TA) on the sixth vertebra; (j to l), unilateral anterior transformation of the seventh cervical vertebrae to a sixth vertebral identity (xe2x80x9cC6xe2x80x9d; panels j to l) inferred from the presence of ectopic tuberculi anterior on the seventh cervical vertebrae (xe2x80x9cTAxe2x80x9d; panels j to l), and either a complete or partial ectopic foramina transversarium (xe2x80x9cFTxe2x80x9d or large arrow respectively; panel j); unilateral anterior transformation of the sixth cervical vertebra to a fifth cervical identity (xe2x80x9cC5xe2x80x9d; panels j to l) inferred from the lack of a tuberculum anterior (arrowhead, panels j to l). Limbs were removed from the skeletons to facilitate analysis. All skeletons were derived from day 18.5 p.c. fetuses with the exception of the skeleton shown in panel h, which was from an 8 day p.p. animal.
FIG. 3 (Panels a-f). Skeletal defects in RARxcex3 null homoygotes at the cranio-vertebral junction and malformation of the cartilaginous tracheal rings.
(a) and (b) frontal histological sections of WT (a) and RARxcex3xe2x88x92/xe2x88x92 day 18.5 p.c. fetus (b) at the level of the rostral border of the foramen occipital magnum. In the RARxcex3xe2x88x92/xe2x88x92 animal, the basioccipital (BO) is attached to the lateral mass of the first cervical vertebra (C1L) by the transverse ligament of the atlas (TL) due to the persistence of the first hypochordal bar (H1 in panel b; compare with a); C1-C2, fused bodies of the atlas (C1) and axis (C2); C3, third cervical vertebra. (c and d), sagittal median sections in the occipital and upper cervical region of a WT (c) or RARxcex3xe2x88x92/xe2x88x92 (d) day 18.5 p.c. fetus. A persistent first hypochordal bar (H1) joins the basioccipital (BO) to the anterior arch of the atlas (AAA) in the RARxcex3xe2x88x92/xe2x88x92 fetus. D, axis dens. (e and f), sagittal median histological sections through the cervico-thoracic region of a WT (e) or RARxcex3xe2x88x92/xe2x88x92 day 18.5 p.c. fetus (f). Some of the tracheal cartilaginous rings (R) in the RARxcex3xe2x88x92/xe2x88x92 fetus are fused ventrally (large arrow in panel f). A, arytenoid cartilage; C, cricoid cartilage; T, thyroid cartilage; S, sternum; V, vertebrae.
FIG. 4 (Panels a-o). Glandular defects in RARxcex3 null homozygotes (a-k), stratified squamous metaplasia of the epithelia of the seminal vesicles and cranial prostates in 3-month-old RARxcex3xe2x88x92/xe2x88x92 mice.
(a), comparison of ventral views of the left seminal vesicle (SV) and of the cranial prostate (CP) of RARxcex3xe2x88x92/xe2x88x92 and WT littermates. The white color of the WT seminal vesicle is due to the secretion products (S in panel d) of the glandular epithelium which accumulate within the lumen of the organ. The RARxcex3xe2x88x92/xe2x88x92 seminal vesicle is atrophic and does not display the characteristic white color of the functional organ of normal, sexually mature, males. The histology of this specimen is shown in panels h and i. (b and c), view from above (b) and dorsal view (c) of the seminal vesicles (SV) and cranial prostates (CP) of two RARxcex3xe2x88x92/xe2x88x92 mice, dissected with the testes (T), epididymis (E), vas deferens (D) and urinary bladder (U), and photographed at the same magnification. The aspect of the seminal vesicle (SV) and cranial prostate (CP) of RARxcex3xe2x88x92/xe2x88x92 mice varies from atrophic (panel b) to hypertrophic (e.g. the cranial prostate on the right of the picture in panel c). Hypertrophy was always associated with histological signs of inflammation (see panel l). In the specimen shown in panel b, the seminal vesicle and the cranial prostate (SV+CP) are indistinguishable by both anatomical and histological criteria (see panels j and k). (d-g), histological sections of the seminal vesicle (panels d and e) and cranial prostate (panels f and g) of a 3-month-old WT mouse. The wild-type seminal vesicle is an elongated hollow organ (panel d) whereas the wild-type cranial prostate (CP, or coagulating gland) is composed of several tubules (panel f). Both glands display very irregular lumens filled with secretion products (S, panels d-g). Their walls consist of a mucosa, forming multiple folds and septa, and in a peripheral layer of smooth muscle cells (MU, panels e, g). The glandular epithelia (GE, panels e, g) are simple columnar. (h-l), histological sections of the seminal vesicle and cranial prostate of three different RARxcex3xe2x88x92/xe2x88x92 mice; panels h and i, correspond to the RARxcex3xe2x88x92/xe2x88x92 specimen displayed in panel a; in the seminal vesicle (SV), the epithelium is stratified, squamous and nonkeratinized (SQ); in the cranial prostate, foci of normal glandular epithelial cells (GE, enlarged in i) and patches of squamous nonkeratinized metaplasia (SQ) coexist; the lumens of both glands are devoid of secretion products; panels j and k correspond to the specimen displayed in panel b; both glands are replaced by large cysts filled with desquamated keratinized cells (K); the cysts are lined by a stratified squamous keratinizing epithelium (SQK) resembling normal epidermis; panel l shows an histological section of the specimen displayed in panel c; the connective tissue of the seminal vesicle is markedly hyperplastic and infiltrated with leukocytes (L) which also occupy the spaces between the desquamated keratinized cells (K) within the lumen of the gland. (m-o), atrophy of the Harderian glands in 3-month-old RARxcex3xe2x88x92/xe2x88x92 mice; panel m, dorsal view of the left (L) and right (R) Harderian glands of two different RARxcex3xe2x88x92/xe2x88x92 mice; the right glands display a normal aspect: conical-shaped with medially-directed tips; the base of the gland on the upper left of the photograph is atrophic and colored in black: this aspect reflects a lack of part of the glandular epithelium without concomitant disappearance of the melanocyte-rich connective tissue of the gland (see panel o); the unformed black structure displayed on the lower left of the photograph replaced the left Harderian gland in the orbit of this mouse; panels n and o, frontal sections through the orbits of a RARxcex3xe2x88x92/xe2x88x92 mouse at the level of the left (panel n) and right (panel o) Harderian glands; panel n, the histological structure of the Harderian gland is normal: it consists of tubules lined by a simple cuboidal glandular epithelium (GE), and of an intertubular connective tissue containing numerous melanocytes (M); in panel o, the epithelium of the Harderian gland is absent. An intraorbital accumulation of closely-packed melanocytes indicates the place where the gland should have been located. MU, orbital muscle. x54 (d, f, h, j); x134 (l); x268 (n,o); x540 (e, g, i, k).
FIG. 5 (Panels a-h). Retinoic acid-induced skeletal malformations.
(a to d), lateral views of untreated WT (a), day 8.5 p.c. RA-treated RARxcex3xe2x88x92/xe2x88x92 (b), RARxcex3+/xe2x88x92 (c) and RARxcex3+/+ (d) skeletons. C, T, L and S; first cervical, thoracic, lumbar, and sacral vertebrae, respectively. (e to h), higher magnification ventral views of the specimens shown in panels a to d; T14* and T15* in panels f and g indicate additional thoracic vertebrae formed by anterior transformation of the first and second lumbar vertebrae, respectively; L1* in panels f and g indicate anterior transformation of the second lumbar vertebrae to a first lumbar identity; L6* and S1* in panel f denote anterior transformation of the first and second sacral vertebrae to a sixth lumbar and first sacral identity, respectively; the arrow in panel h indicates degenerate ribs; T13, L1, L6 and S1 in panel e, normal position of the last thoracic, first and last lumbar and first sacral vertebrae, respectively. Limbs, which were present in all cases, were removed to facilitate analysis.
FIG. 6 (Panels a-d)
(Panel a) Diagram of the RARxcex1 targeting constructs, wild type RARxcex1 locus, and disrupted alleles. The various regions of the RARxcex1 protein (A-F), the DNA-binding domain (DBD) and ligand-binding domain (LBD) are indicated at the top (Leroy et al., EMBO J. 10:59 (1991)). The alternate promoter (P1 or P2) usage and alternate splicing of exons (E1-E8), which generate the xcex11 and xcex12 isoformis, are also shown. The two targeting constructs are drawn above the wild type (WT) RARxcex1 locus. The RARxcex11 targeting construct (left) has the neomycin resistance gene (NEO) inserted into the A1 region encoded by Exon 3 (E3), and has a HSV-thymidine kinase gene (tk) at its 5xe2x80x2 end. The RARxcex1 targeting construct (right; note that it does not include the tk gene) has the neomycin resistance gene (NEO) inserted into the B region which is encoded by Exon 8 (E8).
The plasmid vector sequences are not shown. The structure of the targeted alleles, and the restriction enzyme digests and DNA probes used for Southern blotting, are indicated below. B, BglII; H, HindIII; K, KpnI; N, NotI; R, EcoRI; RV, EcoRV; S, SpeI; Sa, SalI; X, XbaI.
(Panel b) Southern blots of offspring from intermatings of mice heterozygous for either RARxcex1 or RARxcex11 disruptions. The positions of the wild type (+) and mutant (xe2x88x92) alleles are indicated, as well as their size. Genotypes of the offspring are indicated below. +/+, wild type; +/xe2x88x92, heterozygote; xe2x88x92/xe2x88x92, homozygote. The probes indicated (PROBE 1 and PROBE 2) correspond to the probes diagrammed in panel a.
(Panel c) RNAse protection analysis of RNA from day 13.5 p.c. +/+, +/xe2x88x92 and xe2x88x92/xe2x88x92 embryos for either the RARxcex1 (lanes 1-4) or RARxcex11 (lanes 5-7) disruptions. The identities of the protected fragments (RARxcex1, RARxcex2, RARxcex3, CRABPI, CRABPII, and Histone H4) are indicated by the arrows. In the case of RARxcex2 and RARxcex3 only the protected fragments corresponding to the major isoforms RARxcex22 and RARxcex31 are shown; similar results were obtained for the other isoforms (RARxcex21, xcex23 and xcex24; RARxcex32; data not shown). The source of RNAs used in the protection assays was as follows: lane 1, tRNA (negative control); lane 2, RARxcex1+/xe2x88x92; lane 3, +/+; lane 4, RARxcex1xe2x88x92/xe2x88x92; lane 5, +/+; lane 6, RARxcex11+/xe2x88x92; lane 7, RARxcex11xe2x88x92/xe2x88x92. The Histone H4 RNA protection was included as a control for the integrity and quantitation of the RNA samples.
(Panel d) Western blot analysis of day 13.5 p.c. embryo nuclear proteins isolated from RARxcex1+/+, +/xe2x88x92, and xe2x88x92/xe2x88x92 embryos. Embryos from RARxcex1 heterozygote matings were removed at day 13.5 p.c., the yolk sac taken for DNA genotyping, and each embryo was frozen individually on dry ice and stored at xe2x88x9280xc2x0 C. (Lufkin et al., Cell 66:1105 (1991)).
Nuclear protein extracts were derived from: lanes 1 and 2, transfected Cos-1 cells expressing RARxcex11 and RARxcex12, respectively (positive controls); lane 3, RARxcex1+/+ embryos; lane 4, RARxcex1xe2x88x92/xe2x88x92 embryos; lane 5 and 6, RARxcex1xe2x88x92/xe2x88x92 embryos; lanes 7-10, transfected Cos-1 cells expressing either RARxcex21, xcex22, xcex23 or xcex24 (positive control ); lane 11, RARxcex1+/+ embryos; lane 12, RARxcex1+/xe2x88x92 embryos; lane 13 and 14, RARxcex1xe2x88x92/xe2x88x92 embryos. RARxcex1-specific and RARxcex2-specific antisera were used in lanes 1-6 and 7-14, respectively. 1-5 xcexcg of Cos-1 transfected protein extract and 70 xcexcg of embryo nuclear protein extract was loaded per lane (except in lanes 5 and 13 where xcx9c35 xcexcg protein was loaded). Note that the upper band seen in lanes 3-6 corresponds to a non-specific immunoreaction.
FIG. 7 (Panels a-d). Webbed digits of a 2 week-old RARxcex1 null homozygote offspring. Ventral view of left hind-limbs from 2-week old wildtype (a, c) and RARxcex1 homozygote (b,d) animals. Note in b the presence of skin between digits 2, 3, and 4 resulting in a xe2x80x9cwebbedxe2x80x9d limb. Panels c and d correspond to Alizarin red/alcian blue stained skeletons of the limbs shown in panels a and b. Note in d, the absence of any ossified connection between the phalanges indicating that the xe2x80x9cwebbedxe2x80x9d phenotype did not involve any skeletal fusion. Alizarin red/alcian blue staining of skeletons was performed as described elsewhere (Lufkin et al., Nature 359:835-841 (1992)).
FIG. 8 (Panels a-h). Degenerative lesions in testes of four to five month-old RARxcex1 null homozygotes.
Comparison of histological sections through the testes (panels a-f) and epididymal ducts (panels g-h) of wild type (panels a, d and g) and RARxcex1 null homozygote males (panels b, c, e, f and h), both five month-old. Panel a, the parenchyma of wild type testes (RARxcex1 heterozygote testes were identical) is composed of seminiferous tubules (T) with active spermatogenesis and intertubular spaces containing capillaries (CP) and Leydig cells (L). Note that the aspect of the seminiferous epithelium (or germinal epithelium) varies between tubules at different stages of the spermatogenic cycle; however, all tubules contain primary spermatocytes (C, in panel a). Each of these cells will eventually yield four spermatozoids. Panels b and c, the parenchyma of RARxcex1 homozygote testes shows a patchy pattern of seminiferous tubule lesions. These cover a wide spectrum, ranging from rare tubules with complete spermatogenesis (e.g. T1) to tubules containing only Sertoli cells (e.g. T2) which may be enlarged, thus filling the tubules (e.g., T2 in panel c). A majority of tubules lack primary spermatocytes (C). In addition, the seminiferous epithelium shows numerous large, clear, rounded spaces (vacuole-like, V) and occasional clusters of degenerating spermatogenic cells (large arrow in panel c). In the intertubular spaces, focal hyperplasia of the Leydig cells (L) is observed between atrophic seminiferous tubules (panel c). This hyperplasia is likely to result from the decrease in tubular diameter (compare T3 in panel c with T panel a; see ref. 40). Panels d, e and f, correspond to high magnification micrographs of the walls of seminiferous tubules. Panel d, in wild type testes, the seminiferous epithelium consists of supporting cells, the Sertoli cells (S) and spermatogenic cells. The spermatogenic cells proliferate from stem spermatogonia (G), located in contact with the basement membrane (B), and differentiate from the periphery towards the lumen of the seminiferous tubules. This process yields different ontogenetically-related cell types arranged in concentric layers, i.e. spermatogonia (G), primary spermatocytes (C), round spermatids (D), and maturing spermatozoids (Z). Panels e and f, are two different aspects of the seminiferous epithelium in RARxcex1 null homozygote males. Most frequently, the early stages of spermatogenic cell differentiation (e.g. spermatogonia and primary spermatocytes) are missing (panel e: in such a degenerate epithelium spermatogenesis no longer occurs). In rare cases, all stages of spermatogenic cell differentiation, including the round spermatids (D) and maturing spermatozoids (Z) are seen (panel f). Panel g, section through the tail of a wild type epididymal duct; spermatozoids (Z) fill the lumen. Panel h, section through the tail of a RARxcex1 homozygote epididymal duct; the lumen of the duct contains acidophilic (blue) material which is also present within large vacuoles (V) in the epithelium lining the duct (E), possibly as a consequence of extensive cellular absorption; spermatozoids (Z) are occasionally identified in the lumen.
B, basement membrane of the seminiferous tubules; C, primary spermatocytes; CP, capillaries; D, round spermatids; E, epithelium of the epididymal duct; G, spermatogonia; L, Leydig cells; S, Sertoli cells; T, seminiferous tubules; V, vacuoles; Z, spernatozoids. Organs were immersed-fixed in Bouin""s fluid. Paraffin sections, 5 xcexcm thick, were stained with Groat""s hematoxylin and Mallory""s trichrome.
FIG. 9 (Panels a-b). Targeted disruption of the RARxcex22 locus.
(Panel a) A schematic drawing of the RARxcex2 locus is shown at the top, illustrating the alternatively spliced RARxcex2 isoforms and their respective promoters (Zelent et al., EMBO J. 10:71-81 (1991)). Below is shown the portion of RARxcex22 genomic locus (Exon 4, E4). At the bottom, the RARxcex22 targeting vector and the expected structure of the recombinant mutant allele are shown. Note that in the targeting vector the initial 5xe2x80x2 Xhol site has been destroyed, and replaced by a Sall site derived from the HSV-TK cassette.
(Panel b) Southern hybridization experiments employing a probe derived from sequences 5xe2x80x2 to the targeting vector probe 1, FIG. 9a) or a full-length neomycin gene probe are shown as indicated. Genomic DNA prepared from targeted ES cells (BH1, BH32 and BH45) and a cell line harboring randomly integrated copies of the targeting vector (BH68) were digested with Kpnl, BamHl and Xbal, and subjected to Southern hybridization. Note on the left that DNAs derived from the targeted cell lines contain in addition to the normal allele (the 6.5 kb Kpnl fragment, the 20 kb BamHl fragment and the 15 kb Xbal fragment), the fragments representing the mutant allele (4.3 kb for Kpnl, 9.5 kb for BamHl and 9.0 kb for Xbal) which hybridize with probe 1, while the randomly targeted ES cell DNA (BH68) contains only the wild type alleles. B, X, K, Xh, N and S: BamHl, Xbal, Kpnl, Xhol, Notl and Sall restriction enzyme sites, respectively.
FIG. 10. Southern blot analysis.
Southern blot of DNA derived from one week-old offspring of heterozygous RARxcex22+/xe2x88x92 intercrosses showing the presence of homozygous (xe2x88x92/xe2x88x92, containing just the 4.3 kb Kpnl fragment), heterozygous (+/xe2x88x92, containing both the 4.3 and 6.5 kb Kpnl fragments) and wild type (+/xe2x88x92, containing just the 6.5 kb Kpnl fragment) alleles. Below is shown the distribution of wild type, heterozygous and homozygous mutant one month-old offspring from intercrosses of animals heterozygous for the RARxcex22 mutant allele. Note that the homozygous offspring are present at the expected Mendelian ratio (27%) indicating that there is no post-natal lethality associated with RARxcex22 disruption.
FIG. 11. Lack of RARxcex22 protein in RARxcex22xe2x88x92/xe2x88x92 embryos.
Immunodetection experiments employing nuclear extracts prepared from 14.5 dpc wild type (lanes 1 and 6), heterozygous (lanes 2 and 7) and homozygous (lanes 3 and 8) mutant embryos are shown. At left (lanes 1-5), extracts were subjected to direct immunoblotting with a rabbit polyclonal antibody directed against the F-region of RARxcex1. Lanes 4 and 5 contain extracts prepared from Cos-1 cells transfected with RARxcex11 and RARxcex12 expression vectors, respectively. Note that the 51 kd reactive protein corresponding to the RARxcex11 isoform is present at similar levels in all three embryo extracts (lanes 1-3). At right, the nuclear extracts were immunoprecipitated with a mouse monoclonal antibody specific for the A2-region of the RARxcex22 isoform; the protein precipitate was then subjected to immunoblotting with a polyclonal antibody directed against the RARxcex2 F-region (lanes 6-8). A nuclear extract derived from Cos-1 cells transfected with the RARxcex22 expression vector was migrated along side the immunoprecipitates to serve as a size comparison (lane 9). Note the absence of the 51 kd RARxcex22 protein in the mutant embryo.
FIG. 12 (Panels a-b). RARxcex2, RARxcex1 and RARxcex3 RNA analysis.
(Panel a) RARxcex2 isoform RNAs in wild type, heterozygote and homozygote mutant embryos. A scheme and the results of RT-PCR experiments are shown. Total RNA prepared from wild type (+/+), heterozygous (+/xe2x88x92) and homozygous (xe2x88x92/xe2x88x92) mutants. 13.5 dpc embryos were analyzed for the presence of RARxcex21/xcex23 and RARxcex22 transcripts. Following RT-PCR amplification, cDNA products were migrated on 2% agarose gels, and subjected to Southern hybridization employing oligonucleotide probes specific for these RAR isoforms. As a control, a cDNA fragment corresponding to the RARxcex11 isoform was amplified (20 cycles) from the 3 RNA preparations, employing a 5xe2x80x2-primer (primer 5, 5xe2x80x2-ATAGCAGTTCCTGCCCAACAC-3xe2x80x2, spanning nucleotides 589-609 in the RARxcex11 A1-region, Leroy et al., 1991 a) and a 3xe2x80x2 primer derived from the RARxcex1 C-region (primer 6, 5xe2x80x2-GATGCTTCGTCGGAAGAAGC-3xe2x80x2; spanning nucleotides 908-927) to generate an expected product of 338 nt. Note that all 3 embryo RNA preparations contained similar levels of the 338 nt RARxcex11 cDNA (shown at the right). To detect the presence of RARxcex22 transcripts (Zelent et al., EMBO J. 10:71-81 (1991), a 5xe2x80x2-primer corresponding to nucleotides 504-523 in the RARxcex22 A2-region (primer 2, 5xe2x80x2-GATCCTGGATTTCTACACCG-3xe2x80x2) and a 3xe2x80x2 primer spanning nucleotides 716-736 in the RARxcex2 C-region (primer 1,5xe2x80x2-TGGTAGCCCGAGACTTGTCCT-3xe2x80x2) were employed to generate a 232 nt cDNA product (shown at right, note the product is present in equal amounts in wild type, heterozygous and homozygous RARxcex22 mutant RNAs). To test for the possible presence of wild-type RARxcex22 transcripts in RARxcex22 mutants, a 5xe2x80x2 primer (primer 3, spanning nucleotides 369-388 in the RARxcex22 5xe2x80x2-UT, 5-GCGAGAGTTTGATGGAGTTC-3xe2x80x2) and primer 1 located in the RARxcex2 C-region were employed to amplify a 367 nt wild-type product. Note that this product is detectable in wild type embryo RNA, while it is undetectable in homozygote RNAs. To detect the presence of RARxcex21/xcex23 transcripts (Zelent et al., EMBO J. 10:71-81 (1991)) in the 3 RNA preparations, a primer located at positions 469-488 in the common RARxcex21/xcex23 A1-region (primer 4, 5xe2x80x2-GAAGCCTGAAGCATGAGCAC-3xe2x80x2) and primer 1 located in the RARxcex2 C-region were employed in 30 cycles of amplification to generate products of 306 nt for the RARxcex21 isoform and 387 nt for the RARxcex23 isoform (shown at the right). Note that RARxcex21 and xcex23 transcripts were present in all 3 RNA preparations.
(Panel b) Determination of RARxcex1 and RARxcex3 isoform RNA in wild type, heterozygote and homozygote mutant embryos using RNAse protection assays. A schematic illustration of the probes and expected protected fragments are shown. Total RNA prepared from 13.5 dpc wild-type (+/+), heterozygous (+/xe2x88x92) and homozygous (xe2x88x92/xe2x88x92) RARxcex22 mutant embryos was employed in the reactions. Riboprobes were as follows (Lufkin et al., Proc. Natl. Acad. Sci. USA 90:7225-7229 (1993)): the RARxcex12 antisense probe included the region corresponding to the RARxcex12 initiation codon through the RARxcex1 C-region to generate protected RNA fragments of 379 nt for RARxcex12 and 210 nt for RARxcex11; the RARxcex32 antisense probe spanned the RARxcex32 A2-region through the C-region to generate protected RNA fragments of 345 nt (RARxcex32) and 154 nt (RARxcex31). The histone 4 antisense riboprobe used as an internal control generated a 130 nt RNA fragment. Control lanes correspond to the protected RNA fragments obtained with in vitro transcribed sense RNA.
FIG. 13 (Panels a-c). RARxcex22 promoter activity in wild type and RARxcex22 null mutant embryos.
(Panel a) A whole mount of a 10.5 dpc wild-type embryo expressing the RARxcex22/lacZ transgene stained for xcex2-galactosidase activity. Note that promoter activity is visible in the caudal hindbrain up to the rostral boundary of rhombomere 7 (arrowhead).
(Panels b and c) Whole mounts of RARxcex22 null embryos (littermates) expressing the RARxcex22 promoter/lacZ transgene exposed to RA in utero at 10.5 dpc and stained for xcex2-gal activity 4 hours later. Note that the RA-induced rostral shift in promoter activity is similar in the hindbrains of both mutant and wild type embryos (denoted by arrowheads at the approximate level of rhombomere 1 in b and c, see text). Note also the increased labeling of neural crest cells migrating towards the heart (arrow). Abbreviations: ot, otocyst; nt, neural tube.
FIG. 14 (Panels a-h). Alteration of the pre-otic hindbrain in RA-treated RARxcex22 null embryo.
(a-e) show different views of a RARxcex22+/+ (wild type) embryo and of a RARp2xe2x88x92/xe2x88x92 mutant embryo which were exposed to RA at 7.25 dpc, collected at 9.0 dpc and hybridized as whole-mounts to a Hoxb-1 probe. (a) and (b), profile views; (c) and (d), hindbrain viewed from the back; (e), ventral view of the mutant embryo. Note that the forebrain vesicles are partially truncated in the wild type embryo (panel a) and almost lacking in the mutant embryo (panel b). The Hoxb-1 signal extends rostrally almost to the extremity of the neuroepithelium. (f-h), three parallel sagittal sections of a RARxcex22xe2x88x92/xe2x88x92 mutant emryo, which had been subjected to the same RA treatment. The plane of the sections progress from lateral (f) to medial (h) regions. Two neighboring sections hybridized to the Hoxb-1 (middle column) or the Krox-20 (right column) probe are shown under dark-field illumination (signal grains appear white). Only rhombomeres whose boundaries can be tentatively identified are indicated. Hoxb-1 transcripts extend up to the rostral extremity of the neuroepithelium, and a severe truncation of the forebrain is also evident on these sections (panels g and h). ot, otocyst; h, heart.
FIG. 15 (Panels a-c. RA-induced limb malformations in wild type and RARxcex22 null mutant fetuses.
Embryos were exposed to RA by maternal gavage with all-trans RA (80 mg/kg) at 11.5 dpc, fetuses were collected at 18.5 dpc, and stained with alcian blue and alizarin red to visualize cartilage and bone.
(Panel a) A whole-mount of an alcian blue/alizarin red stained forelimb from a wild type 18.5 dpc fetus. Limbs from RA-treated 18.5 dpc fetuses are shown in b and c.
(Panel b) A forelimb from an RA-treated RARxcex22 null fetus.
(Panel c) A forelimb from an RA-treated wild type fetus. Note that the 5th digit is absent in both wild type and mutant RA-treated fetuses, and that the radii and ulnae are truncated and malformed in similar fashions. H, humerus, R. radius, U, ulna, I, IV and V, 1st, 4th and 5th digit, respectively.
FIG. 16 (Panels a-d). Targeting the RXRxcex1 Gene.
(Panels a) A map of the RXRxcex1 genomic region of interest is shown on top. Probe A is a 2 kb BamHl fragment (the 5xe2x80x2 BamHl site corresponds to the 5xe2x80x2 end of the genomic clone (arrow)); probe B is the 2 kb Hindlll-Smal fragment. Bg: Bglll; Sp:Spel; B:BamHl; H: Hindlll; S: Smal; RV: EcoRV; Xb: Xbal.
(Panel b) Detection of wild-type (WT) and mutant alleles by Southern Blot analysis. Placental DNAs from 12.5 dpc embryos recovered from a cross between heterozygote mice were digested with BamHl and Bglll and analyzed by Southern Blot with probe B.
(Panel c) Northern Blot analysis. 30 xcexcg of total RNA from WT (+/+), heterozygote (+/xe2x88x92) and homozygote (xe2x88x92/xe2x88x92) embryos was analyzed by Northern Blotting with a complete RXRxcex1 cDNA probe or the 36B4 constant probe.
(Panel d) Western blot analysis. 15 xcexcg of nuclear extract prepared from +/+ (lane 2), +/xe2x88x92 (lane 3) or xe2x88x92/xe2x88x92 (lane 4) 12.5 dpc embryos were analyzed by Western blot with an anti-RXRxcex1 polyclonal antibody directed against the N-terminal A/B region. The absence of signal in the xe2x88x92/xe2x88x92 lane is not due to an absence of protein in the corresponding extract, since staining of the same membrane with amido black revealed a similar pattern of proteins in all 3 extracts (not shown).
FIG. 17. Distribution of the weight of wild type and heterozygous animals.
Mice derived from crosses between WT and heterozygous parents were weighed at 2-3 weeks of age. The weight of each animal was expressed as the ratio of its weight relative to the average weight of WT animals of the same litter. Weight ratio were then grouped within classes differing from each other by 0.05 ratio increment (abscisa). The numbers of heterozygote (white bars) and WT animals (black bars) in each class is indicated on the ordinate.
FIG. 18 (Panels a-h). External appearance of RXRxcex1 null mutant fetuses.
Comparison of external features between 14.5 dpc WT (a and c), RXRxcex1xe2x88x92/xe2x88x92 (b and d), RXRxcex1/RARxcex3 (d, e and f) and RXRxcex1/RARxcex1(c) mutant fetuses.
(Panels a and b) Lateral view of WT (a) and RXRxcex1xe2x88x92/xe2x88x92 (b). The arrows in the WT (a) indicate blood vessels which are not visible in the RXRxcex1 mutant. The arrow in the RXRxcex1xe2x88x92/xe2x88x92 (b) points out the abnormal eye.
(Panels c-e) In RXRxcex1xe2x88x92/xe2x88x92 (d) and RXRxcex1+/xe2x88x92RARxcex3xe2x88x92/xe2x88x92 mutants (e) the palpebral fissure, limited by the dorsal and ventral eyelids (DE and VE), is smaller than in the WT (c); and the ventral retina is occulted by the ventral eyelid (VE).
(Panel f) In the RXRxcex1xe2x88x92/xe2x88x92RARxcex3+/xe2x88x92 the palpebral fissure is smaller than in the RXRxcex1xe2x88x92/xe2x88x92 (d) and the ventral retina is not visible.
(Panel g) In the RXRxcex1xe2x88x92/xe2x88x92RARxcex3xe2x88x92/xe2x88x92 fetus there is no palpebral fissure and all the eye is masked by the surface ectoderm and a layer of mesenchyme.
(Panel h) In the RXRxcex1xe2x88x92/xe2x88x92RARxcex1+/xe2x88x92 fetus a unilateral coloboma of the iris (CI) is apparent.
Abbreviations: Cl, coloboma of the iris; DE, dorsal eyelid; DR, dorsal retina; P, pupilia; VE, ventral eyelid.
FIG. 19 (Panels a-h). Heart defects in RXRxcex1 null mutant.
Comparison of the heart in WT (a, c, e and g) and RXRxcex1xe2x88x92/xe2x88x92 (b, d, f and h) embryos.
(Panels a and b) Frontal sections of 14.5 dpc fetuses at level of the right atrioventricular canal (AV).
(Panels c and d) Sections of 10.5 dpc embryos at the level of the atrioventricular endocardial cushions (EC).
(Panels e and f) Detail of the ventricular wall in 16.5 dpc fetuses.
Abbreviations: A, atrium; AO, aorta; AV, atrioventricular canal; CL, compact layer; CT, cono truncus; EC, endocardial cushions; VS, ventricular, septum; IVC, interventricular communication; LV, left ventricle; PT, pulmonary trunk; RA, right atrium; RV, right ventricle; T, trabeculation of the myocardium wall; TL, trabecular layer; V, ventricle. Magnifications: 55xc3x97 (a and b), 138xc3x97 (c, d, e, f, g and h).
FIG. 20 (Panels a-d). Electron microscopy of the heart of RXRxcex1 null mutants.
Ultrastructural features of the outer portion of the ventricular myocardium in WT (a and b) and RXRxcex1xe2x88x92/xe2x88x92 (c and d) 14.5 dpc fetuses.
(Panel a) Low power magnification of the outer compact layer of the WT ventricular myocardium.
(Panel b) Higher magnification of the demarcated area in (a), showing that the myofibrils (M) are present in the cytoplasm, but do not display the sarcomeric organization (S in d). These myocardial cells also contain rough endoplasmic reticulum (RER) and abundant polyribosomes (P). No sarcoplasmic reticulum (SR in d) was visible in this outer layer of the myocardium.
(Panel c) RXRxcex1xe2x88x92/xe2x88x92 myocardium comparable area and magnification as shown in (a) for WT.
(Panel d) Higher magnification of the demarcated area in (c). The cells in the outer area of the myocardium exhibit myofibrils; some of them are organized in sarcomeres (S). These cells also display a well developed sarcoplasmic reticulum (SR). Both, sarcomeres and sarcoplasmic reticulum indicate that these cells are at a more advanced stage of differentiation than the compact layer cells in the WT fetus (a and b).
Abbreviations: E, epicardial cell; En, endothelial cell; M, myofibrils; P, polyribosomes; RER, rough endoplasmatic reticulum; S, sarcomere; SR, sarcoplasmic reticulum. Magnifications: 4000xc3x97 (a and c), 25000xc3x97 (b and d).
FIG. 21 (Panels a-f). Eye development in RXRxcex1 null mutants.
Comparison of the eye development in WT (a, c and e) and RXRxcex1xe2x88x92/xe2x88x92 mutants (b,d and f).
(Panels a and b) Frontal sections through the eye of WT (a) and RXRxcex1xe2x88x92/xe2x88x92 (b) 11.5 dpc embryos.
(Panels c and d) Frontal sections of 12.5 dpc WT (c) and RXRxcex1xe2x88x92/xe2x88x92 (d) embryos.
(Panels e and D) Frontal sections of 15.5 dpc fetuses.
Abbreviations: AC, anterior chamber; C, cornea; C*, thicker cornea; CON, coloboma of the optic nerve; DE, dorsal eyelid; DJ, dorsal conjunctival sac; DR, dorsal retina; F, persistent retrolenticular membrane; FE, fused eyelids; J, conjunctival sac; L, lens; ON, optic nerve; RE, root of the eyelid; SE, surface ectoderm; V, vitreous body; VE, ventral eyelid; VJ, ventral conjunctival sac; VR, ventral retina. Magnifications: 55xc3x97 (e and f), 138xc3x97 (a, b, c and d).
FIG. 22 (Panels a-l). Eye defects in RXRxcex1xe2x88x92/xe2x88x92, RXRxcex1/RARxcex3 and RXRxcex1/RARxcex1 double mutants.
Frontal sections through the eye of 12.5 dpc (e), 14.5 dpc (a, b, c, d, f, g h, i and j) and 16.5 dpc (k and l) fetuses. Genotypes as indicated.
Abbreviations: AC, anterior chamber; C, cornea; C*, thicker cornea; CE, corneal epithelium; CL, corneal-lenticular stalk; CON, coloboma of the optic nerve; CS, corneal stroma; EOM, extraocular mesenchyme; DE, dorsal eyelid; DJ, dorsal conjunctival sac, DR, dorsal retina; ER, eversion of the retina; F, persistent retrolenticular membrane; L, lens; ON, optic nerve; PR, pigment retina; RE, root of the eyelid; SC, sclera; SE, surface ectoderm; V, vitreous body; VE, ventral eyelid; VJ, ventral conjunctival sac; VR, ventral retina. Magnifications: 138xc3x97 (a, b, c, d, e, f and g), 280xc3x97 (h, k and l), 550 xc3x97 (i and j).
FIG. 23 (Panels a-i). Three dimensional computer reconstruction of WT, RXRxcex1xe2x88x92/xe2x88x92 and RXRxcex1xe2x88x92/xe2x88x92RARxcex3xe2x88x92/xe2x88x92 eyes.
Three dimensional computer reconstruction from serial frontal histological sections of the eye of WT (a, d and g), RXRxcex1xe2x88x92/xe2x88x92 (b, e and h) and RXRxcex1xe2x88x92/xe2x88x92RARxcex3xe2x88x92/xe2x88x92 (c, f and i) 14.5 dpc fetuses.
(Panels a, b and c) Dorsal views of the eye; the surface ectoderm has been removed.
(Panels d, e and f) External views of the eye in which the surface ectoderm has been rendered transparent.
(Panels g, h and i) Ventral views of the eye without the surface ectoderm. In the RXRxcex1xe2x88x92/xe2x88x92 (b, e and h) it is possible to appreciate the rotation of the eye around the dorso-ventral axis toward the snout. Note in the external view that the dorsal and ventral eyelids (DE and VE) are closer than in the WT eye (d). In the external view of the RXRxcex1xe2x88x92/xe2x88x92RARxcex3xe2x88x92/xe2x88x92 eye (f) it is possible to see the surface ectoderm invagination of the cornea lenticular stalk (CL) and the eversion of the retina (ER).
Abbreviations: CL, corneal-lenticular stalk; DE, dorsal eyelid; ER, eversion of the retina; L, lens; PR, pigment retina; VE, ventral eyelid.
Magnification: 49xc3x97 (a-i).
FIG. 24 (Panels a-c). The RXRxcex2 mutation. (a) Targeting the RXRxcex2 gene. A map of the WT RXRxcex2 gene is shown on top. Black boxes indicate exons (numbering as in Nagata et al., Gene 142: 183-189 (1994)). Probe A is a NotI-KpnI fragment, probe B is a 500 bp BamHI-HindIII fragment immediately upstream of the deleted region; probe C is the HindIII-EcoRI fragment (corresponding to the deleted region). Sp, SpeI; K, KpnI; N, NotI; B, BamHI; E, EcoRI; Nh, NheI; X, XhoI; H, HindIII. (b) Targeted ES cells. Southern blot analysis of SpeI-restricted DNA from wild type (WT), and the HA67 and HA9 ES cell clones, analysed with the 5xe2x80x2 probe A. (c) Southern blot analysis of mutant mice. The top panel shows the analysis of a litter using BamHI restricted DNA and probe B. The lower panel corresponds to an identical blot hybridized with probe C.
FIG. 25 (Panels a-g). Comparison of spermatozoa from the caudal epididymis in wild type (WT) and RXRxcex2xe2x88x92/xe2x88x92 mutants, as indicated. (a,b) are semi-thin histological sections, illustrating the paucity of mutant spermatozoa. (c) corresponds to a smear, showing three spermatozoa displaying coiling of their tails. (d-g) are thin sections illustrating malformations of the acrosome and mitochondrial sheats in mutant spermatozoa; the insert corresponds to a high magnification of the box in (d). A, acrosomes; E, epididymal epithelium; H, head of spermatozoa, M, mitochondrial sheat; N, nuclei of spermatozoa; X, axoneme. The arrowheads, the large arrows and the open arrows point to the coiled tails and the defects of the acrosomes and mitochondrial sheats, respectively. Magnificationxc3x97860 (a-c), xc3x9710.000 (d-g) and xc3x9725.000 (insert).
FIG. 26 (Panels a-j). Failure of spermatid release and progressive testicular degeneration in RXRxcex2xe2x88x92/xe2x88x92 males. Sections through the testes of wildtype (WT; a, c and e) and RXRxcex2xe2x88x92/xe2x88x92 males (b, d, f, and g-j) at 6 months (a-f), 8 months (g and i) and 12 months (h and j). A, acrosomes; B, basement membrane; L, lipid droplets; P, pachytene spermatocytes; R, preleptotene spermatocytes; S, Sertoli cell nucleus; T, seminiferous tubules; V, vacuoles of degeneration; Y, Leydig cells. The numbers in the white dots refer to the degree of maturation (steps) of the spermatids. Arrowheads in b and f, lipid-containing xe2x80x9cvacuolesxe2x80x9d; unlabelled arrows, retained step 16 spermatids. Note that the focal hyperplasia of the Leydig cells (Y) in (g) and the oedema of the intertubular space in (h) represent classical secondary alterations to seminiferous tubule atrophy. Hematoxylin-trichrome (a, b, g and h), alcian blue (c and d) and periodic acid-Shiff-hematoxylin (e, f, i and j). Same magnifications in a, b, g and h (xc3x97260) and in c-f, i and j (xc3x97860).
FIG. 27. Basal portion of a 6 month-old RXRxcex2xe2x88x92/xe2x88x92 mutant Sertoli cell in a stage IX seminiferous tubule. L, lipid droplet; LI, lysosomes; S, Sertoli cell nucleus; arrow, phagocytized elongated spermatid. Magnification: xc3x979000.
FIG. 28 (Panels a-i). Lipids and lysosomal structures in RXRxcex2xe2x88x92/xe2x88x92 mutant testis. (a-e) and (g-i) represent frozen sections through the testes of 6 month-old wildtype (WT; a, c and g) and RXRxcex2xe2x88x92/xe2x88x92 (b, d, h and i) males. (a) and (b), staining with oil red O. (c) and (d), staining with osmium tetroxide. (e), immunofluorescence staining of a RXRxcex2xe2x88x92/xe2x88x92 seminiferous tubule with the anti-phospholipid antibody (MC22-33F, top) or non-immnune rat IgM (0 control, bottom), on consecutive sections. (f), immunostaining of an antral follicle with MC22-33F (top) or non immune rat IgM (0 control, bottom) on consecutive sections. (g-i), detection of acid phosphatase activity [after staining with oil red O in (i)]. B, level of the basement membrane of the seminiferous tubule; G, granulosa cells; L, lipid droplets in Sertoli cells. N, nuclei; T, seminiferous tubules; Y, Leydig cells. Arrows point to lysosomes in (i). Magnifications: xc3x97130 (a-d, g and h), xc3x97200 (e and f) and xc3x97860 (i).
FIG. 29 (Panels a-h). Lipid accumulation in RXRxcex2xe2x88x92/xe2x88x92 mutant testis: early appearance, prior to the completion of spermatogenesis (29 days) and increase with the age of the animal, as indicated. Photomicrographs of semi-thin sections from osmium-fixed testes without (a and b) or with counterstaining (c-h) with alcian blue. Lipids (L) appear as brown or black dots or spots in the seminiferous tubules (T). Y, Leydig cells. V, vacuoles of degeneration. Magnificationxc3x97170.
FIG. 30 (Panels a-g). Comparison of the localization of RXRxcex2 (b, d) and RXRxcex1 (c, e) transcripts in a wild type adult testis and immunoperoxidase staining with an antibody directed against RXRxcex2 of wildtype (WT; f) and RXRxcex2xe2x88x92/xe2x88x92 (g) testis. (a), bright field and (b,c) dark fields. In (d) and (e), the in situ hybridization signal is shown in false colors after computer processing of a bright field view and dark field view of the same section (see Vonesch et al., Dev. Dyn 199: 199-213 (1994) for further details). In (f) and (g) a strong positive signal is exclusively detected in wildtype Sertoli cells. Immunostaining is absent from wild type germ cells and from mutant Sertoli cells and germ cells. The weak staining of the mutant and wild type Leydig cells (L) corresponds to background staining since it can be observed even when omitting the anti-RXRxcex2 antibody in the immunostaining sequence. EP, epididymis; T, seminiferous tubules; S, Sertoli cells or spermatogonia; P, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. Magnifications: xc3x9717 (a-c) and xc3x97250 (d-g).
FIG. 31 (Panels a-b). Comparison of semi-thin sections from osmium-fixed, resin-embedded testes of the seminiferous tubules of 6 month-old RARxcex1xe2x88x92/xe2x88x92 mutant (a) and vitamin A-deficient (VAD) (b) mice. The VAD males were the F1 offsprings of dams fed a VAD diet, and raised on a VAD diet from the time of weaning. The large arrows point to similar tubules having lost their germ cells and thus containing only Sertoll cells (S); the cytoplasm of the Sertoli cells contains large vacuoles (V) which are devoid of lipids. Y: Leydig cells. Magnification: xc3x97520.

Genetically engineered mice containing alterations in the genes encoding retinoic acid receptor proteins

Institut National de la Sante et de la Recherche Medicale

Centre National de la Recherche Scientifique