text:
- Research article
- Open Access
Sex and the single embryo: early deveopment in the Mediterranean fruit fly, Ceratitis capitata
© Gabrieli et al; licensee BioMed Central Ltd. 2010
- Received: 24 June 2009
- Accepted: 26 January 2010
- Published: 26 January 2010
In embryos the maternal-to-zygotic transition (MTZ) integrates post-transcriptional regulation of maternal transcripts with transcriptional activation of the zygotic genome. Although the molecular mechanisms underlying this event are being clarified in Drosophila melanogaster, little is know about the embryogenic processes in other insect species. The recent publication of expressed sequence tags (ESTs) from embryos of the global pest species Ceratitis capitata (medfly) has enabled the investigation of embryogenesis in this species and has allowed a comparison of the embryogenic processes in these two related dipteran species, C. capitata and D. melanogaster, that shared a common ancestor 80-100 mya.
Using a novel PCR-based sexing method, which takes advantage of a putative LTR retrotransposon MITE insertion on the medfly Y chromosome, the transcriptomes of individual early male and female embryos were analysed using RT-PCR. This study is focused on two crucial aspects of the onset of embryonic development: sex determination and cellular blastoderm formation. Together with the three known medfly genes (Cctransformer, Cctransformer2 and Ccdoublesex), the expression patterns of other medfly genes that are similar to the D. melanogaster sex-determination genes (sisterlessA, groucho, deadpan, Sex-lethal, female lethal d, sans fille and intersex) and four cellular blastoderm formation genes (Rho1, spaghetti squash, slow-as-molasses and serendipity-α) were analyzed, allowing us to sketch a preliminary outline of the embryonic process in the medfly. Furthermore, a putative homologue of the Zelda gene has been considered, which in D. melanogaster encodes a DNA-binding factor responsible for the maternal-to-zygotic transition.
Our novel sexing method facilitates the study of i) when the MTZ transition occurs in males and females of C. capitata, ii) when and how the maternal information of "female-development" is reprogrammed in the embryos and iii) similarities and differences in the regulation of gene expression in C. capitata and D. melanogaster. We suggest a new model for the onset of the sex determination cascade in the medfly: the maternally inherited Cctra transcripts in the female embryos are insufficient to produce enough active protein to inhibit the male mode of Cctra splicing. The slow rate of development and the inefficiency of the splicing mechanism in the pre-cellular blastoderm facilitates the male-determining factor (M) activity, which probably acts by inhibiting CcTRA protein activity.
- Miniature Inverted Repeat Transposable Element
- Female Embryo
- Splice Mechanism
- Maternal Transcript
- Zygotic Gene
The onset of embryonic development is the result of a complex interaction between maternal and zygotic genetic information: during the maternal-to-zygotic transition (MTZ) embryos integrate post-transcriptional regulation of maternal transcripts with transcriptional activation of the zygotic genome [1].
In the model insect Drosophila melanogaster maternal transcripts and proteins are deposited in the egg during oogenesis and two waves of zygotic gene transcription occur in the embryos: the first between 1 and 2 h of development and the second major burst between 2 and 3 h of development [2]. For at least one third of zygotically active genes, the expression profile is coupled to the degradation of the corresponding maternal mRNA. It has been hypothesized that the degradation of maternal mRNA could be due to the activity of Pumilio, of Smaug [3, 4] and of miRNAs [5] or to the presence of a general mediator of mRNA degradation in the mRNA itself, i.e. the AU-rich element [6]. The activation of the earliest zygotic genes is linked to the presence of TAGteam sites in their promoter regions; the zinc-finger protein Zelda (zelda, zld) binds to these sites and triggers the expression of the downstream genes. TAGteam sites have been found in the promoters of genes involved in early developmental processes, including cellular blastoderm formation, sex-determination and the dorsoventral-expression pattern [2].
Although the molecular mechanisms involved in the MTZ transition during the first stages of embryogenesis are being clarified in D. melanogaster [4], little is known about embryogenic processes including sex determination in other insect species. This lack of knowledge does not allow us to determine whether there are features common to all embryogenic processes.
The recent publication of ESTs from medfly embryos has facilitated the investigation of embryogenesis in this species [7]. The medfly is a highly invasive agricultural pest species and belongs to the family Tephritidae that is estimated to have diverged from the family Drosophilidae between 80 and 100 mya [8, 9]. Even though the medfly genome is more than three times larger than that of D. melanogaster, a recent analysis has shown that functionally non-coding sequences are conserved between these two species and that homologous genes, such as the even-skipped gene (eve), have the same expression profile in both species [10]. Thus, it is reasonable to hypothesize that medfly embryogenesis may follow a similar sequence of events as in D. melanogaster. If that is the case, C. capitata could be used as a reference species to study the molecular mechanisms that trigger embryogenesis, as a big genome facilitates the identification of cis-regulator elements through comparative genomic methods [10].
It is known that in the medfly the cellular blastoderm formation occurs between 9 and 11 h after oviposition [11] and one of the major events during early embryogenesis, the molecular mechanism that controls sex-determination, is well studied in C. capitata [12]. In order to detect sex-specific differences in the timing of gene expression and/or mRNA splicing in early developmental stages in the medfly, a primary requirement is the identification of the sex of the early life stages. Male and female embryos are not morphologically distinguishable [13]; however the sexes may be recognized in the early life stages using PCR-based approaches. These sexing assays are based on the repetitive nature of the medfly Y chromosome. Several repetitive DNA sequences have been isolated in medfly that are Y-specific or enriched on the Y chromosome [14]. Copies of these repetitive sequences, which appear to occur in tandemly linked arrays, are distributed over 90% of the length of the long arm of the Y chromosome [15]. The basic structure of the repeats is similar: each unit (1.3-1.7 kb) contains an AT-rich (83-87%) internal region (200 bp or more), called the A-T element. Although three previous approaches [16–18], based on the repetitive nature of the Y chromosome, may be suitable for determining the sex of embryos, an improved method has been developed and is reported in this paper. Using this molecular sexing approach it is possible to study the sex-specific expression of genes in early embryos; we have addressed four principal points: i) when the MTZ transition occurs in C. capitata; ii) when the sexual fate is established at the molecular level; iii) how the maternal information of "female-development" is reprogrammed in the embryos; and iv) how similar or different is the regulation of gene expression in early embryogenesis between C. capitata and D. melanogaster.
Individual samples of C. capitata from five laboratory strains and one natural population were considered in this study. The main laboratory strain used in this work was the ISPRA strain, established in 1968 at the European Community Joint Research Centre (Ispra, Italy), with wild flies from Sicily and Greece. The strain has been maintained in the quarantine facility at the Dept. of Animal Biology, University of Pavia (Italy) since 1979. Other laboratory strains used, maintained in the same insectary, were: Egypt II, obtained from the FAO/IAEA Agriculture and Biotechnology Laboratory (Seibersdorf, Vienna, Austria); Guatemala, obtained in 1990 from MOSCAMED (Guatemala); Israel and Hawaii established from flies collected in the corresponding countries, maintained in our laboratory since 1990 and 1992, respectively. In addition, a sample of wild flies collected in May 2007 from coffee drupes in Ruiru near Nairobi, Kenya, part of the ancestral home range of the species, was used in the study.
Egg and embryo collection
To obtain fertilized and unfertilized eggs, two cages were set up using 200 mated or virgin ISPRA females, respectively. When the flies were five-days old, eggs were collected over a 5 min period. The eggs were maintained at 25°C and 65% humidity for set time intervals. After dechorionation (using 1.5-2% hypochlorite solution), the eggs were repeatedly washed in distilled water and individually transferred to 1.5 ml microcentrifuge tubes.
Nucleic acid preparations
DNA and RNA were isolated from individual embryos using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. The extracted RNA was resuspended in RNAse-free water. Genomic DNA was individually extracted from 3rd instar larvae, 7-day old pupae and 3-day old adult flies using the phenol-chloroform extraction method [19]. Following treatment with RNAse A, the DNA was extracted with phenol/chloroform, precipitated with ethanol and resuspended in TE (10 mM Tris-HCl, pH 8, 1 mM EDTA). The DNA concentration was quantified using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA).
PCR amplifications and agarose gel electrophoresis
Oligonucleotide primers CcYf (5'-gctcgaagacatgcattgaa-3') and CcYr (5'-gacggtaagtgccattcgtt-3') were designed on a known Y-specific sequence of C. capitata [GenBank:AF115330] using Primer3 [20]. PCR amplifications were performed in 10 μl reaction volumes using ~50 ng DNA, 1.5 mM MgCl2, Reaction Buffer (10 mM Tris, 50 mM KCl; pH 8.3), 0.2 mM dNTPs mixture, 10 pmol of each primer and 1 unit Taq DNA polymerase (Invitrogen). Amplification was achieved on an Eppendorf Mastercycler Gradient using the following programme: an initial denaturing step at 94°C for 2 min; 30 cycles of denaturation at 94°C for 30 sec, annealing at 55.8°C for 1 min, extension at 72°C for 45 sec; and a final extension at 72°C for 5 min. The PCR products were analysed on 14 × 10 cm 1.5% agarose gel slabs in 1 × TAE buffer together with a 1 kb and a 100 bp ladder standard (Invitrogen). The bands were visualized by ethidium bromide staining and exposure to UV light. Bands selected for further analysis were eluted from the gel using the PureLink Quick Gel Extraction Kit (Invitrogen).
EST bioinformatic analysis and primer design
Medfly homologues of genes involved in sex-determination and cellularization and their function and embryonic expression in D. melanogaster
Gene, (symbol), GenBank Acc.
Drosophilagene, FlyBase ID
Alignment length (aa)
Identity (%)
Similarity (%)
D. melanogastergene functions
D. melanogastergene expression during early embryogenesis
zinc-finger transcription factor involved in MTZ process and neuronal development
maternal origin
Ccsisterless A
sisterless-a (sisA)
transcription factor forming the primary signal of the sex-determination cascade (XSEs) and involved in endoderm development
expressed in female zygote at the end of nuclear cycle 8 (1:12 h)
deadpan (dpn)
transcription factor forming the primary signal of the sex-determination cascade (autosomal) and involved in dendrite morphogenesis
expressed in the zygote starting from nuclear cycle 12 (2 h)
groucho (gro)
corepressor of transcription forming the primary signal of the sex-determination cascade (autosomal) and involved in dendrite morphogenesis
maternal origin, gro transcripts are detectable until stage 11 after complete gastrulation (5:20 h)
Sex lethal (Sxl)
RNA-binding protein, member of the sex-determination molecular cascade
expressed in the zygote starting from nuclear cycle 12-13 (2 h)
transformer (tra)
RNA-binding protein, member of the sex-determination molecular cascade
maternal origin; an increased amount of tra transcripts is detectable 2-3 h after oviposition
transformer2 (tra2)
RNA-binding protein, member of the sex-determination molecular cascade
maternal origin. No change in tra2 transcript amount is detectable after 2-3 h and in chromosome 2R deleted embryos
336a 248b
68 85
77 91
zinc-finger transcription factor involved in sex differentiation process
dsx transcripts are first detectable during stage 11 (9:20 h) in the fat bodies and gonad primordia
sans fille (snf)
U1 snRNA binding protein involved in sex-determination and oogenesis
the SNF protein localizes to the nucleus in all tissues during development
Ccfemale-lethal d
female lethal d (fl(2)d
RNA splicing factor involved in sex-determination and female-germline sex-determination
maternal origin
intersex (ix)
transcription factor binding protein involved in sex-differentiation process
maternal origin; an increased amount of ix transcripts is detectable 2-3 h after oviposition
Rho1 (Rho1)
protein kinase coupled with GTPase activity involved in cellular-blastoderm formation
maternal origin
spaghetti squash (sqh)
myosin heavy chain binding protein coupled with ATPase activity involved in cellularization and ovarian follicle cell development
maternal origin
slow as molasses (slam)
protein of unknown function involved in cellularization
slam expression rapidly increases during cycle 11 (1:45 h) and peaks during cycle 14 (2:30 h)
Similar to serendipity-α gene product, involved in cellularization
maternal origin
cDNA preparation and RT-PCR
cDNA was generated using the cloned AMV first-strand synthesis kit (Invitrogen) according to the manufacturer's instructions. The RT-PCR mixture and the cycling conditions were as reported above, but the annealing temperature was 59°C. Established primers and cycling conditions were used to amplify the Cctra sequences [22]. For each set of RT-PCR amplifications control reactions were performed excluding reverse transcriptase. RT-PCR amplifications were considered only when these negative controls produced no amplification products. Electrophoresis and visualization of amplification products were performed as described above.
Cloning and Sequencing of PCR and RT-PCR Products
Gel eluted PCR and RT-PCR products were cloned into the PCR®2.1-TOPO® vector using the TOPO TA cloning kit (Invitrogen). Positive colonies were selected and the size of the insert quantified by Eco RI digestion and electrophoresis. Clones were sequenced using an ABI-310 automatic sequencer and the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Foster City, CA, USA).
Sequence Analysis
Nucleotide sequences were aligned using T-coffee [23]. Sequence comparisons were performed using the BLAST family of programs from the National Centre for Biotechnology Information [24]. Identity values were calculated using PAUP 4.0b10 [25].
Southern hybridization analyses
Genomic DNA from individual flies (4 μg) was digested with SspI endonuclease. The digested DNAs were electrophoresed on a 20 × 14 cm 1% agarose gel in 1 × TBE and transferred to a positively charged nylon membrane according to Southern [26]. The membrane was hybridized at 55°C with 200 ng of probe DNA labelled with the Gene Images Alkphos Direct labelling system (GE Healthcare, Little Chalfont, UK) using the random primer method. The hybridization and detection protocols were those described by the manufacturer. Signal detection was performed using CDP-star followed by exposure to autoradiographic film (X-OMAT AR, Kodak).
Chromosome preparation and in situ hybridization
Mitotic chromosome spreads were obtained using medfly third instar larvae [27]. Briefly, brain tissue was incubated in 1% sodium citrate for 10 min at room temperature and transferred to methanol-acetic acid 3:1 solution for 4 min. The material was disrupted in 100 μl 60% acetic acid and dropped onto clean slides and dried. Pre-hybridization was performed according to [15]. In situ hybridization was performed using the following protocol: the probe DNA was labelled using the Biotin High Prime kit (Roche, Basel, Switzerland) and detection of hybridization signals was performed using the Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA, USA) and DAB (3,3'-Diaminobenzidine tetrahydrochloride, Sigma-Aldrich, St. Louis, MO, USA) [28].
Results and Discussion
A putative MITE insertion on the Y chromosome permits a fast sex-assignment
The PCR-based sexing method reported here was designed to easily assign sexual identity to the earlier medfly life stages in order to study sex-specific expression and/or mRNA splicing of early, zygotically transcribed genes. This method follows previous sexing assays [16–18] that used primers to amplify a Y-specific repetitive sequence (Y114). In the previous assays, amplification was limited to the male: the presence of a sex-specific pattern only in males may give a false sex assignment should the amplification fail.
The female 242 bp sequence lacks eight base pairs in the 3' region, that are present in both male sequences, while the male 727 bp sequence contains an additional 477-bp sequence, which is absent from the other two fragments. This indel has no homology to the Y-specific repetitive DNA sequence, but it shares 86-89% identity with two medfly ESTs [7], the functional/biological roles of which are unknown. The presence of terminal inverted repeats in these sequences, their small size, the absence of coding sequence, the potential to form a stable secondary structure, the presence of duplicated target regions in the ESTs and the presence of multiple copies in the medfly genome, as confirmed by Southern hybridization (Additional file 1, Figure S1; Additional file 2, Figure S2), led us to hypothesize that the 467 bp indel sequence is a miniature inverted repeat transposable element (MITE) [30]. It is the insertion of the putative MITE into one or more repetitive units on the Y-chromosome that results in the amplification of the male-specific 727 bp sequence (Figure 2A).
The dynamics of the sex determination molecular cascade in early embryos
The MTZ transition process integrates the post-transcriptional regulation mechanisms of the maternally inherited transcripts and the transcriptional activation and control of the zygotic genome. Particular attention has to be focused on the events that regulate the sex determination process, because the zygote has to reset the information of "female development" inherited from its mother to its own sex-development information. This process involves the activities of transcriptional and of mRNA-splicing regulators.
Three of the medfly genes involved in the molecular sex-determination cascade have previously been identified, Cctra [22, 31], Cctra2 [7, 32], and Ccdsx [33]. Cctra is the key-gene of the sex-determination cascade: it generates mRNAs encoding full-length active proteins only in females and displays an autocatalytic function, which guarantees the female-specific development of cell memory. Cctra, in cooperation with Cctra2, determines the sex-specific splicing of Ccdsx, the transcription factor that is the regulator of the sex-differentiation process.
The presence of different splicing transcripts, including male-specific transcripts, in female embryos indicate that the maternal Cctra transcripts do not produce enough active CcTRA protein to prevent the male mode of splicing in female embryos. Concentration-dependant splicing activity is a general property of proteins belonging to the SR-superfamily of splicing factors [34] which includes the products of Cctra and Cctra2. Consequently, we suggest that in female embryos, the Cctra autoregulation loop produces more and more active CcTRA female protein, reaching the threshold concentration that results in the female-specific splicing of all the Cctra transcripts at 8-9 h (Figure 5A). By contrast, in male embryos the postulated Y-linked male-determining factor, M [15, 22, 32], directly or indirectly blocks the Cctra autoregulation loop, inducing the translation of more and more inactive CcTRA protein, resulting in the male-specific splicing of all the Cctra transcripts.
Apart from these sex-specific differences in splicing, as we cannot detect clearcut differences in Cctra transcription levels between male and female embryos, it is unlikely that the M factor interferes with Cctra transcription. Even if we cannot completely exclude this hypothesis, we presume that the M factor acts by affecting Cctra mRNA splicing or CcTRA protein activity (e.g. reducing its phosphorylation).
Sequencing of the Cctra transcripts from embryos also permitted the characterization of the genomic organization of the gene: the exon/intron structure is different from that reported previously [31]. In particular, we found that intron 1 is 50 bp longer than expected and we found no evidence of the presence of a third 52 bp long intron within the genomic portion that we analyzed. This discrepancy could be due to the general genetic variability that occurs between populations of different geographical origins and between different laboratory strains.
In contrast to Cctra, the Cctra2 transcripts appeared in RT-PCR experiments as a single band in both sexes at all the embryonic stages analysed, even though the primers spanned five introns (Figure 1). The presence of only the mature transcript, as ascertained by comparison with the published genomic sequence [Genbank:EU437408], could be due to Cctra2 not being expressed in the zygote during early embryogenesis and that only maternal Cctra2 mRNA supplies the required CcTRA2 protein. This hypothesis is supported by the observation in D. melanogaster that the amount of tra2 transcripts is lower in 2-3 h than in 0-1 h embryos, suggesting that there is no zygotic expression or that the zygotic expression cannot compensate for the concomitant degradation of the maternal mRNAs. Furthermore, in embryos in which the tra2-carrying chromosome 2 is deleted, the quantity of tra2 mRNAs is similar to that in normal embryos, indicating that the maternal tra2 mRNA is stable and that there is no zygotic contribution [6].
As far as it concerns the third medfly sex determination gene, Ccdsx expression begins in 10 h male and female embryos and its transcripts are sex-specifically spliced, as by this stage only female embryos express the functional female-specific CcTRA protein (Figure 5A).
In D. melanogaster sex determination occurs before cellularization as it is coupled with dosage compensation [35]. Our data show that sex determination in the medfly also occurs before cellularization. This is surprising as the medfly X chromosome is largely heterochromatic and contains very few genes, suggesting that there is no need for dosage compensation [35]. In both species, the establishment of this cascade during the first stages of embryogenesis requires the activity of proteins that act in a concentration-dependent manner. These molecular events could be facilitated in a syncitial stage rather than in a cellularized organism.
Our results show that the medfly sex determination cascade is complete at 10 h, before the end of cellularization (9-11 h [11]), whereas in D. melanogaster it ends after gastrulation (stage 13) [36]. A possible explanation for this phenomenon may be the shorter cascade in C. capitata and also the late onset of cellularization (9-11 h) compared to 2.10-2.45 h in D. melanogaster.
Possible medfly sex determination-related genes
In D. melanogaster the ratio of X-linked and autosomal gene products represents the primary signal of the sex determination cascade. Among these genes, sisterless A (sisA) is one of the X-linked genes and deadpan (dpn) and groucho (gro) are autosomal genes. Early during development, the ratio of their product concentrations, together with those of other genes, determines the expression status of the key gene Sex-lethal (Sxl) starting from an early promoter (SxlPe) [37, 38]. In females the Sxl early mRNA encodes an active SXL protein. At later developmental stages a maintenance promoter (SxlPm) triggers Sxl transcription in both males and females, but this pre-mRNA requires the presence of an active SXL protein before it can mature into full-length SXL-encoding mRNA. Only in female embryos does the SXL translated from SxlPe mRNA act as the memory device for female sexual development via its auto-regulatory function. The function of SXL requires the activity of cofactors, such as sans-fille (snf) and female lethal (d) (fl(2)d). When Sxl is activated, it sets in motion a cascade of regulatory genes, transformer (tra), transformer-2 (tra2) and doublesex (dsx) [39]. In female embryos the DSXF protein cooperates with the product of the intersex (ix) gene to drive the expression of the genes responsible for sexual differentiation [40].
Even though the C. capitata sex determination cascade differs from that of D. melanogaster, several medfly EST transcripts with homology to the sisA, dpn and gro genes were identified (Table 1). Their expression in the embryos comply with the classification of their D. melanogaster homologues: CcsisA and Ccdpn are expressed by the zygote earlier than 4 h after oviposition while Ccgro is a maternal gene, as its transcripts are inherited from the mother (Figure 5B). There were no apparent differences in expression between male and female embryos, apart from an apparent lower amount of Ccdpn transcripts in the 8-9 h female embryos, compared to their male counterparts. This difference has to be confirmed with further analyses and its biological significance has to be investigated. As the medfly primary sex determination signal is not based on the ratio of X and autosomes gene products, the presence of transcripts of these genes during embryonic development opens the interesting prospect of identifying the roles that they play during early medfly development.
As expected, the CcSxl gene has no sex-specific splicing variants and it is inherited maternally as a mature transcript. This difference with respect to D. melanogaster was also reported for MdSxl in Musca domestica [41], a species that appears to have a sex determination system similar to that of the medfly [42]. This is indicative of a fundamental difference in transcriptional regulation of this gene and shows that this gene has a different function in drosophilid and non-drosophilid species.
The medfly embryos also inherit transcripts of the Ccsnf, Ccfl(2)d and Ccix genes. These genes could have the same functions in the medfly as their Drosophila homologues, as co-regulators of Cctra splicing (Ccfl(2)d and Ccsnf) or as a co-factor of Ccdsx (Ccix).
Like Cctra, the Ccgro, CcSxl and Ccfl(2)d maternal genes present at least two PCR products in 5 to 8 h old embryos in both sexes (Figure 5B). Sequencing demonstrated that the larger fragment corresponds to the pre-mRNA, while the smaller fragment is the spliced/mature mRNA. This suggests that the newly transcribed mRNAs are not immediately processed into mature forms.
To conclude, we can hypothesize that two waves of zygotic expression occur in the medfly: the earliest genes, which include CcsisA and Ccdpn, are expressed before 4 h after oviposition. The second major burst of transcription occurs at 5 h after oviposition and the Ccgro, CcSxl, Cctra and Ccfl(2)d genes belong to this second wave.
Towards the cellular blastoderm formation
A second major event during the first stage of embryogenesis is the cellular blastoderm formation, which in C. capitata occurs between 9 h and 11 h after oviposition [11].
In D. melanogaster a set of genes is involved in this process that includes maternal, maternal-zygotic and zygotic genes. In the medfly EST database we identified two sequences that are similar to the D. melanogaster maternal genes, Rho1 and spaghetti-squash (sqh). These genes are also maternal in the medfly and the immature form of CcRho1 is present in 5-8 h old embryos, indicating that this gene is expressed during the second major wave of transcription in the embryo (Figure 5C).
The slow as molasses (slam) and serendipity-α (sry-α) genes are expressed only in the zygote in D. melanogaster. The Ccslam gene complies with this classification, whereas the Ccsry-α gene is maternally inherited in the embryos as a mature transcript. Again, in 5 to 8 h old embryos, the immature form of the Ccsry-α mRNA was detected. The recent embryonic in situ detection of Ccsry-α did not reveal the presence of maternal transcripts of this gene [43]. This discrepancy may be due to the higher sensibility of a PCR- compared to a hybridization-based approach. What is more, the Ccsry-α gene showed greater amino-acid similarity to the D. melanogaster CG8247 gene than to sry-α. CG8247 is a sry-α-like gene that is also involved in cellular blastoderm formation but is, unlike sry-α, inherited maternally in D. melanogaster
The putative CcZelda gene
In D. melanogaster the presence of TAGteam sites in promoter-sequences is a feature of genes that are expressed early during embryogenesis [44]; these sites are bound by the zinc-finger transcription factor Zelda [2]. zelda (zld) transcripts are present in the germ line cells of the ovary, in unfertilized eggs and throughout early embryonic development, while later on in development zld becomes restricted to the nervous system. The Zld protein is a general activator of early genes and it is responsible for the MTZ transition. It regulates the activation of genes involved in the three early processes that occur in the embryos, sex determination (including sisA, Sxl and dpn), cellular blastoderm formation (including slam and sry-α), and the dorsoventral genes.
In the medfly EST database we identified a sequence that encodes for a protein with a high degree of identity with Zelda (Table 1). Like its putative homologue zld, Cczld transcripts are maternally derived and they are present throughout the syncitial period (Figure 5D). The promoter regions of two of the medfly genes putatively regulated by Zelda, Ccslam and Ccsry-α, are known, but it was not possible to identify conserved TAGteam sites between the medfly and Drosophila homologues of these genes. In the D. melanogaster slam gene the TAG sites are present only in the first intron, as we do not have the genomic sequence of Ccslam we cannot confirm that these sites are conserved in the medfly gene. In the sry-α gene the TAG sites are located in the promoter, whereas in Ccsry-α the promoter lacks these sites. This lack of TAGteam sequences in Ccsry-α could be a further demonstration that this gene has a different transcription-control molecular mechanism with respect to sry-α in Drosophila. The sequencing of the entire medfly genome, currently in progress, will provide a powerful tool to understand the molecular mechanisms of the MTZ transition process in this species.
The maternal information of female development is reset in the medfly embryos through the reprogramming of Cctra mRNA splicing and the degradation of the maternal Ccdsx mRNA. The heterogeneity of the Cctra mRNA population during the "splicing-resetting" phase (5-8 h after oviposition) is indicative of a threshold-dependent activity of the CcTRA protein. Thus we suggest that the maternally-inherited Cctra transcripts in female embryos are not sufficient to produce enough active protein to inhibit the male mode of Cctra splicing. These data suggest that the M-factor [15] acts by inhibiting the CcTRA protein activity, so that it does not reach the threshold concentration required for female-specific splicing of all the Cctra transcripts. The slow rate of development and the apparent inefficacy of the splicing mechanism in the pre-cellular blastoderm, exemplified by the presence of immature mRNA precursors of many genes (Ccgro, CcSxl, Ccfl(2)d, CcRho1 and Ccsry-α), could facilitate the M-factor activity. This model of sex determination in the medfly differs from that previously proposed [12].
Operationally, the slow rate of early development in the medfly may facilitate the study of the expression and splicing mechanisms that occur in slow motion compared to D. melanogaster, thus making C. capitata a suitable candidate to deepen our knowledge of the molecular mechanisms involved in the MTZ transition in Diptera.
As a practical consequence, promoter and enhancer sequences that are active in early stages of development will become available as tools for population control strategies against pest insects.
We thank the two anonymous referees for their constructive comments and suggestions. We thank Saverio Brogna for critically reading the manuscript. This work was supported by the grant PRIN 2006, #2006050870_004 (Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale) from the Italian Ministry of University and Research.
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text: Garden Day is a special event at MSR and everyone in our community loves to pitch in to make the campus beautiful. Not everyone gets to see the rewards of all that hard work, unless they come on campus for SummerScape. So, for those that don't attend SummerScape, we thought we'd share some pictures of the beautiful gardens in their summertime splendor.


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Jerusalem (CNN) -- The Elders, an independent group of leaders brought together by former South African President Nelson Mandela to promote Mideast peace, travel to Israel on Thursday.
On Tuesday, the group -- including former U.S. President Jimmy Carter-- met in Syria with the exiled leader of Hamas, the Palestinian movement that runs Gaza.
Carter, former Irish President Mary Robinson, former U.N. envoy Lakhdar Brahimi, and Ela Bhatt, founder of the million-strong Self Employed Women's Association of India, were the four "Elders" who met with Hamas' Khaled Meshaal.
The group's Middle East tour has taken them to Gaza and Egypt. It is expected to visit the West Bank as well.
The Elders said in a statement that talks with a range of people in Gaza, Egypt and Syria led them to back a "more comprehensive approach to the Israeli-Palestinian peace talks."
The Elders have repeatedly heard that there are low expectations that the current U.S.-led talks between Israel and the Palestinian Authority will succeed.
"A far greater sense of urgency is needed. People are tired after almost two decades of talks," Robinson said. "They keep telling us that there is too much focus on process and not enough on results. They are asking themselves whether the United States and the Quartet are more interested in managing the conflict than resolving it. As Elders, we believe the two-state solution has the potential to deliver peace -- but a more energetic and comprehensive approach is needed."
The Quartet is made up of the United Nations, the United States, the European Union and Russia, which has backed the so-called road map to a Middle East peace.


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Calling all tomato lovers in Utah!
I mistakenly planted literally 30+ cherry tomato plants–pale yellow, yellow pear, butter yellow, orange, super orange, and plenty of red. They are gorgeous and yummy. Please come to my house and pick and eat them whenever you want.
This morning, other people we know had no problem with WaMu accounts, so we are wondering if he was placed on fraud alert because he's spending money in four different states, some of which on the same day.
Still, it was freaky. And it is. But isn't it nice that with modern science and whatnot we can have our banks fail and not even feel it? Gosh, I'm sure everything is going to be just fine, aren't you?


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I can't believe CEP 812 is coming to a close. Sometimes it feels like the past 8 weeks flew by, and other times it feels like they were the slowest 8 weeks of my life. This course has been the most challenging thus far….although it is only my third course in the program. In the other two classes my learning was much more physical- i learned to play the guitar and make circuits out of play-dough. The first two courses presented me with problems to solve, or so it seems, but this course really made me identify the problem.
Reflecting back on the past year, I am reminded of the course of events that led me to the MAET program. Almost a year ago now, I emailed past professors asking for their suggestions on master's programs focusing on educational technology. At the time, my passion for implementing modern tech tools in my classroom was conflicting with my school's technology policy. I hated telling my students to use resources and tools, and then have to punish them if they got out their cell phone to look something up on the Internet, snap a picture of the whiteboard work, or video something they heard and want to remember…especially because I felt like a hypocrite for telling them not to use a tool I regularly use: my cellphone. I was looking for a program to help me reconnect learning and life. I was curious as to why my learners were highly adaptive to rapidly evolving technology outside of school, but struggled to use technology in the classroom. Now, finishing up CEP 812, I am still reaching out to professors and others within my growing PLN (professional learning network). It seems that everything I do regarding teaching and learning is driven by my passion and curiosity and remediated by reaching out to individuals in my PLN or searching for the answer on the web, which are appropriate closing thoughts considering our final learning task.
For our final task in CEP 812 we were asked to read the article It's P.Q. and C.Q. as Much as I.Q. written by Thomas Freidman. In the article, Friedman (2013), says that in our hyper-connected, technology-driven world, the individuals who will succeed "won't just be those with more I.Q. It will also be those with more P.Q. (passion quotient) and C.Q. (curiosity quotient) to leverage all the new digital tools to not just find a job, but to invent one or reinvent one, and to not just learn but to relearn for a lifetime." That is, intelligence alone will not prepare individuals for an unknown future with unknown problems and jobs, but rather a combination of passion, curiosity and intelligence is necessary for success in a rapidly changing economy. After reading and reflecting on this article we had to create a representation of how we embody and envision PQ and CQ in both our present and future work as educator. I created a video remix using YouTube and iMovie to hopefully help views see that passion and creativity go hand in hand in my instructional practices and learning experiences. Enjoy.
Friedman, T. L. (2013). It's p.q. and c.q. as much as i.q. The new york times. Retrieved from http://www.nytimes.com/2013/01/30/opinion/friedman-its-pq-and-cq-as-much-as-iq.html


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1964 Triumph 650, Just had a top end rebuild and new charging system installed, including rotor and tympanium. Pipes were just wrapped and the bike has newish tires. Runs good and starts easily. This bike has a California title and is ready to go.
Call Jeff at 7I4 4O4 4419
Saturday, January 12, 2013
For Sale
Posted by J.j.Rowe at 12:03 PM 1 comment:
Labels: bike for sale, triumph
Wednesday, January 2, 2013
Happy new Year!
Follow Hell on Wheels on facebook, it's the easiest way to get updates on event's, shows, ride's and other shenanigans!
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Navy Petty Officer 3rd Class Ryan J. Carlson, son of Patricia Carlson of New Port Richey and Kirk Carlson of Mount Sterling, Ill., recently graduated from the U.S. Navy's Nuclear Power School at Naval Nuclear Power Training Command in Goose Creek, S.C.
Nuclear Power School is a rigorous six-month course that trains officer and enlisted students in the science and engineering fundamental to the design, operation and maintenance of naval nuclear propulsion plants. Graduates will have additional instruction at a prototype training unit before serving as a surface warfare officer aboard a nuclear-powered surface ship or as an electronics technician aboard a nuclear-powered submarine.
Carlson, a 2008 Dunedin High School graduate, has served in the military for one year.
We need your news! Hometown publishes Wednesdays and Sundays with your good news. Tell us about your club or thank someone for a job well done. Keep it under 100 words. E-mail photos in JPEG format. Contact Hometown news coordinator Gail Diederich at [email protected]; by phone at (813) 909-4600, ext. 4660; by fax at (727) 869-6233 or (813) 909-4611, or mail Pasco Times, 11321 U.S. 19, Port Richey, FL 34668, or at 26240 Golden Maple Loop, Wesley Chapel, FL 33543.


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- This event has passed.
City of Austin Music Office to Host Revenue Development Workshop
July 28, 2016 @ 10:00 am - 5:00 pm - FREE
Event Navigation
The City of Austin Economic Development Department / Music & Entertainment Division will host a Revenue Development Workshop for Austin musicians from 10am-5pm on Thursday, July 28, at Soundcheck Austin (1901 E. 51st St Ste #45, Austin, TX 78723).
This comprehensive workshop will provide musicians with expert level insight on various aspects of the industry including: merchandise sales, establishing a music business, licensing music, collecting royalties, branding, marketing, public relations and establishing a social media presence. Featured presenters for this engagement are Jason Feehan and Randy Chertkow, authors of The Indie Band Survival Guide and the DIY Music Manual.
"Helping Austin's musicians get expert-level industry support to learn how to make more money in their chosen profession is just one way we are showing that we are committed to keeping Austin the Live Music Capital of the World," said City of Austin Mayor Steve Adler.
Attendance is free of charge and open to Austin area musicians. Attendees may RSVP to Stephanie Bergara with the City of Austin Economic Development Department at [email protected] or 512-974-7804.
About The City of Austin Music & Entertainment Division: The City of Austin Economic Development Department / Music & Entertainment Division is highly engaged in developing and executing initiatives that help accelerate the growth of the music industry infrastructure by focusing on job creation, talent export, trade development, and industry revenue growth. The Music & Entertainment Division has also organically developed an important role for the Austin music industry by facilitating introductions, communication, and shaping discussions between music business operators who may not otherwise have knowledge of each other, resulting in new business and connections. For more information check out www.atxmusic.org.
EDITOR'S NOTE: Listings on the Austin.com calendar contain user-submitted information. Make sure to double check the date, time, and location of your event!


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Kildare Derby Person of the Year nominees honoured
Sally Purcell was crowned Kildare Derby Festival Person of the Year at a special ceremony on September 10.
The nominees for the title were also honoured at Tigerlilies.
Paddy Phelan, Ann O Neill, Margie Sheridan, Kim Kelly, Amanda McCannon, Mary Kavanagh, Fred Archibold, Sr. Brid Kenny, Josie Farrell, and Charlene Bennett were all nominated.
The Kildare's Got Talent competition was also held on the night and prizes were awarded for the best entries for the Derby Festival Parade. Last year's winner, Paddy Phelan will go forward to the County Kildare Person of the Year Awards.


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Published on May 22nd, 2019 | by gupta-dasa | Full size image13
Srila Prabhupada's books Should Not be changed!
Post-disappearance editorial revisions by conditioned souls to the sacred, memorialized realizations of an exalted Acarya are nothing less than crude, insulting and offensive. Yet, this is precisely what has taken place for two decades and continues to take place by those who, ironically, have a special spiritual duty to preserve Srila Prabhupada's literary legacy at all cost.
Hare Krishna Govinda devi. All glories to Srila Prabhupada.
Yes, I certainly share your deep concern over the ongoing editorial changes being made to Srila Prabhupada's literary legacy. And here is the gist of it: while an author might, during his or her lifetime, re-edit and then re-print a previously published book, we would be hard pressed to find anyone, anywhere, who would entertain the idea that, once published, an author's work may later be edited posthumously and then presented anew in a 'revised and enlarged edition' as if it were the author's original work!
And, for those who have any sensitivity or knowledge of spiritual etiquette regarding the proper post-disappearance handling of an Acarya's sacred adi-vani, or original spiritual instructions, such a disturbing editorial scenario is wholly unacceptable.
In the Western tradition, the posthumous bastardization process is referred to as "bowdlerizing" a work (named after an English editor, Thomas Bowdler, famously involved in an editorial scandal involving an expurgated edition of Shakespeare). In the Eastern tradition the principle of not changing an Acarya's work post-disappearance is enshrined in the sastric principle referred to as arsa-prayoga. Clearly, post-disappearance editorial changes to an Acarya's transcendental literary work is not done and should not be done. Sastric authority on this point abounds.
What really strikes me is how unfortunate it is that there is need for this discussion at all. Indeed, it should not have to be pointed out that post-disappearance editorial revisions by conditioned jivas to the sacred, memorialized realizations of an exalted Acarya is nothing less than crude, insulting and offensive. Yet, this is precisely what has taken place for two decades and continues to take place by those who, ironically, have a special spiritual duty to preserve Srila Prabhupada's literary legacy at all cost. Certainly those entrusted with the fiduciary duty of preserving Srila Prabhupada's adi-vani, or original spiritual teachings, should know this.
As regards the rationale and reasons cited for making post disappearance changes to Srila Prabhupada's adi-vani, His original instructions, those flimsy arguments completely miss the mark.
A threshold argument made is that continuing changes are authorized merely because certain editors were permitted, during Srila Prabhupada's manifest presence, to make editorial adjustments to punctuation, spelling and grammar, and because, at that time, Srila Prabhupada expressed a certain degree of confidence in their editorial ability.
That pre-1978 service engagement was not a blank cheque, however, to continue the editorial process unabated for all time. Just as you never ask a barber if you need a haircut, you cannot reasonably expect an editor to make an objective decision whether or not to continue editing a book! There is no written authority whatsoever that Srila Prabhupada expected or wanted the editorial process to continue post disappearance on books that he already approved and that were already published. In fact, the unequivocal evidence is inapposite — Srila Prabhupada did not want his books continuously changed and edited after they were published.
It is one thing for an understudy (an apprentice) to contribute to a Master's work, be it artistic or literary, during that Master's lifetime and under the Master's supervision and approval. However, once a Master signs off on a work (that is, approves the work as finished), it is unheard of in either the Western or the Eastern tradition that the apprentice would then take the liberty sua sponte to change the painting or book and publish it posthumously AS IF that painting or book had been approved by the Master during his lifetime! Using such logic, a one percent per year change to the enigmatic Mona Lisa could wind up being, in a short hundred years, a picture of MAD Magazine's demented Alfred E. Newman!
Moreover, we should not overlook the fact that Srila Prabhupada personally lectured on most every verse in the 1972 Bhagavad-Gita As It Is, other than perhaps the first chapter, and as far as I am aware, He only asked that three words be changed. If those pre-1978 books were good enough for the author and Founder/Acarya, they should certainly be good enough for us! If those books were considered transcendental by the author and by the entire movement then, why should we not continue using those books now?
And, if Srila Prabhupada had authorized continuing editorial changes, as is being intimated, then why is it that permission from the GBC was sought in 1983 for the 'revised' edition? The reason, of course, is that such post disappearance changes were never authorized by Srila Prabhupada and therefore institutional approval was sought by the editors. By the way, the GBC agreed to allow the changed book in 1983 by only one vote — not exactly a landslide.
The main argument being asserted for changing the books is as deceptive as it is subtle. The argument is that changes made to the 1983 edition of the Bhagavad-Gita As It Is brings the book closer to the manuscript (or galley proofs) than was the original 1972 edition. As noted above, however, this argument completely fails to take into account the absolute prohibition, in both the Western and Eastern tradition, against posthumous and post disappearance changes, respectively.
It is critical to understand that the issue is NOT whether the manuscript is closer to the first or second edition (or perhaps in fifty years closer to the tenth edition). If that were the issue we could then expect who knows how many more editors making who knows how many more changes to Srila Prabhupada's books under the guise that their editorial changes were, in fact, closer to what Srila Prabhupada really meant or really said. A misguided publishing policy which approves post disappearance changes to Srila Prabhupada's works will only result in an unending series of changed books for many more years to come. Is that what Srila Prabhupada wanted? Certainly not!
Instead, the actual issue at hand is that once an author has personally approved his work and it is published, it cannot be changed posthumously or post-disappearance by a non author and then presented AS IF it were the actual author's originally approved work. Doing so runs directly afoul of both Western and Eastern traditions.
Srila Prabhupada's literary legacy must be preserved, frozen in time so to speak, for posterity. Continuing changes over the upcoming decades and even centuries will only serve to incrementally denigrate the potency of His gift. The world wants and needs to hear Srila Prabhupada's transcendental voice As It Is — not the prissy sterility of an editorial cottage industry run amok. Certainly, if the authorized and approved pre-1978 books were good enough for Srila Prabhupada during his manifest presence, they should be good enough for everyone right now!
About the only way out of this editorial and publishing morass is to firmly re-establish the 1972 edition of the Bhagavad-Gita As It Is which was approved and authorized by its author, His Divine Grace, as the literary standard, and then, some time later, to publish the original manuscript so that scholars and spiritually-minded persons can study and comment as they see fit. The editors are free, of course, to print their own books and to make a record as they want, but not under the guise that the original author approved their particular post disappearance word craft.
On the other hand, to continue along the "I know better what Srila Prabhupada really meant" route by allowing post disappearance editorial changes to Srila Prabhupada's authorized and approved pre-1978 editions can only be seen for what it is — an ongoing, blatant violation of the sastric principle of arsa-prayoga.
At bottom, it is Srila Prabhupada's transcendental vision the world wants and needs — not the conditioned viewpoint of some editor's temporal sense of grammar or correctness that we seek.
With regards,
Gupta das
Joseph Fedorowsky
Some quotes:
Yasodanandan: Sometimes they appeal that "We can make better English," so they change like that, just like in the case of Isopanisad. There are over a hundred changes. So where is the need? Your words are sufficient. The potency is there. When they change, it is something else.
Svarupa Damodara: That's actually a very dangerous mentality.
Yasodanandan: What is it going to be in five years? It's going to be a different book.
Srila Prabhupada: So you… What you are going… It is very serious situation. You write one letter that "Why you have made so many changes?" And whom to write? Who will care? All rascals are there. Write to Satsvarupa that "This is the position. They are doing anything and everything at their whim." The next printing should be again to the original way. (Srila Prabhupada conversation, June 22, 1977, Vrindaban)
Srila Prabhupada: … So you bring this to Satsvarupada. They cannot change anything. (Srila Prabhupada conversation, June 22, 1977, Vrindaban)
"It is not possible for a common man to write books on bhakti, for his writings will not be effective. He may be a very great scholar and expert in presenting literature in flowery language, but this is not at all helpful in understanding transcendental literature. Even if transcendental literature is written in faulty language, it is acceptable if it is written by a devotee, whereas so- called transcendental literature written by a mundane scholar, even if it is a very highly polished literary presentation, cannot be accepted . The secret in a devotee's writing is that when he writes about the pastimes of the Lord, the Lord helps him; he does not write himself. As stated in the Bhagavad-gita, dadaami buddhi-yogam tam yena maam upayaanti te. Since a devotee writes in service to the Lord, the Lord from within gives him so much intelligence that he sits down near the Lord and goes on writing books. Chaitanya Charitamrita Adi 8.19
"So unless one is self-realized, there is practically no use writing about Krsna. This transcendental writing does not depend on material education. It depends on the spiritual realization. You'll find, therefore, in the comments of Bhagavatam by different acaryas, even there are some discrepancies, they are accepted as asat-patha. It should remain as it is." (Srimad-Bhagavatam 7.5.23-24, March 31, 1976, Vrindaban)
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13 Responses to Srila Prabhupada's books Should Not be changed!
Hare Krishna !
My full respect to Prabhu Madhuvidsa Dasa for write this article. My apologize to comment on 2 years old article, but I want and need to ask several things, Prabhu.
#1. Why do they change Prabhupada's works? What's their goals? Money?
#2. I'm still confused about Arjuna in Bhagavad Gita, Why is there no Arjuna's name on any parampara ? Is there any explanation Prabhu ? I already try to find explanatiom from ISKCOM, but I'm afraud that it is conditioned for their motives.
I hope for your guidance, Prabhu.
Sincerely, lowest Krishna devotee of all times.
Hare Krishna,
Are all of the Sri Prabhupada books that are sold on krishnastore.com the original pre 1977 versions?
Hare Krishna
Haribol, can any one please confirm if the set of books available from Bhaktivedanta Manor is the original books or not….
From Bhaktivedanta Manor website it says:
Srimad Bhagavatam Pocket Edition Cantos 1-10 (30 Volume Set) This edition is a 2004 reprint of the 1972 Hamsaduta pocket edition
Dear Madhudvisa dasa:
All glories to Srila Prabhupada
First let me congratulate you for your courageous preaching in preservation of literary legacy of our beloved spiritual master Srila Prabhupada; on the other hand, do not remember where, but I read that there are 4 Prabhupada's books that cannot fail to be read (of course it should be authentic and original books of Prabhupada, as you preach and defend admirably), of which two are the Bhagavad-Gita and Srimad Bhagavatam, for what you ask in the most attentive way, remember me what are those 4 books and the order they should be read (I think the Bhagavad-Gita is the first).
Your unworthy servant
Padma Carana Dasa
How can i know the original Bhagavad-gita without changed?


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Due to vandalism in the area, the Stone Mother has been closed to general public by the Pyramid Lake Paiute Tribe until further notice.
The Great Stone Mother is a remarkable tufa rock formation that resembles a hooded Indian woman seated with an open basket lying next to her. The site is considered sacred to the Paiute People and is associated with traditional stories that explain the creation of Pyramid Lake. There are many versions to the Stone Mother story and vary amongst Paiute families.
Following is the most commonly told story about Stone Mother:
One day the father of all Indians came to this area and lived on a mountain near still water. It is said that he was created near Reese River. He was a very great and good man. He was very lonesome and wished he had someone to keep him company.
One day, much later, Woman heard about Man. She was married to Bear. She wished that someday she might see Man, and this made Bear very jealous. One day Woman and Bear had a fight. They fought for a long time and finally she knocked him down and killed him with a club. She decided to leave the country and go north in search of Man. She had many interesting experiences on her trip. Even today, her footprints can be seen along Mono Lake.
Near Yerington, she fought a giant who tried to eat her. She managed to kill him and his body turned to stone, where it can also be seen today.
She arrived at Stillwater Mountain at last. There she saw Man who was so handsome. She hid from him in for fear he might leave. One day, as Man was walking around he saw Woman's tracks. He started to look for her, and called out, saying that he knew she was around. At last she came out from hiding. She was nervous and very tired from her trip. He noticed this and spoke to her kindly. He asked her to go with him to his camp where he would give her food. She meekly followed him.
After they finished eating, Man asked Woman to stay with him. That night she stayed near the fire. The next night she slept by the door. Each night she moved a little closer. On the fifth night they were married. They had many children.
Their first born was a boy who was very mean. He was always causing trouble among the other children. One day when they were fighting, the father called the children together to talk to them. He told them that if they continued to fight he would have to separate them. They started fighting before he finished talking.
Man became very angry. He stopped them and said, I am going to separate you now. I shall go up to my home in the sky. When you die you will come up to me. All you have to do is follow the dusty-road (pointing to the Milky-Way). You will reach my home where I shall be waiting. Some day I hope that you will all come to your senses and live together in peace
Slowly he called the oldest boy and gave him one of the girls. He sent them west. They became the Pitt-Rivers. The other children who were peaceful, he kept at home. He told them that they were to take good care of their mother whom he was leaving with them. They became the "Paiutes". Then he went up into the mountains then up to the sky.
The Paiutes grew into a strong Tribe, but woman still grieved for her other children. Woman was so sad that she began to cry bitterly. She missed her other children very much. She cried more and more each day.
One day she decided to sit near a mountain where she could look toward Pitt River country. She sat there day after day crying. Her tears fell so fast that they formed a great lake beneath her. This became "Pyramid Lake". She sat so long that she turned to stone. There she remained to this day, sitting on the Eastern shore of Pyramid Lake, with her basket by her side.
The "Kuyuidokado" (Pyramid Lake Paiutes/Cui-ui eaters) call her "Stone Mother."


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*****Support Our Riverfront Businesses and Organizations!*****
As we head into our busy winter holiday season and end-of-the-year charitable decisions, please remember that your decisions can help support the riverfront organizations and businesses that you love and that make it such a special place. There are so many wonderful stores where you can find special gifts (and gift certificates for riverfront dining and activities are always a great idea, too), plus there are so many wonderful nonprofits within (and supporting) our riverfront that would truly appreciate your support.
*****Events and Activities*****
15th Annual JA Holiday Variety Show
Friday, December 4, 9 – 11 pm
Aster Café, 125 Main Street SE
Jeff Arundel will host his annual holiday variety show, featuring Ann Klein, Doug Macleod, Steele Arundel, the Willmar Women's Ensemble, and Jeff Arundel and the Mammsmen. Pleasure guaranteed, holiday spirit promised and kaleidoscope of experiences possible! $10, Reservations recommended; call 612-379-3138.
The American Craft Council and RSP Architects present:
Holiday Craft Hop
Saturday, December 5, 12 noon – 5 pm
Grain Belt Brewhouse, 1224 Marshall Street NE
Find the best in handmade holiday gifts at the Holiday Craft Hop. The fine folks at Ship and Shape, Minnesota Jewelry Arts Guild and Gather Art Registry have joined the American Craft Council to co-curate a holiday market with more than 50 Minnesota artists and artisans – See more at: http://craftcouncil.org/event/holiday-craft-hop#sthash.JrAqE0kk.dpuf.
A SIP OF SCIENCE – If We Found Rock Evidence for Life on Mars, What Might It Look Like? Microbialites from Ancient Lakes
Wednesday, December 9, 5:30 pm
Aster Café, 125 Main Street SE
A SIP OF SCIENCE bridges the gap between science and culture in a setting that bridges the gap between brain and belly. Food, beer and learning are on the menu in a happy hour forum that puts science in context through storytelling. The Mars orbiter has used remote sensing techniques to demonstrate the presence of active groundwater seeps on our planetary neighbor. The Curiosity Rover has sent back stunning images of deposits that indicate flowing water and, from Gale Crater, what look like lake deposits. If standing water had been present on Mars, it is more likely to have been in the form of lakes, not widespread oceans. If life inhabited these lakes, it would be more likely to be microbial in origin. Geologists have studied marine microbialites (rocks that formed under the influence of microbes), mainly stromatolites, for many decades. However, these marine microbialites differ in important and significant ways from those formed in modern and ancient lakes. We should expect to find lacustrine (lake-influenced) microbialites on Mars if life had established itself there. Ancient lacustrine microbialites here on Earth, mainly from the Cenozoic, provide excellent examples of what to expect if we encounter them on Mars. In this talk, University of St. Thomas geology professor Tom Hickson will cover the basics of microbialite formation, the macro- and microscopic textures of lacustrine microbialites and some basic microbialite chemistry. We will then focus on ancient and historical lakes from southern Nevada, with examples from other units in the Western U.S. The talk takes place during happy hour at the Aster Café. No cover; food and drink will be available for purchase. Thomas Hickson is professor of geology and environmental science at the University of St. Thomas. He graduated from Stanford University in 1999 and completed a post-doc at the St. Anthony Falls Lab immediately before coming on board the St. Thomas faculty in 2000. He is engaged in active collaborative scholarship with undergraduate students to understand the tectonics, paleoclimate and landscape evolution of the Lake Mead region (Nevada) during the Miocene. A SIP OF SCIENCE is a science happy hour sponsored by the National Center for Earth-surface Dynamics (NCED). It is a chance to hear about new and exciting research over beer, in a cool bar. Come talk with the experts about their efforts to address some of the Earth's most pressing problems. NCED's A SIP OF SCIENCE brings the wonder of science to happy hour. Please RSVP!
Mill City Farmers Market
Saturday, December 12, 10 am – 1 pm AND
Saturday, December 19, 10 am – 1 pm
Mill City Museum, 704 Second Street South
The Mill City Farmers Market has moved inside the Mill City Museum every second Saturday (and more). Visitors can stroll inside the cozy and lively marketplace and choose from an abundance of fresh, local fruits and vegetables, freshly baked bread, pastured meats and cheeses, meatless meat, expertly crafted ceramics, kitchenware, jewelry and much more. Stop by the Info Booth for free, seasonal recipes to inspire your winter meal planning. Each Winter Market brings a unique celebration of seasonal offerings. At the December 12 and 19 markets, they will have tables full of produce for your holiday meals in addition to an expansive selection of unique holiday gifts and décor, including sustainable Christmas trees! Free gift-wrapping will be available at both December markets. Winter markets are a great opportunity to stock up on storage crops like squash, potatoes, garlic, onions and apples and nonperishable goods like wild rice, maple syrup, pickles and preserves to help you eat locally all winter long. Make sure to check out the Recipe Archive for seasonal meal ideas. For information:http://millcityfarmersmarket.org.
Kroening Interpretive Center Programs (Three Rivers Park District)
North Mississippi Regional Park, 4900 Mississippi Court
For information: 763-694-7693; for map and driving instructions: https://www.minneapolisparks.org/parks__destinations/parks_and_lakes/north_mississippi_regional_park/. For programs that require reservations, call 763-559-6700 or register on-line:https://threeriversparks.org.
My Preschooler and Me: Puppet Escapades – Winter at Kroening
Thursday, December 17, 10 – 11 am
Animal puppets introduce your child to nature's wonders. After the show, enjoy a short hike, craft or other themed activity. This session's theme is "River Puppets and the Perfect Gift." Restricted to participants age 2 and older. Children must be accompanied by a registered adult. Reservations required. $4. 20% discount for groups of four or more. Ages: 2-6; both adult and child pay. For information and to register: https://threeriversparks.org/events/M/my-preschooler—me–puppet-escapades—winter-at-kroening–.aspx.
Winter Solstice Celebration
Saturday, December 19, 2 – 4:30 pm
Celebrate the shortest day of the year with light, music and cheer! Explore holiday traditions from around the world. Hike or snowshoe to "tie down" the sun in the prairie. Enjoy warm refreshments by a bonfire, and create crafts to take home. Children 17 years and younger must be accompanied by a registered adult. Reservations required by December 16. $5. 20% discount for groups of four or more. For information and to register: https://threeriversparks.org/events/W/winter-solstice-celebration–.aspx.
Free Family Fun Day: Paws and Claws
Sunday, December 20, 1 – 3 pm, ongoing
Drop in anytime! Explore a different topic each week through hands-on activities that everyone can enjoy at their own pace.This program is free and open to all ages, but children younger than 17 must be accompanied by an adult.
Mississippi River Forum: "Maximizing the Efficiency of Seneca's Wastewater Effluent"
Friday, December 18, 8 — 9:30 am
Minneapolis: McKnight Foundation, 710 Second Street South, Suite 400
You are invited to join the National Park Service for the Mississippi River Forum–your opportunity to learn about issues impacting the Mississippi River and to make sure other decision makers understand your perspective. We water our lawns and wash our cars with water that is cleaner than many people in the world have access to for drinking, and the sustainability of our water supply is a growing concern. That's one reason Minnesota communities are increasingly looking for ways to use their water resources more efficiently and wisely. The City of Eagan is partnering with Metropolitan Council Environmental Services (MCES) and Barr Engineering to study the feasibility of using reclaimed water from MCES' Seneca Wastewater Treatment Plant. The idea is to shift non-potable demands (like irrigation, firefighting and vehicle washing) from the City's treated, potable water supply to reclaimed wastewater. In addition to reducing demand on the groundwater aquifer the city uses for potable water supply, this could reduce treatment and water supply costs for users, improve regional source water sustainability and may even provide a recharge source for groundwater. Come learn how projects like this get off the ground and could help us all build more sustainable water use systems! The presenters will be Steve Klein and Michelle Stockness (engineers with Barr Engineering Company) and Jon Eaton (Utilities Superintendent for the City of Eagan). Light breakfast provided. Free. RSVP to firstname.lastname@example.com.
Mississippi Park Connection presents:
Winter Hike/Snowshoe
Saturday, December 19, 10 am – 12 noon
Coldwater Spring
Winter snowshoe/hike at Coldwater Spring! National Park Service park rangers will have snowshoes available for people to borrow, weather permitting, at Coldwater Spring. No experience or registration required. This is a guided geocaching tour of Coldwater Spring. Free. For information: http://parkconnection.org/event/winter-snowshoe-hike-2/.
Sample of Performances at MacPhail Center for Music
501 Second Street South
For more information about events (including student performances) and classes: http://www.macphail.org or 612-321-0100.
Giving Voice Chorus Final Performance
Saturday, December 19, 2 pm and 4 pm2015
Antonello Hall
Come enjoy an uplifting and heart-warming performance by the Giving Voice Chorus! General admission: $10. For information:http://givingvoicechorus.org.
Samples of Classes and Activities at the Minnesota Center for Book Arts
1011 Washington Avenue South, 612-215-2520, http://www.mnbookarts.org.
Hours: Monday 10 am — 5 pm, Tuesday 10 am — 9 pm, Wednesday-Saturday 10 am — 5 pm, Sunday 12 noon – 4 pm
Class details, instructor biographies, registration details and online registration are all available at above web site. You may also register by calling 612-215-2520. Note: This is only a sampling of classes, focusing on those that are less expensive and that don't require experience.
Family Class: Sunday Wee Book Series
Sunday, December 20, 1 – 3 pm
Love miniature books? In this new series of playful, hands-on classes, learn how to make a bunch of them in no time at all! Great for families with kids, or for kids-at-heart! Four classes will be offered, each of which will focus on different binding styles. This class will include the following styles: Mini Hardcover Sketchbook with Pockets and Mini Pop-Out Book. For grades 1 and up. Adult/child pair: $32 ($29 members) + $6 supply fee; additional participant (adult or child): $16 ($14.50 members) + $3 supply fee. Individual adult participant: $20 ($18 members) + $3 supply fee. To register: http://www.mnbookarts.org/event-registration/?ee=824.
*****Input, Participation and Stewardship Opportunities*****
Downtown Survey Input Sought
Your time is important and so are your opinions. That is why the Minneapolis Downtown Improvement District (DID) wants to know what you think about downtown Minneapolis. Survey responses about safety, cleanliness and other issues will help make downtown Minneapolis a better place to live, work and visit. The survey will take 15-20 minutes to complete, and your responses are confidential. After completing the survey, you can enter to win a $50 gift card to a downtown business. Visitwww.minneapolisdid.com to learn more about DID.
*****Reminder of Ongoing Activities*****
Classes at Surdyk's
Mondays and Tuesdays
Surdyk's Liquor and Cheese Shop, 303 East Hennepin Avenue
Surdyk's offers an extensive and fun choice of classes to help you learn about wine, beer and cheese (both in general and specific types). Check out their calendar at http://surdyks.com/calendar/.
National Park Service "Ranger on Call" Virtual Tour of the Mississippi River
Explore the river with a National Park Service ranger, any time you like, just by using your cell phone. Ranger on Call is an audio tour that explores the Mississippi River's exceptional places. Three tours are geared for bicyclists, with tour stops at Nice Ride stations by the Stone Arch Bridge/Saint Anthony Falls area, downtown St. Paul riverfront and Fort Snelling/Minnehaha Falls. Three additional Ranger on Call walking tours are at Coldwater Spring, Bruce Vento Nature Sanctuary and Hastings' Jaycee Park. For more information, visit: http://www.rangeroncall.com/
Horse and Carriage Tours
Enjoy a tour of the riverfront via a horse-drawn carriage ride. Regular tours are available from both the Nicollet Island Inn and VIC's Restaurant on Main Street. For information: http://www.thehitchingcompany.com.
Live Music at Day Block Brewing
Various days/times during the week
Day Block Brewing, 1103 Washington Avenue South
Day Block Brewing offers a variety of live music a few days each week. For a full calendar: https://www.dayblockbrewing.com/events#calendar.
Acme Comedy Club
Tuesdays through Thursday, 8 pm; Fridays and Saturdays, 8 and 10:30 pm
708 North First Street
Enjoy stand-up comedy at one of the top five comedy clubs in the country. For more information:http://www.acmecomedycompany.com.
Murder Mystery Dinner
Old Spaghetti Factory, 233 Park Avenue
The Murder Mystery Company offers a murder mystery performance to accompany your dinner at the Old Spaghetti Factory.
Snag your seat with The Murder Mystery Company by clicking or calling (888)643-2583 (CLUE) today. For information and tickets: http://www.grimprov.com/Murder-Mystery-Dinner-Minnesota/.
Classes Available at the Lundstrum Center
Lundstrum Center for the Performing Arts, 1617 North Second Street
Dream of performing on Broadway? Preparing for your college audition? Looking to get back into class? Let the Broadway veteran faculty at the Lundstrum Center help develop your gifts. Fall session registration is now open. Children and adults, all levels welcome. Programs include:
- Children: After school programs; age 3 and up
- Preteen/Young Adults: Broadway Musical Theatre — Serious training in dance, voice and theater
- Adults: Ballet, tap, jazz, Pilates, voice
- Special Needs Children: "Dance Mania"-ages 6 to 12
The center offers state of the art, spacious, sun lit studios, with free parking and #14 Metro Transit bus-accessible. Class cards available! For a class schedule please check out the center's website http://www.lundstrumcenter.org or call 612-521-2600 to be put on their mailing list.
Keegan's Pub Entertainment
Keegan's Irish Pub, 16 University Avenue NE
Keegan's Pub offers a full schedule of live music, Irish lessons, pub quizzes and other activities each month. For more information and a full event calendar, visit http://www.keeganspub.com.
Food Building Tours
Food Building, 1401 Marshall Street NE
Mondays, 10 am – 12 noon
The Food Building in Northeast started as a conversation about pigs. Kieran Folliard and Mike Phillips were discussing the absurdity of shipping Minnesota pigs to California for processing and then shipping the salumi back to Minnesota to sell. What if salumi made from Minnesota pigs was made in Minnesota? So they found a building on a desirable corner in the working heart of Northeast Minneapolis, along the Mississippi. The building had more space than Mike needed to make delicious salumi, so Kieran decided to create a space where several expert makers could craft their food. Enter cheesemaker Rueben Nilsson. Rueben is fired up about making cheese using milk from Minnesota grass-fed cows. So with Kieran's help, Mike created Red Table Meat Co. and Rueben created the Lone Grazer Creamery. These brands are connected by a "Taste of Place." Simply put, Minnesota's distinct seasons and savory characters combine to make one-of-a-kind foods. Their motto is "Framer Near, Made Here." Everyone is invited to come see the process – "it's like a brewery tour, only with food. Open tours are now offered on Mondays, with expanded hours planned for spring 2016. For private tours and events, firstname.lastname@example.com. For information: http://www.foodbuilding.com.
*****Informational Resources*****
Minneapolis Historical Mobile Tour Smartphone App Now Available
The Minneapolis Historical Mobile Tour app is now available–for free!–for iPhones and Androids. There are two tours so far: The Old Highland tour (created in partnership with the Old Highland Neighborhood Association) and the Marcy-Holmes tour (created with the Marcy-Holmes Neighborhood Association). The app offers users an on-the-go look into Minneapolis's past, from the well-known headlines to the forgotten tales and people. Learn more and download it now atwww.minneapolishistorical.org. The Marcy-Holmes tour tells stories of the Marcy-Holmes neighborhood—what was once the town of Saint Anthony Falls—from 1849 through the early twentieth century, using images, text and recorded commentary from 27 neighbors and friends. Created using material drawn from Penny Petersen's book, Hiding in Plain Sight: Minneapolis' First Neighborhood, the tour focuses on 24 historical sights depicted in the bronze sculptures on the Sixth Avenue Stroll, an outdoor gallery created by artist Aldo Moroni in 2004 that extends from University Ave SE to Main Street. Each stop on the tour consists of a present day image of the site, the corresponding bronze sculpture, an historic image (when available), brief text and two audio recordings by an individual somehow related to the site.
Park Board Event Calendar
The Minneapolis Park and Recreation Board (MPRB)'s website features a multi-month event calendar. The current event calendar is now available at https://www.minneapolisparks.org/events/ and is a quick guide to MPRB-sponsored and co-sponsored events as well as outside organizers' permitted events in the parks. Check it out, mark your calendars and enjoy the many events occurring in Minneapolis parks!
Mill City Times
This web site covers "life, work and play in the Historic Mill District" and offers videos of many important presentations and meetings related to riverfront topics. It also includes an event listing that is more expansive than the Mill District and that would be a useful resource for you in between issues of the RiverCurrent (or if, as sometimes happens, you accidentally deleted it).
River Hub Event Site
The folks at Mill City Times also offer this extensive listing of events related to the Minneapolis riverfront. The goal with River Hub is to serve the large community of people passionate about our riverfront by helping you stay up-to-date on events impacting the river AND to serve event planners by helping to avoid scheduling conflicts with other river-related events to the extent possible. River Hub is a very simple site – just an activities list and a page for readers to submit events. To check it out:http://www.riverhub.org/
Mississippi River Trail
The Mississippi River Trail bikeway (MRT) follows the course of the mighty Mississippi from its headwaters to the Gulf. Find information for the Minnesota segment on the MRT website http://www.dot.state.mn.us/bike/mrt/, complete with an interactive Google Earth based map, and a complete series of maps printable at 8 1/2" by 11".
1 Mississippi Campaign
This site includes information on the entire Mississippi River and how you can help protect and enhance it.
*****Riverfront News Articles*****
Nye's Site Redevelopment Proposal
FujiYa Demolition
Eastside Restaurant Reviews
University of Minnesota's Old Main Heating Plant
Riverfront Business Duffy's Ignite Program
MWMO Stormwater Park and Education Center
Misfit Coffee and Izzy's
Bohemian Flats


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Sting was honored at the 2016 American Music Awards and he took to the stage at the show to perform a medley of his hits before accepting the award.
The 65-year-old entertainer was presented with the American Music Award of Merit during the event held at the Microsoft Theatre on Sunday (November 20) in Los Angeles.
"I'm deeply moved by this extravagant gesture, and it's important for me as a musical migrant to acknowledge the very real debt I already owe to American music because after all American music that lit the musical spark in my mind, this uniquely American experiment by perhaps America's greatest and most influential export, rock and roll," Sting said during his speech.
"A giant musical jam session where the doors are always open, open to all comers, and the spirit of welcome and inclusion, the mixing of culture, rhythm and passion is what made this country the greatest country in the world," he added. "While I may be an Englishman in New York, I promise you that my musical soul is all American."
Also pictured inside: Tim McGraw and Florida Georgia Line winning awards in the country categories at the show.


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General Forum
Jun 5, 2014 - 3:52PM
Hey, thanks for the prompt response. There's a Chaplin festival happening this weekend near me, (Chaplin filmed quite a few of his films where the festival is at here in Northern California) and I wanted to get a shirt made up of Robert as Chaplin to wear.
Country USA
Jun 5th, 2014 - 3:52 PM
Jun 5, 2014 - 7:19PM
I travel to Northern Cali quite a bit. Always in Napa, Fairfield, Sacramento and Suisun. Where are you located?
Country USA
Age (opt) Roberts
Jun 5th, 2014 - 7:19 PM
Jun 6, 2014 - 7:41PM
Let me know if you have luck with these T-shirts. I've had problems putting Robert's likeness on a T-shirt due to copyright infringement issues.
Country USA
Jun 6th, 2014 - 7:41 PM
Jun 8, 2014 - 12:58PM
In this day and age of social media its not as hard as it once was to get a tshirt, blanket, coffee mug or whatever you want printed anymore. I've gotten several things done for my own personal use and honestly so have thousands of others. I think it all depends on the photo though. Some photographers want their images out there. Then there are some who have extreme copyright infringement laws or watermarks that cannot be removed unless you purchase the picture directly. Even then you would have to take a photo of the photo. Its all fairly confusing but more and more social media is to thank (or not thank) for breaking through those barriers. I get offended when I see so much of DUF's content stolen and used on the internet but Ive gotten used to/over it. You have to. People want and will take. I don't mind at all for personal use. What I mind is when credit is not given of the source or the artist. Much of DUF's content was purchased. That's the sad part. Everyone takes and no one supports the website. Try Café Press JudyG
Country USA
Age (opt) Roberts
Jun 8th, 2014 - 12:58 PM
Jun 9, 2014 - 9:34PM
Will do.
Country USA
Jun 9th, 2014 - 9:34 PM
Jun 11, 2014 - 7:19PM
That you, Gina I had tried Zazzle and they were very strict.
Country USA
Jun 11th, 2014 - 7:19 PM
Jun 13, 2014 - 1:52PM
I think I tried them way back too. There's also a body pillow company and a cardboard standup company that are REAL STRICT! Tried both at one time. Sorta glad I never was able to do it, lol
Country USA
Age (opt) Roberts
Jun 13th, 2014 - 1:52 PM


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Now enjoy this splendid combination of fish with cheese. This aromatic dish will surely hypnotize you and your friends!
1 x pack fish pie mix (cod, salmon, smoked haddock etc, weight around 320g-400g depending on pack size)
3 spring onions, finely chopped
100ml milk
450g potato, peeled, large ones cut in half
75g frozen sweet corn, defrosted
Handful of grated cheddar cheese
1 large egg, beaten
Flour, for dusting
Olive oil, for frying
Cook the potatoes in boiling water until just tender. Drain well and return to the pan on a low heat.
Heat for another minute or two to evaporate excess liquid. Mash the potato with a small knob of butter. Allow to cool.
Put the fish spring onions and milk in a shallow dish, cover with cling film and cook in the microwave for 1 ½ – 2 mins until just cooked.
If you don't have a microwave, put everything in a saucepan and gently cook until just opaque and cooked through.
Drain the fish and spring onions through a fine sieve.
Gently mix through the potatoes, avoiding breaking up the fish too much, along with the sweet corn, cheddar and a generous grind of black pepper. Form into 6 – 8 patties.
The cooler the mash potato is when you do this; the easier it will be to form the patties as the mixture will be very soft when warm.
Pour the egg on one plate and scatter flour on the other. Dip the patties in egg and then flour and arrange on a sheet of baking paper on a tray.
Put the patties in the fridge for at least half an hour to firm up if the patties feel very soft. At this point you can freeze the patties, wrapped individually. Defrost thoroughly before moving onto the next stage.
Heat a large frying pan with a generous glug of olive oil. When the oil is hot, carefully lower the fish cakes into the pan.
Cook for 5 – 7 minutes or until golden brown underneath and then carefully flip them over.
Fry for another 5 – 7 minutes or until golden on the bottom and heated all the way through.


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Create Room for Imagination
Let your children's imagination run wild with the different textile themes from FLEXA
A FLEXA Bunk Bed is the perfect bed for your children. It is a high quality bunk bed, safe and secure and versatile (did you know you can separate the bunk bed into twin beds). And the FLEXA Classic Bunk Bed is made from the very best pine tree and comes in three lacquers: Natural, Whitewashed and Terra.
With our FLEXA textiles, you can create lots of room for imagination and play – customized to the age, gender and personality of your child. Check out the different textiles themes from the cute Princess, adventurous Pirate and brave Knight to the cheeky Jungle, elegant Circus and the fun Forest
Play in the castle and hide away small treasures in the bed pockets. The princess theme is super girly with cute pink and red colors.
From a cheeky monkey to a dangerous lion. The Jungle theme is filled with fun animals that kids love.
The Circus theme is an elegant modern theme in cute pastels that both young and older kids love.
The Knight theme transforms the bed into a fortress that brave little knights can defend.
Change your child's bed into a Pirate Ship with the Pirate theme and let them set sail into exciting adventures.
Explore a forest full of friends and fun with the Forest theme. Our funky and colorful forest friends are ready to play and bring nature indoors.