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Honda Odyssey Under NHTSA Scrutiny, Might Roll Away
Broken bones, bum knees and smashed mailboxes — these number among the accusations owners are leveling at Honda for a supposed flaw with the 2003 and 2004 Odyssey.
Drivers who forget to shift their vehicle into "park" might still be able to remove their key – something meant to be prevented by the ignition cylinder park-shift interlock feature. NHTSA's Office of Defect Investigations reports receiving 42 complaints so far.
In several cases, those reports cited personal injury including one person who said they tried to catch their van before being knocked down and having their leg run over and fractured.
In another case, someone reported a knee injury after trying to stop their Odyssey. Many other reports detailed the vehicles crashing into parked cars, mailboxes and even travelling multiple blocks before stopping in come cases.
A defect investigation is only one preliminary step on the way to a possible recall. Should it move forward, an engineering analysis would follow.


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Product Information
This is a graded product. A1 products have no obvious scratches, marks or dents. It will be fully bubble-wrapped for transportation and comes with the full manufacturer's warranty.
GRADE A1 - As new but box opened
Clear simple layout and easy to clean
- John from Alton
Indesit I6G52X Single Oven Dual Fuel Cooker - Stainless Steel
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Thanks to the removable pan supports allow for quick and efficient cleaning after cooking; letting you spend more time on the important things in life.
Key Features
- B energy Rating
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- 2x Enamelled Pan Supports
- 5 Multifunction: Gratin, Grill, Multilevel, Top Heat Only, Traditional

subset: commoncrawl


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Empanada Nation
Pioneer Centre, Brixton St., Kapitolyo, Pasig, Metro Manila
- 1 Reviews
- 0 Recommend
- 1 Reviewers
Primary Details
- Category:
- Snack
More Info
Most Recent Reviews
I had the King's meal and an empanada.
The meal was ok. I liked the bagnet the most. Was not too keen on the empanada though.
Self-serve, parking is almost always easy at Pioneer Center.
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The Joker residence is situated near the entrance of resort in the Arolles area, next to the bus station and the Caron sport and shopping centre. Access to the slopes at 30 meters. Resort map n° 41/C3.
South facing studio of 21m² with balcony. 5th floor slope side (2nd floor from building entrance).
Kitchenette with microwave/ grill combi, refrigerator, electric hobs and dishwasher. Dining area.
Bathroom with bath. Separate toilet.
Cabin room with single bed (separated by sliding door).
Living room with double sofa bed.
Resort WIFI network payable on condition that apartment has sufficient coverage.
Join us on Facebook :


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Microsoft's software donation program, which has already provided more than $3.9 billion worth of software to nonprofits around the world since 1998, is being expanded to enable more nonprofit organizations to request software donations when they need them at any point in the year.
"Every day we see how technology is helping nonprofits to operate more effectively and ultimately do their best work," said Rebecca Masisak, co-CEO of TechSoup Global. "The updates to the Microsoft Software Donation Program provide nonprofits increased flexibility in requesting software, which we believe will greatly benefit many nonprofits in the TechSoup Global community that rely on a range of Microsoft products to run their organizations effectively."
The updates to the Microsoft Software Donation program, which is currently available in more than 100 countries around the world, include the following:
- Number of different Microsoft software products that can be requested has been expanded from six to 10 to allow nonprofits to get the software they need, such as Windows 7, Office 2010 and SharePoint 2010.
- Three new categories of nonprofit organizations are now eligible for software donations, including medical research organizations, private foundations, and amateur sports and recreational organizations.
- Donations program includes a new Get Genuine offering so nonprofits can ensure their existing computers are running genuine versions of Microsoft operating systems to help keep their software up-to-date and security-enhanced.
- Nonprofits can now request a software donation from Microsoft through the TechSoup Global Network whenever they need it instead of the previous limit of only one request per year.
- Nonprofit organizations ordering their donations through the TechSoup Global Network can now easily get key donations details in one place with the Microsoft Donations Center, a new website where organizations can review their donation history and identify products their organizations can reque
Eligible nonprofit organizations can choose from a wide range of Microsoft desktop and server products (including Office and Windows). Check out www.microsoft.com/nonprofit to see what's available and learn how to apply. In fact, if you are in one of these 35 countries with a local TechSoup program, you may be able to get technology donations from other companies too, like Cisco, Symantec, or Adobe.
You can also read more about the program changes in detail on TechSoup's site: Overview of Microsoft Software Donations, and you can review our Frequently Asked Questions.
[Source: Microsoft Press]


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AMS is now a Qualified Integrator and Reseller Company (QIR).
The PCI Security Standards Council accreditation, allows qualified companies to implement, configure, and/or support validated PA-DSS Payment Applications on behalf of merchants.
At AMS, we are constantly exploring new technologies for our clients which has fueled substantial growth in our suite of solutions that includes a comprehensive merchant services program and a full service PCI DSS compliant payment gateway.
Secure, stackable solutions provide the power behind your payments with leading data security technology including Point-to-Point security for the ultimate in authentication technology for government.
AMS utilizes only the industry's most secure and reliable processing networks, all of which are compliant with the PCI DSS. We continuously educate and update our merchant community with all industry regulatory and security announcements.


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Curtain falls on Dame Joan Sutherland Award
- Details
- Published: 05 April 2017
- Written by NFA Editor
Regional Australia's richest opera award, the Dame Joan Sutherland Vocal Competition Award (DJSA), has taken its final breath. For nearly three decades, the finals of the prestigious national opera award were held in Noosa annually (and later bi-annually), proudly hosted by its patron Noosa Federation of the Arts Inc. (NFA).
Since 1999, the DJSA has helped 13 past winners plus numerous other young Australian singers aged 20-34 to reach dizzying heights performing with illustrious opera houses nationally and abroad. Past winners who have achieved international fame include Mirusia Louwerse, who sang under contract to Andrei Rieu, and Kang Wang who is under contract to the New York Metropolitan Opera. Guy Booth, Virgilio Marino and Shaun Brown currently have major roles with Opera Queensland.
Founding chairman of NFA, Meg Lawrence OAM, said it was with deep regret that the Federation had to shut down in April due to a continuing lack of funding and a paucity of interest by a younger generation.
The NFA was founded in 1989 to promote the arts within the local community and to elevate Noosa as a cultural destination. The Federation was based on four pillars: music, art, dance and avant garde. It became a cultural force to be reckoned with when Professor Anthony Camden, the then provost and director of the Queensland Conservatorium, became NFA's artistic advisor. The following year (1990), Prof Camden invited founder and music director of St Martins in the Fields, Sir Neville Marriner, to be the NFA's patron, which he was until his death in 2016. Marriner and his Academy hold a record for the largest number of classical music recordings in the world – more than 500 albums – but he was most famous for his soundtrack to the 1984 Oscar-winning film, Amadeus.
"As a champion of the arts, we really were unique in Australia," Meg said.
"We were a museum, art gallery, film club, dance organisation, music, theatre and more, all rolled into one. Anthony was instrumental in our being able to present, here in Noosa, the entire Queensland Philharmonic orchestra, conducted by Nicholas Braithwaite, and featuring Xong Dong Kong, the winner of the 5th Sydney International Piano Competition."
Other luminaries brought to Noosa by NFA include international pianist Piers Lane, renowned flutist Jane Rutter, Sir Neville Marriner, two 13-year-old pianist prodigies from the Hong Kong Performing Arts Academy as well as the Queensland Philharmonic Orchestra, which played on four different occasions at NFA's Strauss balls. NFA also screened the Queensland premiere of the film,' Love's Serenade', winner of a Camera D'Or in Cannes (1996-7), '32 films by Glen Gould' and the iconic 'Tales of the Vienna Woods'. At the Noosa Regional Art Gallery it held an exhibition curated by Arthur Boyd and other specially-curated art exhibitions.
"One of the most magical experiences was the John Nixon singers with the Camerata of St John singing the Maria Mass," Meg said. "I recall looking around me and seeing the audience on their feet, hands clasped in rapture with tears of joy streaming down many of the faces. That experience has never left me."
Noosa high schools were enriched by the annual Year 12 graduate incentives, each receiving an award valued at $300; the $600 Rick Farbach jazz scholarship (awarded to an Immanuel College student for the past 14 years); and the Schools' Instrument Bank, which loaned three cellos, a violin and the Hofmann pearl flute for talented school children to use.
"We found the kids who were playing stringed instruments were absolutely fantastic musicians but because the instrument and bow they were using were inferior, they thought they couldn't play," Meg said.
"We gave them quality instruments and when they heard the 'colour' of the music, they were assured it wasn't their bad playing."
NFA also created the Anthony Camden Fund, which lent exceptional quality instruments to older talented musicians. Its assets include a $37,000 Capellini cello, the last one made by Virgilio Capellini in Cremona, Italy, when he was 80, and a $25,000 Primavera violin. The Capellini is to be donated to the Queensland Conservatorium of Music, Griffith University in memory of Anthony Camden.
Meg said she would like to express her heartfelt thanks to the Coast community because without their moral and financial support over the years NFA wouldn't have existed.
"The most important people in the world are an audience, because without them there is no point in staging a performance," she said.
Pictured: NFA founding members. Left-to-right Hillary Roots, Albert Thoma.
Dame Joan Sutherland Award Finals 2016
- Details
- Published: 10 August 2016
- Written by NFA Editor
The finals of the 2016 NFA Dame Joan Sutherland Award were held on Friday 22nd July at the Good Shepherd Lutheran Hall in Noosaville.
Results as follows:
- First : Iain Henderson
- Second / Margreta Elkins Award : Tabatha McFadyen
- Judges Encouragement Award : Anastasia Bickel
- Peoples Choice Award : Iain Henderson.
Congratulations to all participants and finalists.
Pictured: Finalists and Judges. More pictures below...
Afternoon Delight - Feeling the Pinch of Dad's Shoes
- Details
- Published: 02 June 2016
- Written by NFA Editor
Guest speaker: Famous opera tenor Robin Donald Smith (retired), son of Australia's greatest operatic tenor, the late Donald Smith OBE
When: Friday 24 June 2.30-4pm
Venue: 131 Shorehaven Drive, Noosa Heads (private home, bookings essential)
Passion is set to soar at Noosa Federation of the Arts' Afternoon Delight where famous opera tenor Robin Donald Smith (retired) will regale the audience with tales of his uber-famous father, Australia's greatest operatic tenor, the late Donald Smith OBE.
Hear spellbinding stories about opera duets the father-and-son from Bundaberg, Queensland, performed and Robin's struggle to assert himself as a world-renowned tenor in his own right. Brace yourself for surprise bursts of song by Robin along the way and (as rumoured) a duet with NFA's artistic director, Brad Jarrett, whose illustrious titles include former artistic administrator Opera Queensland.
Donald and Robin made history as the first father-and-son tenors to perform together in Wagner's Opera, Der Fliegende Holländer (The Flying Dutchman), with the then Australian Opera Company, (now OA – Opera Australia). They also appeared on the same stage as an operatic double bill at the Sydney Opera House with Donald performing his famous role as Canio in Leoncavallo's Pagliacci and Robin singing Turridu in Mascagni's Cavalleria Rusticana. Hear some hilarious tales of how they both prepared for a life on stage by studying the acting skills required of their profession.
Robin will also speak candidly of his journey to find his own voice by leaving Australia to forge a stellar international career with the Sadler's Well Opera Company (now the ENO – English National Opera). Despite dropping "Smith" from his name, the comparisons with his famous father were inevitable as the two mostly performed the same roles and both voices had a very similar vocal timbre.
Robin made his professional debut at the Coliseum Theatre, London, in 1968, in the role of Rodolfo in Puccini's La Boheme. He toured Britain and Europe with Sadler's Well Opera Company for a decade performing leading tenor roles in Verdi's Rigoletto and La Traviata; Donizetti's L'elisir D'Amore and Massenet's Manon.
Career highlights include performing in operas celebrating the opening of the Sydney Opera House in 1973: Puccini's Gianni Schicchi in the role of Rinuccio, and as Walther von der Vogelweide in Wagner's Tannhäuser (Opera Australia); and signing with Dame Kiri Te Kanawa as Ferrando in Mozart's opera Cosi Fan Tutte, at the London Opera Centre.
In 1974 the prodigal son returned to Australia permanently. For more than 10 years Robin was Opera Australia's major principal tenor singing in La Boheme, Der Fliegende Holländer, Cavalleria Rusticana, Madam Butterfly and Fledermaus. An exciting raconteur with a great sense of humour, Robin will talk about working with many famous singers, conductors and producers including Dame Joan Sutherland and Janet Baker; Sir Charles Mackerras and Richard Boynage; and Sir Robert Helpmann and John Copley.
Noosa's search for 'The Voice'
- Details
- Published: 15 March 2016
- Written by NFA Editor
Regional Australia's richest opera award calls for contestants
Are you an emerging opera diva or male vocalist looking for the next big break? Regional Australia's richest and most prestigious opera prize, the Dame Joan Sutherland Vocal Competition Award 2016, could help you hit a career high note.
Founded by Noosa Federation of the Arts (NFA) in 1994, the Dame Joan Sutherland Award (DJSA), has helped 12 winners plus numerous runners-up to reach dizzying heights performing with illustrious opera houses nationally and abroad.
If you're an amateur or professional soprano, mezzo, contralto, baritone, bass, tenor or counter-tenor singer aged 20-34, and an Australian resident, you're eligible to apply for this coveted national award.
Applicants will audition live before a panel of adjudicators on Sunday 5 June at Griffith University Conservatorium of Music, Brisbane. Contestants at the heats will sing their song of choice plus either an aria, a song from the classical repertoire or an operetta / music theatre piece chosen for them by the panel. The top six will then battle it out at the final in Noosa, Friday 22 July.
This year's panel for the heats and final are Brad Jarrett (NFA artistic advisor and former artistic administrator Opera Queensland), Andrew Collis (performer with major opera houses in Germany, US and Australia) and Margaret Russell (a long-standing soloist and guest in more than 25 German opera houses).
Jarrett said at the heats and the final, judges would be looking for voice quality, natural ability and vocal technique; repertoire choice, language ability and understanding of the text; performance; musical style; and a well conceived plan about how they would spend the prize money.
At the final (Noosa) the audience votes for the People's Choice Award and the judges also assess how the finalists communicate with the audience emotionally and musically.
"We're looking for someone with that indefinable star quality," Jarrett said. "The best performance on the night, taking into account the judging criteria, wins."
NFA past chairman Meg Lawrence said the DJSA was a tremendous opportunity for young singers to receive feedback from a panel of accomplished judges.
Over the past 15 years, 500 young singers have auditioned to some of the world's most seasoned opera divas and artists. Past adjudicators include Marilyn Richardson (who opened Sydney Opera House), James Christiansen (Chorus Master Queensland Opera and one of Australia's foremost voice teachers), Jillianne Stoll (repetiteur Opera Queensland and Principal/Guest Vocal Coach for its Young Artist Program), Lisa Gasteen (1991 winner Cardiff Singer of the World competition) and the late Margreta Elkins (who sang with her friend Dame Joan Sutherland at Covent Garden).
Jarrett said the DJSA was a "stepping stone" to becoming a successful opera singer in today's world. The winner could travel anywhere to pursue the best opportunities to either continue their vocal development, immerse themselves culturally in preparation for an operatic career, or to access opportunities to get work in major international opera houses.
Jade Moffatt, winner 2014, advises young opera singers to "go for it!".
"It's not only a wonderful opportunity to perform for an incredibly supportive and encouraging panel and audience, but you might just find yourself with some much needed cash to help you on your way," Moffatt said.
After her win, Moffatt travelled to the UK for auditions and to be coached by David Harper, have lessons with opera diva Yvonne Kenny and work with the great Welsh tenor, Dennis O'Neill. She's currently undertaking her Masters on a full tuition scholarship at the Guildhall School of Music and Drama, London. She said signing for her "opera idol" Renee Fleming in London in February was a career highlight.
"I remember listening to her (Fleming's) recordings over and over, and going to see her in MET live screenings at the cinema," Moffatt said.
"I never thought I would ever get the opportunity to meet her! She was so incredibly warm and welcoming, and I came away from the masterclass with newfound motivation. She was also really funny. She realised I was Australian, and told me stories about hanging out with Hugh Jackman."
Moffatt and other past star winners expressed their gratitude for the moral and financial support of NFA members and friends.
"I am incredibly grateful to the NFA for its amazing support of young opera singers, and would not be where I am today without their generosity," Moffatt said.
Winner 2008 Russell Harcourt said being awarded the DJSA was "a great encouragement, both financially and professionally, and a recognition of the countertenor voice".
"Without it, my postgraduate studies at The Royal Academy of Music (2008-2010) would not have been possible," Harcourt said.
"I now study with Yvonne Kenny in London, who continues to encourage me in every aspect of fine bel canto technique – beauty of tone, ease of vocal production and delivery of text. Qualities Dame Joan made appear effortless!"
Where are they now?
• Amy Wilkinson (winner 2001) (soprano) won a three-month scholarship to study at the NY MET and was invited to audition for Opera Paris. After living and working in the USA for some years she has now returned to Australia.
• Mirusia Louwerse (winner 2006) at 21-years-old was the youngest DJSA winner. Since 2007 she has toured the world as the star soprano of Andre Rieu and as a solo artist.
• Virgilio Marino (winner 2007), tenor, will be performing Pinkerton in Opera Queensland's production of Madama Butterfly in May, followed by Count Almaviva in The Barber of Seville in July.
• Victoria Lambourn (People's Choice Award winner 2007) (mezzo soprano) will perform Oberto in Handel's Alcina at the Hessisches Staatstheater Wiesbaden, Germany in April and Smeraldina in The Love of Three Oranges for Opera Australia in May-July.
• Kang Wang (People's Choice Award Winner 2010), tenor, is on the Lindemann Young Artist Development Programme at the MET this year. He's a regular soloist with the Queensland Symphony Orchestra and returns in March for the Mozart Requiem. He was also a principal singer at Theater Basel in Switzerland.
About Noosa Federation of the Arts
NFA was founded in 1989 to promote the arts within the local community and to elevate Noosa as a cultural destination. The bi-annual DJSA is integral to NFA's mission to encourage youth involvement in the arts and relies on donation for its continuation.
First prize $15,000, Margreta Elkins Award $4000, Judges Encouragement Award $1000, People's Choice $750 and an Audition Encouragement Award $300.
Photo: L-R (centre) Renee Fleming, Jade Moffatt (on Fleming's right). Russell Harcourt, Kang Wang & Mirusia Louwerse.
For further information, please contact:
King Arthur - at the Brisbane Baroque
- Details
- Published: 10 February 2016
- Written by NFA Editor
Brisbane City Hall
Monday 11th April – 7.30pm
Noosa Federation of the Arts package includes: Bus transport - Noosa / Brisbane return and reserved tickets.
Semi-opera by Henry Purcell to an original text by John Dryden Adapted by Terence Clarke Directed by Mark Gaal with chorus, soloists and members of the Orchestra of the Antipodes conducted by Brett Weymark
Stirring choruses, sparkling solos and delicious incidental music combine to make King Arthur a memorable musical epic with the widest possible appeal.
This specially commissioned edition directed by Mark Gaal includes distinguished international and local recitalist, concert and stage performer Greta Bradman.
Greta Bradman – recipient of Australian International Opera Award, critics choice award APRA/ AMCOS Performance of the year 2013, numerous Ozcart awards. Helpmann award nominee.
Recordings for Sony Music, ABC Classics – latest album "My Hero" debuted at No.1 on the ARIA classical and crossover charts.
Other soloists include: Jacqueline Porter, Nicholas Scott, Brenton Spiteri, David Greco and Samuel Piper.
All proceeds contribute to fundraising for the Dame Joan Sutherland Award.
Numbers are limited to 24-33 people due to available bus size.
Reserve your tickets by contacting:
Noosa Federation of the Arts Ph: 0447 280 521 (close off date for reservation - early Feb 2016) Information regarding bus departure / return times will be provided closer to the date.
Cost: $150 single, $280 couple.
Young Musician Recognised For Excellence
- Details
- Published: 03 November 2015
- Written by NFA Editor
Multi-talented Year 12 student at Immanuel College, Jarvis Miller, has been awarded the prestigious 2015 Rick Farbach Jazz Scholarship for outstanding work with cello, trombone and piano. Throw in an original composition or two for school musicals.
Pictured left to right: NFA Treasurer, Joanne Daley, Jarvis Miller, NFA Deputy Chairman, Juanita Overson
His Principal, Colin Minke, described him as "an accomplished cellist, trombonist and pianist of many years standing who performed with numerous ensembles during his time at Immanuel". "Jarvis is a dedicated musician who has consistently excelled and this year, he composed an original song for the student devised musical, Teenstars.
"In Music Extension, he achieved at the highest level possible."
Noosa Federation of the Arts established the Rick Farbach Scholarship for a Young Jazz Musician in 2001 to honour Rick's deep love and lifelong service to music. It is presented each year to a Year 12 Immanuel student who has displayed excellence, leadership and commitment to music and intends to pursue musical studies following their time at Immanuel. This year's winner, Jarvis Miller, hopes to study at the Queensland Conservatorium in 2016.
Rick, who sadly passed away in 2005, was considered by his peers to be the greatest exponent of the jazz guitar in Australia. His book "Cleftomania – a disease that strikes all musicians" is a testament to his life as a musician.


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During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity.
Citation: Sebastián-Serrano A, Sandonis A, Cardozo M, Rodríguez-Tornos FM, Bovolenta P, Nieto M (2012) Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1. PLoS ONE 7(2): e31590. https://doi.org/10.1371/journal.pone.0031590
Editor: Amit Singh, University of Dayton, United States of America
Received: June 15, 2011; Accepted: January 16, 2012; Published: February 16, 2012
Copyright: © 2012 Sebastián-Serrano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the (MICINN) Ministerio de Ciencia e Innovación grants (SAF2008-00211; PIE-200820I166; BFU2007-61774), and a grant from the Spanish Comunidad de Madrid CCG08-CSIC/SAL-3464. ASS from the MICINN (BES-2006-13901). FMR holds a fellowship from La Caixa foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The selective response of axons to elongation and guidance cues encountered along their paths enables the precise formation of neuronal circuits and the formation of topographic maps. During development, mechanisms that specify neuronal subclasses are coupled to those that specify their axonal response through the selective expression of transcription factors.
Pax6 is a homeodomain transcription factor expressed in several territories of the developing nervous system, mostly in the proliferative regions containing neural precursors [1]. The vast majority of the neural progeny of these precursors including neurons of the dorsal cortical plate or neural stem cell (NSCs) derived neurons, shut down Pax6 expression upon exiting the cell cycle. Consequently, very few postmitotic populations express Pax6. Notable examples are the retinal ganglion cells (RGCs), the dopaminergic neurons of the olfactory bulb and certain neurons in the basal telencephalon and midbrain [2], [3], [4], [5]. While the role of Pax6 in neural precursors has been widely explored, revealing a central function in cell fate specification and cell cycle regulation [5], [6], [7], [8], [9], less attention has been paid to its functions in postmitotic neurons, with the major exception of a recent study demonstrating that Pax6 is needed for the survival of dopaminergic neurons in the olfactory bulb [10].
RGCs are one of the best-characterized postmitotic populations that express Pax6 [11]. Pax6 expression in both developing and mature RGCs is graded, with higher levels in the ventro-temporal distal cells and lower in the proximal domains [11]. These two differentially expressing Pax6 populations project to distinct non-overlapping and complementary topographic regions of the superior culliculus and lateral geniculate nucleus (LGN), which suggests that Pax6 may contribute to control axon targeting. Indeed, RGCs of mice overexpressing Pax6 show disrupted axonal trajectories and abnormal bundle formation [12], whereas changes in Pax6 expression in the RGCs correlate with axonal regeneration in the optic nerve of lizards and zebrafish [13]. Still, there is no information on whether Pax6 is directly involved in the control of axon growth and what guidance cues, if any, depend upon its activity.
Secreted Frizzled-Related Protein 1 (SFRP1) is one of the factors known to stimulate the directional growth of RGCs axons in Xenopus and chick retina. This activity is independent of Wnt signaling and modulated by extracellular matrix molecules [14], [15]. Mouse Sfrp1 is expressed in several structures of the embryonic and adult eye and brain, including the pigmented retina, cornea, ciliary bodies, lens epithelium, the prospective thalamus and the proliferative regions of the telencephalon [14], [16], [17], [18], [19]. The SFRP1 distribution often coincides with that of Pax6 or decorates the axonal pathways of Pax6 expressing neurons [14], [16], [17], [18], [19], raising the possibility of a functional relationship between the two molecules.
Here we show that expression of Pax6 in neuronal postmitotic populations is necessary and sufficient to confer neurons with a positive response to SFRP1. Over-expression of Pax6 rendered cultured cortical neurons competent to respond to endogenous and exogenous SFRP1. Conversely, knock-down of Pax6 in mouse retinal explants abolishes RGC axonal growth induced by SFRP1 without affecting RGC differentiation or their axonal response to a different guidance cue like sonic hedgehog (Shh) or Netrin. These results suggest that Pax6 and SFRP1 are pair regulators of axonal connectivity in the retina and reveal a new function of Pax6 in the postmitotic populations.
1. Pax6 induces axonal growth in postmitotic neurons
The majority of postmitotic neurons, including those derived from telencephalic Pax6 positive precursors, such as cerebral cortical neurons or neurons derived from cortical neural stem cells (NSCs), do not express Pax6 [2] (Fig. S1a). To test the consequences of Pax6 expression in postmitotic populations, we forced its expression in neurons derived from mouse NSCs. NSCs grown as neurospheres were nucleofected with CAG-empty vector or CAG-Pax6, together with a CAG-GFP that allowed the visualization of transfected cells. GFP staining demonstrated efficient transfection by FACs cytometry (Fig. S1b) and immunostaining of plated cells confirmed that cells transfected with CAG-Pax6, but not those transfected with the empty vector, had nuclear Pax6 protein expression (Fig. S1a). Plated transfected cells were then allowed to differentiate for 9 days to analyze their differentiation and morphology. GFP neurons identified by β-tubulin III expression show an advance differentiated asymmetrical polarized morphology, both in control and Pax6 transfected cells (Fig. 1 c), and the longer neurite was identified as the axon by MAP1b staining (not shown), a selective marker of the distal part of the growing axon [24]. Overexpression of Pax6 alone was sufficient to promote axonal extension compared to control neurons (Fig. 1a). Quantitative analysis of β-tubulin III positive-projections confirmed a significant increment of the axonal length from neurons transfected with CAG-Pax6 both at 6 and 9 days of culture (Fig. S2 and Fig. 1a, respectively). Similar results were obtained upon quantification of MAP1b projecting axons (not shown). The total number and extension of the additional neurites was not affected (Figure S1c).
NSCs were nucleofected with CAG-empty vector or CAG-Pax6 and co-electroporated with CAG-GFP. a) The graph shows the percentage of nucleofected neurons with respect to their axonal length after 9 days of differentiation. Over-expression of Pax6 increments the axonal length compared with control neurons. b) The graph shows quantification of the axonal length of a cohort of BrdU positive neurons. c) Nucleofected neurons were identified by the specific expression of β-tubulin III and GFP. BrdU staining allowed us to compared neurons of similar birth dates. The effect in axonal growth is unrelated to an early exit from the cell cycle. Arrows point to BrdU stained nuclei. Bar indicates 50 µm. d) Cortical primary neurons were also transfected with CAG-empty vector or CAG-Pax6, and with CAG-GFP. Transfected primary axons were identified by GFP and MAP1b staining. Arrows indicate the distal part of the axon stained by MAP1b. Bar indicates 70 µm. e) The graph shows the percentage of primary neurons with respect to their axonal length. Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01.
Several reports have shown that Pax6 over-expression induces precursor cells to exit the cell cycle and differentiate [9], [25]. To discard the possibility that increased axonal length was a consequence of a premature exit from cell cycle and, thus, of an extended time of differentiation, we restricted our study to those cells that were leaving the cell cycle at similar times. To visualize cohorts of nascent neurons, BrdU was added on the second day of culture and extensively washed after 12 h. Analysis of BrdU-positive neurons on the ninth day of culture confirmed that the axons of the Pax6 expressing population were significantly longer than those of cells transfected with mock vector (Fig. 1b and c). Restricting to this cohort of BrdU neurons correlated with a higher increment in axonal length as expected from the analysis of a more homogenous population (compared Fig. 1a and b). This demonstrates that the effect in axonal growth is unrelated to an early exit from the cell cycle. To strengthen this conclusion, we over-expressed Pax6 in non-proliferating primary cortical neurons and quantified the length of MAP1b projections, obtaining similar results (Fig. 1d and e), although the increment of outgrowth was less evident than that observed in NCSs, likely owing to the heterogeneity of primary differentiating neurons. Together, these experiments demonstrated that Pax6 expression in differentiated neurons stimulates process elongation by a cell cycle independent mechanism.
2. Pax6 mediates the axonal response to SFRP1
SFRP1 is expressed by the neural precursors of the ventricular zone (VZ) of the dorsal telencephalon, which give rise to both cortical neurons and NSCs [2]. We confirmed that neural precursor cells cultivated in vitro also express detectable levels of the SFRP1 protein (Fig. 2a). We therefore postulated that SFRP1 produced and secreted in the culture by undifferentiated cells was responsible for the selective axonal growth of Pax6-transfected neurons. Addition of antibodies against SFRP1 to the culture media two days after NSCs transfection completely blocked Pax6-induced axonal growth, leading to processes with a length undistinguishable from that of control cells (Fig. 2b). This result was specific since addition of control unrelated antibodies had no effect (Fig. 2b). This data indicated that Pax6 stimulated the growth of NSCs derived neurons in response to endogenous SFRP1 produced in the in vitro culture.
a) Immunofluorescence shows expression of SFRP1 (red) in differentiating, NSCs cultured for 5 days. Cells were double labeled with anti-nestin, GFAP and β-tubulin III. SFRP1 expression is found in the cytoplasma of Nestin-positive cells or in some with a low GFAP expression (arrowheads). No SFRP1 expression was found in cell with high GFAP levels or in β-tubulin III-positive cells. Bar represents 20 µm. b) The graphs show the percent of axons longer than 100 µm upon Pax6 overexpression in NSCs when cultured in the presence or absence of control or anti-SFRP1 antibodies. Note that antibody against SFRP1 blocks axonal growth stimulated by the ectopic expression of Pax6. c, d) Response to increasing concentrations of purified recombinant SFRP1 in neurons derived from NSC c) or primary cortical neurons (d). NSCs (c) and primary neurons (d) were transfected with control empty CAG and CAG-Pax6 vectors and stimulated with different concentrations of SFRP1.Addition of recombinant SFRP1 only stimulates the growth and elongation of axons of neurons ectopically expressing Pax6. Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01; (***) p<0.001. Number of axons per condition >50 (n = 3).
To support this conclusion, we next assayed the effects of exogenously added SFRP1 upon forced expression of Pax6 in primary cortical neurons or neurons derived from NSCs. Purified, recombinant SFRP1 was added to the neuronal culture after transfection and the early axonal response was evaluated after two or three days in cortical neurons and NSCs, respectively. Notably, addition of exogenous SFRP1 to control neurons had no effect on the length of their axons, but stimulated, in a dose dependent manner, the elongation of those belonging to Pax6-positive neurons (Fig. 2c, d and Fig. S3). Therefore, Pax6 appears to be necessary and sufficient to confer cortical neurons with the competence to respond to SFRP1 of exogenous or endogenous origin.
Postmitotic RGCs express Pax6 [4] and their axons respond to SFRP1 [14]. We therefore asked whether this response was similarly dependent on Pax6. To this end we decided to knock-down Pax6 using shRNA lentiviral constructs. Effective knock-down of the Pax6 protein by the shRNA lentiviral constructs was first evaluated in cultured mammalian CHO cells transfected with Pax6 (See Methods and Fig. S1d). Selected constructs were thereafter targeted to the retina of E13.5 embryos by in utero electroporation. A CAG-GFP plasmid was co-electroporated to visualize targeted cells and their axons. The expression of Pax6 and the fate of the targeted cells were analyzed at E19.5 in histological sections. In retinas targeted with either control or Pax6 shRNA, the majority of GFP-positive cells were located in the inner layers, corresponding with the normal location of RGCs (Fig. 3a and b). Double staining with anti-Pax6 and -GFP antibodies demonstrated strong expression of Pax6 in GFP-positive control cells but undetectable levels in the majority of cells electroporated with the Pax6-shRNA (Fig. 3a). Quantification of the number of GFP and Pax6 positive cells demonstrated that the selected shRNA resulted in efficient down-modulation of Pax6 in the mouse retina cells (Fig. 3a, graph). No significant changes were observed in the number of apoptotic, Caspase3-positive cells [26] (Fig. 3a). Because Pax6 is involved in cell fate decisions and cell cycle exit [4], [9], [27] we next analyzed the molecular identity of the GFP electroporated cells using two RGC specific markers: Islet-1/2, that marks most of RGCs [28], and Brn3, which labels only a subpopulation [29]. In control retinas, an important proportion of GFP positive cells showed expression of Islet-1/2 and Brn3, with the expected pattern. Down-regulation of Pax6 did not block RGC generation but somehow favored the increase of the number of Islet-1/2 positive cells, which was less clearly correlated with that of Brn3 positive cells (Fig. 3b). This moderated shift in the proportion of RGCs cells might be associated to the reported effects in cell cycle exit [4], [9], [27], rather than on selective cell death, which was invariant (Fig. 3a).
Frontal cryostat sections of E19.5 retinas after electroporation at E13.5 with shRNAs constructs control and targeting Pax6 and immunostained (red) for Pax6 and Caspase 3 (a) or Islet-1/2 and Brn3 (b) as indicated in the panels. Electroporated cells are visualized with GFP (green). The graphs show the quantification of the proportion of the double positive cells for the indicated marker. Note that shRNA efficiently target Pax6 without inducing cell death (a) or changes in expression of markers for postmitotic RGCs (b). Bar indicates 10 in (a) and 40 µm in (b). Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01. Number of axons per condition >60 (n = 3). INL, inner layer; VZ, ventricular zone.
Given that shRNA-mediated knock-down of Pax6 in E13.5 retinal precursors did not interfere with RGC generation and organization, we examined the response of Pax6 deficient RGCs to SFRP1 [14]. Explants from E13.5 retinas electroporated ex vivo with control shRNA or shRNA constructs targeting Pax6 were seeded on laminin-coated coverslips, stimulated with SFRP1 and fixed 24 h after. Staining with anti-β-tubulin III confirmed that, as in chick and Xenopus [14], SFRP1 stimulated axonal outgrowth and extension from control mouse retinas, increasing the proportion of axons longer than 900 µm (Fig. 4a). Similarly, addition of SFRP1 to control electroporated retinal explants doubled the proportion of GFP-positive electroporated RGCs with axons longer than 200 µm (Fig. 4b), although the average length was appreciably shorter than that of axons from non-electroporated retinas (compare Fig. 4a and 4b). Likely, this is because targeted cells only account for newly generated RGCs [30]. Knock-down of Pax6 in RGCs prevented their response to SFRP1 (Fig. 4b) but did not interfere with axonal initiation because the total number of axons per explants was not significantly different in control and Pax6 shRNA electroporated retinas in the presence or absence of SFRP1 (not shown). Furthermore, in non-stimulated explants, the length of GFP positive axons was similar in both control and Pax6 shRNA electroporated retinas (Fig. 4b). Notably, axonal growth was restored when knock-down was attempted in the presence of a silent mutant form of Pax6 resistant to shRNA, excluding off-target effects of the shRNA constructs (not shown). Furthermore, Pax6 knock-down had no effect on the response of dorsal retinal explants to exogenously added Shh (Fig. 5a and 5b) or Netrin1 (Fig. 5c), additional axon guidance cues known to modify RGC axon growth [23], [31], [32]. These results indicated that the expression of Pax6 in mouse RGCs prompt axon extension specifically in response to SFRP1, whereas it does not influence the response to Shh and Netrin1.
a) Low magnification images (a) and confocal micrographs (b) of explants from control (a) or electroporated retinal explants (b) seeded onto laminin coated coverslip and cultured in the absence or presence of recombinant SFRP1 as indicated in the panels. Explants in (a) were stained with β-tubulin III whereas those in (b) were co-electroporated with CAG-GFP and shRNA control or shRNA targeting Pax6 and axons were visualized by GFP expression. Graph shows quantification of the proportion of total axon longer than 900 (a) or 200 (b) µm. Note that knocking-down Pax6 inhibits the axonal response stimulated by SFRP1. Bar indicates 300 (a) and 150 µm (b). Data are expressed as the mean ± SD. (*) p<0.05 comparing stimulated populations; (**) p<0.01 and (***) p<0.001 compared with control.
a) Micrographs show confocal images of GFP positive axons of retina explants co-electroporated with CAG-GFP and control shRNA or shRNA targeting Pax6. Explants were seeded onto laminin coated coverslip in the presence or absence of recombinant Shh. Bar indicates 100 µm. b) Graph shows quantification of the proportion of GFP positive axons longer than 100 µm in the presence or absence of recombinant Shh. c) Graph shows quantification of the proportion of GFP positive axons longer than 200 µm in the presence or absence of recombinant Netrin 1. Data are expressed as the mean ± SD. (*) p<0.05; (**) p<0.01.
The Fz2 receptor appears to mediate the axonal response to SFRP1 in RGCs [14]. Thus, the selective induction of the expression of this receptor appeared as possible feasible mechanism of the action of Pax6. However, equal expression of Fz2 protein was detected in the axons of RGCs electroporated with either control or shRNAs targeting Pax6. Similarly quantitative PCR analysis did not reveal significant differences in the mRNA levels of Fz2 from control and Pax6 overexpressing neurospheres or primary neurons (Fig. S4). Similarly, there were no significant changes in the expression of other members of the Frizzled receptor family expressed in the retina, [33], including Fz3, Fz5, Fz6 and Fz7 (Fig. S4). We therefore consider the possibility that Pax6 could regulate the expression of members of the family of Unc receptors, Unc5a, Unc5b, Unc5c and Unc5d, that contain SFRP1 binding domains [34] or that of other receptors implicated in axonal guidance like Neuropilin1 or PlexinD1 [35]. However, Pax6 overexpression in primary cortical cells did not induce significant changes in the expression of any of these genes (Fig. S4), suggesting that other factors might be involved.Hence, the regulation of the expression any of these receptors does not underlie the specific response to SFRP1 triggered by Pax6. Altogether, our results demonstrate that Pax6 is necessary and sufficient to confer postmitotic differentiating neurons with the competence to respond to SFRP1, revealing a novel non neurogenic function of this pleiotropic transcription factor.
Pax6 expression is crucial for dorsal-ventral and anterior-posterior patterning of the neural tube and controls neural precursor proliferation and fate decisions [13], [36], [37]. We herein demonstrated that expression of Pax6 in postmitotic neurons is necessary and sufficient to provide axons with the competence to respond to SFRP1, a secreted molecule that can control growth cone movements [14]. This conclusion is based on the observation that abrogation or activation of Pax6 expression in neuronsthat, respectively, normally do or do not express Pax6, correlates with the response of their axons to SFRP1. This novel activity adds up to the very limited examples of Pax6 functions in postmitotic neurons, namely the control of dopaminergic neuron survival via the regulation of Crystallin αA [10]. In progenitors, other recent studies have also pointed out non-neurogenic roles of Pax6, such as the downregulation of the neurotrophin receptor p75NTR [38]. Together, these studies consistently highlight alternative functions of this pleiotropic factor.
Although much progress has been made in defining the mechanisms directing the formation of brain circuitry, there is still much to learn about the precise coordination between axonal connectivity and the acquisition of neuronal identities, which, through the selective expression of certain specific transcription factors, specifies the interpretation of these signals. Indeed, the reported number of such transcription factors is still limited and needs to be expanded to obtain a truly comprehensive landscape of the determinants of neuronal connectivity. Moreover, the mechanisms that accurately synchronize acquisition of neuronal subtype identity with guidance molecule expression deserve further elucidation. Examples of this coordination are offered by the LIM homeodomain protein control of EphA receptor and ephrin-A ligand expression during motor neuron innervation of limb muscle [39]. Similarly, Lhx2/9 in the dorsal spinal cord directly regulates the expression of Rig-1 [40], a divergent member of the Robo family, required for midline crossing of commissural axons [41]. In the retina, expression of Zic2 in the ventro-temporal retina determines ipsilateral identity of RGCs [42] and controls the behavior of their axons likely by regulating the expression of EphB1 [43], a specific guidance receptor implicated in ephrinB2-mediated repulsion of uncrossed axons at the optic chiasm midline [44]. However, genetic control of receptor expression is not always an evident explanation, and there are also multiple transcription factors shown to be critical in directing axonal trajectories by still unknown or alternative pathways. In the cerebral cortex for example, Ctip2, and Satb2, determine the final targets of corticospinal motor neurons and callosal projecting neurons, respectively [45], [46], [47], [48], [49] and recent studies report that these two cell populations differentially respond to Sema3A, a potent repellant of cortical axons [50]. Surprisingly, similar levels of Sema3A receptors are found in cells expressing Satb2 or Ctip2, and the selective response is proposed to be dependent on the control of the receptor's endocytic and signaling pathway [51].
In this scenario, our data provide an additional example of a transcription factor that selectively controls the response to axon guidance cues. Although we clearly demonstrated a relationship between Pax6 expression and response to SFRP1 in different neurons, we did not found an obvious mechanism to explain this relationship. In chick and Xenopus, SFRP1 binds to Fz2 to mediate axonal response [14]. Our experiments indicate the unlikely involvement of the Fz2 receptor and suggest other unknown mechanisms that we have been so far unable to decipher. There are several possible scenarios. Other yet undefined SFRP1 receptors or co-receptors might be involved. Pax6 might also control the expression of other molecules involved in the pathway downstream of SFRP1 response. Alternative Pax6 may regulate the expression of ECM molecules known to be needed to control SFRP1 response, such as laminin and fibronectin [14]. Previous microarray analysis of Pax6 mutants and cells over-expressing Pax6 makes way to these possibilities and have shown regulation of guidance molecules receptors such as the Netrin receptor Unc5h, PlexinA2 and EphA5, signal transduction molecules of the Wnt pathway such as Gpc3, or cell adhesion molecules such as tenascin C or cadherin 11 [52], [53]. Notably, the screening of a phage display peptide library revealed that SFRP1 binds with high affinity to the peptide motif L/V-VDGRW-L/V, which is present in Unc5H3 [34], although, whether the two proteins actually interact is still unknown.
Understanding the role of Pax6 and SFRP1 in the selective connectivity of neuronal subclasses may contribute to untangle a piece of the puzzle of brain circuits. In particular, in the retina, developing RGCs express different levels of Pax6 protein [11] and their axons travel in the vicinity of several anatomical domains of expression of SFRP1 that may serve as cue to establish their connectivity [15], [18], [54], [55]. In regard to other Pax6 positive postmitotic populations in the brain, as the ones referred above, Sfrp1 expression patterns would be consistent with a possible role of the Pax6-SFRP1 pair in circuit formation [16], [19]. However, although Sfrp1 knock out mice have been generated and are viable, defects in axonal connectivity have not yet been investigated [16] and will require attention in the future, especially in relation to Pax6, as our results indicate.
Besides axon guidance, an additional interesting implication of our study is raised by the blocking experiments using anti-SFRP1 antibodies. Our data evidenced the secretion of endogenous SFRP1 by NSCs and differentiating primary cortical cells, which is consistent with the reported Sfrp1 expression patterns in the developing and adult brain [19], [56]. NCS express Pax6 both in vivo and in vitro, which suggests possible roles for the coordinated functions of both molecules in the physiology of NSCs and neural niches.
All animal procedures were approved by the National Center for Biotechnology Animal Care and Use Committee, in compliance with National and European Legislation (ID 080016). C57BL6 mice were obtained from Harlan Laboratories, Inc. (Indianapolis, US). Morning of the day of the appearance of the vaginal plug was defined as embryonic day (E) 0.5.
Antibodies, immunohistochemistry, histology
Mouse embryos were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS; pH 7.4) and cryoprotected in 30% sucrose in PBS. 10–20 µm horizontal sections were produced and mounted on Superfrost plus microscope slides (Fisher Scientific, Pittsburgh, PA). Inmunostaining of tissue sections and explants, or cell cultures was performed as described [20] using the following antibodies: rabbit anti-GFP (Molecular probes, Eugene, OR, 1∶500), mouse anti-β-tubulin III (Sigma-Aldrich, ST Louis, MO, 1∶1000), rat anti-Bromodeoxiuridine (BrdU) (BD Biosciences, San Jose, CA, 1∶250), mouse anti-Islet-1/2 (39.4D5 1∶10) developed by T. Jessell and the monoclonal antibody anti-Nestin (Rat 401, 1∶100) developed by S. Hockfield, were obtained from the Development Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, rabbit anti-SFRP1 (Abcam, Cambridge UK, 1∶500), goat anti-Brn3 (C-13) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, 1∶250), rabbit anti-Caspase3 (Cell Signaling Tech Inc, Danuers, MA, 1∶500), rabbit anti-Pax6 (Covance, Princeton, NJ, 1∶500), mouse anti-MAP1b (Covance, Princeton, NJ, 1∶500), rabbit anti-Fz2 (Abcam, Cambridge, UK, 1∶200), mouse anti-GFAP (Sigma-Aldrich, ST Louis, MO, 1∶400), and Alexa Fluor 488-, 594- or 647-conjugated fluorescent secondary antibodies (Molecular probes, Eugene, OR, 1∶500).
Epifluorescece and confocal microscopy
Confocal microscopy was performed with a TCS-SP5 (Leica Microsystems GmbH, Welzlar, Germany) Laser Scanning System. Tissue sections of 50 and 20 µm were analyzed by taking 0.2 µm serial optical sections with the LASAF v2.2.1 software (Leica Microsystems GmbH, Welzlar, Germany). Fluorescence microscopy was performed with a Leica DMRXA and images were captured with Leica CW4000 FISH Version V1.2 software.
DNA constructs and recombinant proteins
The hairpin sequences targeting Pax6 were: TRCN0000075377:ICCGGCCTGAAGCAAGAATACAGGTACTCGAGTACCTGTATTCTTGCTTCAGGTTTTTG;ITRCN0000075376:ICCGGCATGGCAAACAACCTGCCTATCTCGAGATAGGCAGGTTGTTTGCCATGTTTTTG;ITRCN0000075375:ICCGGGACGGCATGTATGATAAACTACTCGAGTAGTTTATCATACATGCCGTCTTTTTG;ITRCN0000075374:ICCGGCCACTTCAACAGGACTCATTTCTCGAGAAATGAGTCCTGTTGAAGTGGTTTTTG.
Pax6 and green fluorescence protein (GFP) cDNAs were under the cytomegalovirus enhancer, chicken β-actin promoter, and rabbit β-globin poly (A) signal (CAG) (cytomegalovirus [CMV] and beta-actin) promoter. For knock-down experiments, a mixture of the selected shRNA lentiviral constructs (1 µg/µl) and pCAG-GFP (1 µg/µl) was used. For testing off-target effects, we co-electroporated a construct driving expression of a silent resistant form of Pax6 (1 µg/µl; CAG-mutPax6; see Text S1) and shRNA targeting Pax6 (1 µg/µl) plasmids. Pax6 lentiviral shRNA constructs were obtained from Sigma-Aldrich Inc. (St. Louis, MO). Purified recombinant SFRP1 protein was generated as previously described [21] and recombinant purified N-Shh (2.5 µg/ml) was a kind gift from Dr. Sebastian Pons [22].
In utero and ex vivo electroporation
In utero and ex vivo electroporations were performed as described previously [23]. E13.5 pregnant C57BL6 female mice were anesthetized by continuous inhalation of Isoflurane (Baxter. Deerfield, US). The abdomen was opened and the uterine horns exposed. The DNA solution containing 0.03% fast green in PBS was injected into one eye of each embryo using a pulled glass micropipette. The head of each embryo was placed between tweezer type electrodes (CUY 650 P5 Nepa GENE, Chiba, Japan) and five square electric pulses (38V, 50 ms) were passed at 1 sec intervals using an Electro Square Porator ECM 830 (BTX, Harvard Apparatus, Holliston, MA, USA). The wall and skin of the abdominal cavity were sutured and the embryos were allowed to develop normally until E16-P0. For the ex vivo electroporation the head of the E13.5 embryos were transfer to a dissecting dish where they were electroporated with the above conditions. Then, electroporated retinas were dissected and incubated in DMEM-F12 (Gibco, Invitrogen, Carlsbad, CA) supplemented with N2 (Gibco, Invitrogen, Carlsbad, CA) medium at 37°C, 5% CO2 for 24 hours.
Retinal explants and analysis of axonal growth
Dorsal retinal microexplants were plated on glass coverslips coated with poly-D-Lys (50 µg/ml; Sigma-Aldrich, Co. St. Louis, MO) and laminin (10 µg/ml; Invitrogen, Carlsbad, CA) and grown for 24 hours in DMEM-F12 medium supplemented with N2 in the presence or absence of SFRP1 (2 ng/µl) or N-Shh (1 ng/µl). After 24 hours, explants were fixed in 4% PFA at RT for 30 minutes and immunostained with anti-β-tubulin III and anti-GFP antibodies. Axonal length was determined in confocal images employing LaserPix v.4 image software (Bio-Rad). In non-electroporated explants, axonal length was estimated by counting the number of β-tubulin III positive axons crossing 600 and 900 µm radius circumferences of a minimum of 100 axons in each of three independent experiments (n = 3). For electroporated explants, we measured the length of each individual GFP positive axon from the edge of the explant up to the distal tip of the growth cone. Knock-down of Pax6 in RGCs did not interfere with axonal initiation because the number of total axons per explants was not significantly different in control and Pax6 shRNA electroporated retinas (not shown). It was also not affected by treatment with SFRP1, Netrin1 or Shh. For SFRP1 experiments at least 50 axons per condition in each independent experiments (n = 6) or 60 neurons per condition (n = 4) in Shh and Netrin1 experiments. We chose to specifically study SFRP1 response in dorsal explants because this population expresses high levels of Pax6 and is homogeneously composed of contra-laterally projecting axons facilitating the analysis of SFRP1 response.
Neurosphere culture and nucleofection
Dorsal telencephalons of E13.5 WT embryos were dissected and cells were dissociated employing trypsin and a flamed-tip, glass Pasteur pipette. Dissociated cells were cultured in DMEM-F12 supplemented with N2 (Gibco, Invitrogen, Carlsbad, CA), heparin, recombinant human basic fibroblast growth factor (FGF-2) (10 ng/ml; R&D Systems, Minneapolis, MN), and epidermal growth factor (EGF; 10 ng/ml; Gibco, Invitrogen, Carlsbad, CA). Neurosphere cultures were passage every 3 days by mechanical dissociation. Dissociated cells were transfected using the nucleofector (Amaxa Biosystems, Gaithersburg, MD) with 5 µg of a 1∶1 mixture of CAG-GFP and CAG-Pax6 or CAG- empty vector. Nucleofection efficiency and GFP positive cells were analyzed by flow cytometer (Coulter Epix XL) and data were analyzed with the Win MIDI software. Nucleofected neurospheres were seeded on glass coverslips coated with poly-D-Lys and cultured in DMEM-F12 supplemented with N2, heparin and 1% fetal calf serum (FCS). Cells were fixed in 4% PFA at RT for 30 minutes, then immunostained with anti-β-tubulin III and anti-GFP antibodies. Axonal length was determined with the LaserPix v.4 image software (Bio-Rad, Richmond, CA) over images taken with a fluorescence microscopy Leica DMRXA. For analysis of the effects of Pax6, cells were fixed after 6 or 9 days in culture. To compare neurons that exit at the same time from the cell cycle, postmitotic neurons were labeled with a 12 h pulse of Bromodeoxiuridine (BrdU, Sigma-Aldrich, ST Louis, MO) (0.5 ng/ml) in the media and subsequently extensively washed. For blocking the endogenous SFRP1 activity a mix of anti-SFRP1 antibodies (4 µg/ml dialyzed in PBS): rabbit anti-SFRP-1 and goat anti-SFRP-1 (H-90 and C-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a control IgG (4 µg/ml, dialyzed in PBS; rabbit anti-p-αPAK Ser 199/204; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to the culture media after nucleofection. For stimulation experiments, SFRP1 was added to the media 24 h after the nucleofection and the early axonal growth measured 3 days after the addition of SFRP1.
Primary neurons culture and transfection
Dissociated cells from the dorsal telencephalon of E13.5 embryos were seeded onto 24 well Poly-D-Lys coated plates in Neurobasal media supplemented with B27 complement (Gibco, Invitrogen, Carlsbad, CA) and BrdU (0.5 ng/ml) to test proliferation. 24 h after seeding, cells where co-transfected with CAG-Pax6 or the empty CAG- vector and CAG-GFP using lipofectamine 2000 (Invitrogen, Carlsbad, CA) and following manufacture procedure. Three days after transfection, cells were fixed with PFA 4%, and immunostained with anti-β-tubulin III and anti-GFP antibodies to measure their axonal length as describe next. GFP positive neurons did not incorporate BrdU demonstrating an effect on posmitotic neurons independent of cell cycle exit. As mentioned, SFRP1 was added to the media 24 h after transfection and the axons measured 2 days after the addition of the protein.
Quantification of axonal length
In neurons differentiated from NSC or primary neurons, we identified the axon by morphological criteria and the specific staining of MAP1b, selectively localized to the distal part of axonal projections [24], but not in neurites. As illustrated in Fig. 1d, staining with MAP1b allowed us to identify the axon in primary neurons, while GFP staining reveals transfected cells. In neurons derived from NSCs after 6 and 9 days of differentiation, a long projection neurite was always clearly observed (Fig. 1c) and quantified as an axon. Specific staining with MAP1b demonstrated that the axon was always the longest neurite projection (99% Map1b positive projections were the longest neurite in the cell (>100 cells counted). Accordingly, experiments in which we measure MAP1b or the morphological identified neurite gave equivalent results (compared results shown in Fig. 1e and Fig. 2). We measured the axonal length from the nucleus of the neurons to the distal end of the axon where the growth cone is localized. We analyzed a minimum of 50 axons per condition in four independent experiments (n = 4).
Supporting Information
Figure S1.
Efficiency of Pax6 overexpression and inhibition. a) Transfection of pCAG-Pax6 in NSCs results in efficient overexpression of Pax6 proteins in NSC derived neurons. Pax6 proteins are not expressed normally in GFP positive CAG-control targeted neurons, but are ectopically expressed (yellow) in cells transfected with CAG-Pax6 plasmid. Bar indicates 20 µm. b) Fluorescent-activating cell sorting (FACs) analysis demonstrates more than 20% transfection efficiency. c) Ectopic Pax6 expression in NSCs does not alter the total number of secondary and primary neurites per cell. The number of neurites, excluding the axon, was quantified 9 days after transfection. d) Transfection of shRNA lentiviral constructs efficiently suppresses the ectopic expression of Pax6 in CHO cells. CHO cells co-trasfected with CAG-Pax6; control shRNA and CAG-GFP show expression of Pax6 protein (red). Pax6 is down-modulated in cells transfected with CAG-Pax6 and shRNAs targeting Pax6. Number of cells >100 (n = 3). Bar indicates 30 µm. Graph represents the proportion of GFP positive cells expressing Pax6 protein. Data are expressed as the mean ± SD. (***) p<0.001.
Figure S2.
Ectopic expression of Pax6 stimulates axonal growth in cortical neurons. NSCs were nucleofected with CAG-empty vector or CAG-Pax6 and co-electroporated with CAG-GFP. Graph shows the percentage of nucleofected neurons with respect to their axonal length after 6 days of differentiation. Results are equivalent to those obtained at 9 days. Over-expression of Pax6 increments the axonal length compare with control neurons. Data are expressed as the mean ± SD. (**) p<0.01.
Figure S3.
SFRP1 stimulated axonal response in Pax6 over-expressing neurons but not in the control population. Graph represents the population distribution of neurons with axons longer than 100 µm or more. Data are expressed as the mean ± SD. (**) p<0.01; (***) p<0.001.
Figure S4.
Knock-down of Pax6 did not affect the expression of Fz2 receptor. a) Micrographs show confocal images of GFP positives axons in retina explants that were electroporated with shRNA control or shRNA targeting Pax6, and with CAG-GFP. Both populations present low levels of Fz2 expression and a positive dotted pattern of Fz2 staining. GFP negative mature axons presented a stronger signal. Bar indicates 3 µm. b) Q-PCR detection of the relative expression of the mRNA of Fz1, Fz3, Fz5, Fz6, Unc5a, Unc5b, Unc5c, Unc5d, Neuropilin1 and PlexinD1 from primary cultured neurons. Expression levels are relative to GAPDH transcript and normalized to one control sample (see Text S2). There are no differences in the relative mRNA expression of these receptors in control and Pax6 transfected cells.
Text S1.
cDNA sequence for the silent mutant form of Pax6 resistant.
Text S2.
Methods for real-time quantitative RT-PCR (Q-PCR).
We are grateful to S. Gutierrez-Erlandsson, R. Gutierrez, A. Morales and T. Poderoso for technical assistance.
Author Contributions
Conceived and designed the experiments: PB ASS MN. Performed the experiments: ASS MC AS FMR. Analyzed the data: ASS MC FMR. Contributed reagents/materials/analysis tools: AS. Wrote the paper: MN.
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- 7. Hack MA, Sugimori M, Lundberg C, Nakafuku M, Gotz M (2004) Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol Cell Neurosci 25: 664–678.MA HackM. SugimoriC. LundbergM. NakafukuM. Gotz2004Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6.Mol Cell Neurosci25664678
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What is Brockhanger Lane like ( good area?)
We are interested in a house up at Brockhouse Hanger Lane in Kennington, Ashford, TN24, not sure about the area, any advice welcome
Asked on Mar 23 2011, General in Ashford | Report content
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YMCA Hamilton City took out the national YMCA Award at its annual meeting on October 6.
The award comes in recognition of community excellence for the Hamilton centre's work with children in their after school and holiday programmes, as well as integrating children with challenges into their care.
Only one of the awards is given out each year in the federation of YMCAs in New Zealand.
YMCA Hamilton centre manager Graeme Mead said there is a lot involved in deciding who receives the YMCA Award.
"We have to go through a submission to the YMCA board on why we think we're deserving the award, why we think the staff are deserving of the award, what have they done, what makes their contribution to the community excellent."
Mead said the award was given to the Hamilton centre because of the volume of staff they have, the way they run their programmes in conjunction with Childsafe and the regimented, structured and process driven nature of its operation.
"Also, the type of care that we give and the type of programmes - whether it's interactive, whether the kids are always made to feel safe, whether the instructors are trained in first aid and child protection," Mead said.
"It's a whole raft of things, it's a whole range where you go right through the whole process to say these are all the things you can do that will enable you to be better than we currently are. We're striving for excellence."
YMCA Hamilton lays claim to being the biggest provider of after school care of any YMCA around New Zealand.
The centre employs 70 staff, all of whom are paid. Mead said most of them are either young people going through tertiary education seeking good, stable work, or older people who want to work part-time.
The facility is also in the national finals of the New Zealand Fitness Awards. The two other finalists are larger centres, in Auckland in Christchurch.
Mead said added the Jumpstart Diabetes programme has been a great success in Hamilton.
"What we run is a programme that's being driven and organised by YMCA Hamilton and Auckland. It's doing exceptionally well for people with Type 1, Type 2, or pre-diabetes.
"We take them through supermarkets and we have nutrition visits in which they actually come and read labels and learn what they're eating."
Sometimes, YMCA Hamilton even manages to make a small profit.
"We're competitive in what we do - we're proud to make a profit if we can - but we tip a heap back into the community.
"I think we've given in excess of $50,000 last year from fees we didn't charge or fee assistance," said Mead. "For us, we think we've got a hell of a good facility. For a fitness centre it's as big and as up to date as anything in Hamilton. It's just not a brand new building."


text:
Posts by tamy
Total # Posts: 25
how do I change this 2 3/6 to an improper fraction????
1.5 ???
they need 11/2 cups of sugar.how many 1/4cups should they measure?
yes thanks :)
don't have an idea that's why im asking ???
they need 2 cups of flour. how many 1/4 cups should the measure?
31/2 =48 ounces ???
at the beginning of the school year,jared;s dog weighed 46 1/2 pounds.at the end of the school year,it weighed 50 pounds.how much weight did jered's dog gain during that time ? how many ounces is this ?
It does't said
susanna and her sister had a bake sale ,and earned 2/9 of the money.how much did they earn ?
convert the given angle measure into degrees and decimal fractions of a degree. 112degree 20' 54"
facility planning
How have utilization and design of healthcare facilities changed over the last 150 years? What key factors led to these changes?
How many grades will you have for your research project? Notes its like points or grades
if Maalox is mixed with 40ml of 0.1 M HCI and isn't neutralized, how much extra help with this antacid pill need to neutralize 40ml of 0.1 M HCL by adding 0.05 M NaOH drop by drop to back-tritrate the solution until the pH is neutral?
english vocab
i told you i was really bad at this also i got lazy on the last question so i guessed on that one
english vocab
ok I finished my essay now and I got this vocabulary finished. I really tried my best using a dictionary website but like i said earlier i just cant seem to tell the difference between some words meanings. all i can say is i tried my best on this. the answers that i chose have...
english vocab for sue
well i have used the dictionary websites and they don't helkp much because some of these definitions are almost the same definition as the other words. do you think you can jst answers a few questions you know because it woulf really help. :-) 1. Students found her ...
english vocab
well i do use the dictionary websites they have out but i don't find them very helpful. some of the words almost have the same meaning as the other words. do you think you can answer just some of the questions you know because any amount of help is great for me.
english vocab
hey this is alans sister and i need some help with some of my homework also and the questions below i don't understand either. we both are always assigned big huge packets from our mean teachers and we don't want to get a low grade from some of our assignments so when ...
english vocab
ok i have been working on my english homework for some time now. i was assigned a 400 question vocabulary packet. here are just 15 questions i coudnt find out while doing the entire packet. I couldn't even find any answers using the dictionary website. even if someone only...
ok i have been working on my english homework for some time now. i was assigned a 400 question vocabulary packet. here are just 15 questions i coudnt find out while doing the entire packet. I couldn't even find any answers using the dictionary website. even if someone only...
Does anyone know where Johannas Keppler did most of his work? (like what area) Any help would be great!


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New Delhi, Aug 8: Just after winning the tri-series trophy in the Caribbean last month, MS Dhoni had put the "DND" sign as he went on vacation. Now, he has returned home but is not reachable on his phone even to his team-mates.
India's stand-in skipper Kohli tried to contact Dhoni on phone but he could not be reached. Kohli and his team won the five-match ODI series 5-0 against Zimbabwe recently.
"Well, when he (MS Dhoni) goes away on holidays, it is very hard to contact him. I...I tried to message him on the phone but I couldn't reach him. But I'll be seeing him soon I guess. And I'll speak to him about it," Kohli was quoted as saying in the media on Wednesday (August 7).
This is not the first time, Dhoni has gone incommunicado. Last year, when VVS Laxman wanted to talk to him about his retirement plans, he wasn't available.
Dhoni has been criticised by many for not being available to his team-mates. But he continues to remain that way.


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Another early morning.
My poor baby bug woke this mommy up at 4 this morning. I know he feels miserable poor thing. Those molars coming in are rough plus him and his bed were both soaked. I know his mouth hurts because he woke up and all I heard in the moniter was "owie mamma" over and over before I got to his room.
My son is getting his molars too. It's so hard on them. I hope T feels better soon.
The molars are definitely harder on him. His first one popped through yesterday and he has been walking around holding the side of his face saying owie. Thank God for tylenol, it's the only thing that helps.
Comment deleted
Me too! Especially since he's such a tough kid I know it has to hurt. He seems to be a little better now. He's just been sitting on me since he got up which is not like him.
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About Amy
Birth: September 14
On Moms.com since: Mar 19, 2014
Stay at home wife and Mom. I have an older step son that is an amazing young man. My husband and myself have a little boy and newborn girl. Balancing life isn't always easy but it's always worth it :-)
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can you advise me of an efficient and secure remote desktop connection software;
9 Replies
Nov 14, 2009 at 4:06 UTC
hello Khalid
i think it would be helpful when you describe more in detail what you wanna do. should it be easy to use or powerful and feature rich. used for remote management or remote user support?
with more information we could provide you a good answer.
Nov 14, 2009 at 5:31 UTC
IT Journey, LLC is an IT service provider.
If you are trying to connect to your desktop at your office from a remote location without the need of a VPN, take a look at LogMeIn. They offer several remote desktop access solutions (Free, Pro, Rescue, Reach) that are all good. They encrypt the communication path for you and are basically making an SSL VPN between the software you install on the remote computer and the computer you are using to access it.
Nov 14, 2009 at 9:26 UTC
Niagara Technology Group (NTG) is an IT service provider.
Big fan of LogMeIn here.
Nov 14, 2009 at 11:03 UTC
Yup. Logmein rocks.. What's awesome is even with the free version you can install it on multiple PC's under your one login and have access to them all..
Nov 15, 2009 at 9:07 UTC
If you're looking at something for remote assistance, I used TeamViewer last week and was pretty pleased with it's performance. If you use the free version (free for non-commercial use), then it pops up a little thing about upgrading to the helpee when the connection is closed, but other than that it cleans up when it's done, and the conneciton was pretty quick.
Other than that, LogMeIn or VPN and Remote Desktop/Windows Remote Assistance.
Nov 15, 2009 at 9:33 UTC
Logmein, Hamachi with RDP, depends on the client.
Teamviewer is not bad.
I use webex for commercial which i dont really enjoy. Not simple to get user connected up they way my company is setup.
Nov 24, 2009 at 10:26 UTC
A personal favorite of mine is Symantec PCAnywhere. I know people cringe when they hear the word "Symantec" but this program is very user friendly and very reliable. I has many useful features like Remote PC Restart, File transfering system, and ability to be a service for ease of access.
Nov 24, 2009 at 11:01 UTC
IT Journey, LLC is an IT service provider.
Symantec = not in my shop
Nov 24, 2009 at 11:38 UTC
I use Proxy's remote PC access software because it is fully-featured, has a multi-layered security system, and has a simple-to-use interface. Installation is a snap and you can try it for free. TechInLine and LogMeIn also have options to try for free. Shop around and use whichever free version works best for you.
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non-premixed combustion source terms
i'm using non-premixed combustion model and i need to set a massflow source of fuel in a subdomain. Under Source terms in Cell Zone Conditions i see that the "Mean Mixture Fraction" can be set in kg/m^3/s. But how can I specify if it's value is 1 (fuel) or 0 (oxidant)? In fluent guide it's not explained. Maybe I'm doing it in the wrong way? Thank you
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A PARTY is being held at Rooftop's Ferry View in Boat Lane, Evesham, on Wednesday (Aug 13).
The Summer Fun Day will take place in aid of Sight Concern and Rooftop's Can-Do Group for people with disabilities.
The day will kick off with a mega sale of jewellery from Hey Hey Good Looking, handcrafted products from Panache Gifts and JET Gifts, sweets from Gummy Gumdrops, homemade cakes, stationery and a great selection of prizes to be won in the tombola from 10amuntil 12pm.
For those who would like a spot of lunch after, there will be a fish and chip van on-site providing a delicious meal at just £5 per person. Tickets for lunch must be pre-ordered.
There will also be a chance to enjoy some live music from the Harvington Ukulele Band with refreshments and raffle from 2pm until 4pm.
If you would like to purchase any tickets contact Kim Skipsey on 07854717430 or Leah Jones on 07969236924.
For more info on Sight Concern, visit sightconcern.co.uk or call 01905 723 245.