text: Copying and distributing are prohibited without the permission of the publisher. If you wish to distribute this content please contact Christopher Hunt (firstname.lastname@example.com) for a quote. Subscribe today: Order Euromoney by contacting us on +44 (0)20 7779 8999 (UK) or +1 212 224 3570. Quotes of the month By: Published on: September 2014 Print Order Share Share on facebook Share on twitter Share on linkedin Share on googleplus "I thought the US authorities knew of our existence and purposely were not adding us to the list as we were focusing on humanitarian goods. It now seems that I was mistaken" Parviz Aghili, founder and CEO of Iran's Middle East bank, shows there is no escape from US sanctions "You can't get a baby to pole vault, it needs to walk first. The government cutting down its stake to 51% will deal with my capitalization issue for two years. After two, two-and-a-half years, there will be a need for more capital infusion, but this will get resolved before it becomes a problem" Arundhati Bhattacharya, chairwoman of State Bank of India, urges patience when it comes to reforming the country's public lenders "If any investment bank says they are investing in big data, ask them to pull up their aggregate exposure to Apple and ask them to show you how it breaks down by product. I'd bet that most investment banks would not be able to answer that question" Basil Qunibi, a former investment banker and founder of fintech firm Novus, says the industry has a long way to go to meet the simplest of big data challenges "There has not been enough regulatory attention on the buy side versus the sell side. As the regulators have been paying limited attention, funds have been allowed to grow and grow. The high yield market is concentrated in too few players" Fraser Lundie, co-head of credit at Hermes Investment Management, worries about the impact of a sell-off in high yield markets Add Your Comment All fields are compulsory All comments are subject to editorial review as we are subject to the same regulations adhered to in publishing our own content. For this reason, your comment may not be live immediately, or may not be published. Your name: Your email: (will not appear) Publish your name? Comments: I have read and agree to the Terms and Conditions


text:
Psalm 13New International Reader's Version (NIRV)
Psalm 13
For the director of music. A psalm of David.
1 Lord, how long must I wait? Will you forget me forever?
3 Lord my God, look at me and answer me.
5 But I trust in your faithful love.
New International Reader's Version (NIRV)
Copyright © 1995, 1996, 1998, 2014 by Biblica, Inc.®. Used by permission. All rights reserved worldwide.
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subset: commoncrawl


text:
Home >> 9.4" Height Manmade Fuchsia Flower Green Leaf Aquatic Fish Tank Plants Decoration
|Product Name
|Aquarium Plastic Plant
|Plastic, Ceramic
|Base Size (Approx.)
|5.7 x 4.6cm / 2.2" x 1.8"(L*W)
|Total Size(Approx.)
|24 x 12cm / 9.4" x 4.7" (H*W)
|Main Color
|Fuchsia, Green
|Net Weight
|Package Content
|1 x Aquarium Plastic Plant
- Designed with simulated grass plant and ceramic base.
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text:
:: Events
Northville Road Runner Classic - Evening Event!
Date: Jul. 22, 2017
Contact for Race Timing
|5k Run/Walk, 8k Run & One Run/Walk
Family fun during the race includes: story hour, face painting and hands-on activities.
Join us for post-race activities: pizza, live music, libations, home made baked goods, and much more.
Awards: overall male and female, masters male and female, first, second, and third place awards in the following age groups: 14 and under, 15-19, 20-24, 25-29, 30-34, 35-39, 40-44, 45-49, 50-54, 55-59, 60-64, 65-69, over 70.
5k course has been altered slightly to include small portion of easy trail.
Participant numbers are up.
Hoping for best Classic ever.
The Maybury hills are calling. Hope to see you there.
Mayor of NRR,
For more information please contact email@example.com
|Maybury State Park - Northville, MI.
|Bart Gregoroff
|Timing System:
|Pull Tag
|Website Link:
|Visit site
|Registration Form:
|Download Form
XC Registration Form
Recent Results
Upcoming Events
- Mar. 11, 2017
St. Paddy's Dash (formerly lucky dash)
- Mar. 12, 2017
35th Annual Saint Patrick's Parade Corktown Races Detroit 2017
- Mar. 14, 2017
Run for PI
- Mar. 18, 2017
Lucky Leprechaun


text:
With Instructables you can share what you make with the world, and tap into an ever-growing community of creative experts.
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Atomic Zombie's Granny's Nightmare bicycle chopper
Join 2 million + to receive instant DIY inspiration in your inbox.
© 2016 Autodesk, Inc.


text:
The Triad Orbit IO-W Wallplate/Desktop Mount provides a stable platform for mounting Triad-Orbit IO-equipped adaptors, boom arms, tablet holders and assorted camera accessories to flat surfaces including walls, ceilings and desktops.
- Footprint Width: 4.72 in.
- Hole Spacing: 3.14 in. OC
- Height: 2.76 in.
- Weight: .44 lbs


text:
Battleheart incorporates elements from RTS, tactical RPGs, and the simple "line drawing" games that the App Store has popularized. You take control of a party of heroes of various classes and engage in hectic real-time battles with evil creatures.
Eyyyyyyyy!!!! I play so many games just msg me up! We can play anytime ^_^


text:
This download looks like an unpacked addon. You can either convert the files to an addon or merge the folders in the zip with the folders in your Garry's Mod folder.
You should end up with a directory structure similar to below:
This option makes it difficult to remove the content should you no longer want it. Merge any folders from the zip with the equivalent folders in your Garry's Mod folder.
Don't know where the Garry's Mod folder is or how to extract zip files? See more useful installation tips.
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Fantech 5ACC01GC - Grease Collection Box, 24" Long
DetailsTrap leaking grease from the ventilator with the Fantech5ACC01GC 24" Long Grease Collection Box. It diverts and collects the residual grease. Made from heavy-duty galvanized steel, it offers durability. You can mount it to the base of ventilator and remove it easily for cleaning.
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text: We have been going to this restaurant for many years now and again we were not disappointed. It is clean and the staff are very welcoming and the food is first class. We all had a lovely meal in this restaurant from starters through to the desert. Thank you Le Blanz Nez for a wonderful evening again


text:
Trophy Tom is an application that allows hunters to use the National Wild Turkey Federation formula to get a score estimate for their potential trophy turkey. Just enter each of the trophy measurements and Trophy Tom will output your turkey's score.
If you have an atypical bird, just add the length of all the beards and enter the information into the application.
To find out where you can get your bird officially scored, go to http://www.nwtf.org/all_about_turkeys/slam_directions.html.
You are allowed to use this product only within the laws of your country/region. SharewareOnSale and its staff are not responsible for any illegal activity. We did not develop this product; if you have an issue with this product, contact the developer. This product is offered "as is" without express or implied or any other type of warranty. The description of this product on this page is not a recommendation, endorsement, or review; it is a marketing description, written by the developer. The quality and performance of this product is without guarantee. Download or use at your own risk. If you don't feel comfortable with this product, then don't download it.


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Search by
Sugar Free Allstars
This event has already taken place. Click here for the latest events.Add to Calendar 23-03-2014 23-03-2014 15 Sugar Free Allstars The Sugar Free Allstars are an award-winning family rock band from Oklahoma City. Chris �Boom!� Wiser (Hammond B-3 organ, lead vocals, saxophone) and Rob �Dr. Rock� Martin (drums, backing vocals) make up the dynamic duo. They mix blends of New Orleans funk, Memphis soul and Hammond organ-drenched gospel, wrapped in a colorful rock and roll package. Their latest CD, �All On A Sunday Afternoon,� features guest appearances by notable Kindie artists such as Keller Williams, Marty Beller, Jack Forman of Recess Monkey, Shawana Kemp of Shine and the Moonbeams and Trout Fishing In America. So bring your family for a Rock-n-Roll good time. http://www.nymetroparents.com/2015neweventinfo.cfm?id=157167 Long Island Children's Museum true DD/MM/YYYY
If you go: Please tell them you saw this event on
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text: This is the closest Chinese restaurant to our home. It has been open as long as I can remember. I feel like it has declined. The places is dirty. They have these plastic covers for the table and then plastic over that. The white plastic covers need to be whipped down badly. Honestly we almost left as it was so... More


text:
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Forum indexSport and Action PhotographyChange forum
Photographing swim meets, Tips please.
Started Jan 14, 2013 | Questions thread
Shop cameras & lenses ▾
TTMartin • Veteran Member • Posts: 7,304
Re: Photographing swim meets, Tips please.
In reply to cnwphotocom • Feb 13, 2013
cnwphotocom wrote:
Definitely jumping up to a f/2.8 lens will make a huge difference. I would also keep the camera on full manual since lighting won't be bouncing around indoors. Set your ISO to what you are comfortable with and work around that. Have you considered a 50mm f/1.8 or 1.4? The 1.8 is typically under $250 brand new! But then you are stuck with that focal length....
Flash may work as well, check out alienbees.com and set it up with the long cord it comes with away from you.
Good luck!-- hide signature --
Connor Walberg
Learn how to shoot action/sports at http://www.actionphotoschool.com
Flashes are definitely a NO GO at a swim meet as the start indication is a combination of an audible horn and a strobe light.
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I'm looking for a: Woman
meet a nice lady for fun and sight seeing, iam living in dubai looking for a serious relation with a lady travelling to dubai
Purpose of Travel: Beach break, Concert/festival, Road trip, Shopping trip
Finances: I can pay for everyone
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Hotel Accommodation near Mar Bella Beach
Search 18 hotels near Mar Bella Beach
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Trending now in Barcelona
Hotels within 0.5 kilometer of Mar Bella Beach
- Most bookedHotel 4 Barcelona
167 people looked at this hotel in the last hour
Most recent review
"Hotel staff recommended a fantastic Spanish restaurant within a short walking distance from the hotel and the food is excellent and not too costly. Value for money hotel and is conveniently located."
A Traveller, Nov 2016, SG
153 people looked at this hotel in the last hour
Most recent review
"We stayed here for 10 days and highly recommend it. The lodgings were in perfect shape and comfortable, the location is convenient and interesting, and we were especially happy with the service of the office staff. They were helpful and friendly. For example, one of the girls spent a lot of ti..."
A Traveller, Jan 2017, US
24 people looked at this hotel in the last hour
Most recent review
"We had a two bedroom, two bathroom apartment for a week. Very clean and well equipped. Welcome pack and water were very welcome. Reception staff were really helpful on arrival and afterwards too. It's on a popular street, so noisy in evenings if windows are open, but a small price to pay. Good lo..."
A Traveller, Oct 2016, GB
22 people looked at this hotel in the last hour
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"The apartment was walking distance to the beach, with lots of restaurants and very kid friendly. Only 15 min tram to La Rambla and short Walk to the metro. My family really enjoyed staying here, lots of shots, cafes, places to eat, small supermarket across the street for snacks, water, basic food..."
A Traveller, Jun 2016, CA
Hotels within 1 kilometer of Mar Bella Beach
90 people looked at this hotel in the last hour
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"lovely hotel, will definitely go back in the summer"
A Traveller, Jan 2017, GB
54 people looked at this hotel in the last hour
Most recent review
"Pillows were not clean You must clean the place I'm in vacation not cleaning"
A Traveller, Oct 2016, US
42 people looked at this hotel in the last hour
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"The stay was okay. The address reflected on Hotels.com is different from the actual address and made the first day a nightmare. The address on Hotels.com is the office address and not the apartments address"
A Traveller, Dec 2016, ZA
19 people looked at this hotel in the last hour
Most recent review
"This place is great for any business or leisure. The location is amazing close to Metro station and touristic areas"
A Traveller, Nov 2016, US
18 people looked at this hotel in the last hour
17 people looked at this hotel in the last hour
Where to stay near Mar Bella Beach
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A city packed full of attractions, Barcelona is bursting at the seams with things to do. Visit the ...


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Crest Cadillac Collision Center
ReScore Reviews™ (1)
93% Would Recommend
Latest Review 10 months ago
Review for Crest Cadillac Collision Center
Rod C. on 01/30/2015
Rod said this business was very nice and good, and he has recommended them already. They did an excellent job. The representative did a really good job and he was very consciousness.
Category: Collision
Service Date: 01/28/2015
Review Created: 01/30/2015
This review was collected via phone interview by Customer Research Inc.


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Is Metaphor Design + Architecture Pte Ltd incorrect or missing key business information? Update your Hotfrog listing today.
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We are design oriented award winning architects
Our design philosophy is to understand clients' needs, value add into the design concept and create architecture
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David Beckham's eldest son Brooklyn has reportedly been signed to a short-term deal by Arsenal.
The 15-year-old went on trial with the Gunners and after impressing the coaches at the Emirates has signed a deal until the end of the season, according to the Daily Star.
Brooklyn has been attracting attention from a host of sides, with his father's former club Manchester United thought to be among them. Chelsea have also shown interest whilst he went on trial with Championship side Fulham late last year.
A source has been quoted by the Daily Star as saying: "Brooklyn is talented and he has stood out in all of the training sessions and games he has played.
"He has had other teams sniffing around and it was always going to be a case of when, not if, he was signed up by a big-name club.
"Next summer we may see him sign a big-money deal and with Arsenal."
If things don't work out for Brooklyn, father David still has two more chances to see one of his sons follow in his footsteps. Younger brothers Romeo and Cruz, aged 12 and nine respectively, have also been training with Arsenal's appropriate youth teams.
Video: Beckham with Arsenal in 2013
Beckham Snr trained with Arsenal in January last year prior to joining Paris St Germain, the club that would turn out to be his last before retirement last summer.
Arsenal are unable to comment on the situation due to Brooklyn's age. Meanwhile, professional contracts cannot be signed until a player is 17, whilst a scholarship cannot be signed until a player is 16. That however, does not rule out a club and player reaching a pre-agreement.Reuse content


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Terms and Conditions:
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text:
The explanation in the previous section should help you understand how and why support-vector machines work, but the algorithm for training a support-vector machine involves mathematical concepts that are very computationally intensive and are beyond the scope of this chapter. For these reasons, this section will introduce an open-source library called LIBSVM, which can train an SVM model, make predictions, and test the predictions within a dataset. It even has built-in support for the radial-basis function and other kernel methods.
You can download LIBSVM from http://www.csie.ntu.edu.tw/˜cjlin/libsvm.
LIBSVM is written in C++ and includes a version written in Java. The download package includes a Python wrapper called svm.py. In order to use svm.py, you need compiled versions of LIBSVM for your platform. If you're using Windows, a DLL called svmc.dll is included. (Python 2.5 requires that you rename this file to svmc.pyd because it can't import libraries with DLL extensions.) The documentation for LIBSVM explains how to compile the library for other platforms.
Once you have a compiled version of LIBSVM, put it and svm.py in your Python path or working directory. You can now import the library in your Python session and try a simple problem:
from svm import *
The first step is to create a simple dataset. LIBSVM reads the data from a tuple containing two lists. The first list contains the classes and the second list contains the input data. ...
No credit card required


text:
BoD NominationsPosted on February 11, 2017
Be a part of social and environmental justice in action! Deadline to apply is March 12
NSPIRG is Hiring!Posted on March 4, 2016
NSPIRG is hiring a new Summer and Part Time STUDENT ENGAGEMENT OFFICER Student Engagement Officer 2016 (PDF) See Full Posting on www.goodwork.ca
Rad Frosh 2015!!!Posted on September 10, 2015
Please invite your pals, share with your networks, and come out to an event as we link classrooms to community.


text:
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text:
Today, it was still easier for me to get an engineering PhD than it is to get a girlfriend. FML
By still single - / Thursday 6 August 2015 01:22 / Brazil - Santa Maria
Top comments
If it worked like that I bet there'd be a lot more people trying to get into engineering programs.
Too many negative votes, comment buried. Show the comment
Girl friend is not a toy
Like how Howard built a sex robot on The Big Bang Theory
In fact, it is kind of like the opposite of expensive because it means you can earn more money.
By herpderp1234
Construct a girlfriend to put that degree to use! She'll be just the way you want! Lol
If a girl is only interested in you because of the money you have.. let me put it this way: Bye, Bitch!
Looks are important too. If you're not physically attracted to your spouse, then your relationship will eventually crumble, no matter how much you like their education. But stability helps in a relationship. (Stability can sometimes equal money.)
By nomoreparty
Too many negative votes, comment buried. Show the comment
Write a paper on 69... Will help u in scoring high.
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text:
is th 1998 dodge cummins engine the same as the 1997?
does anyone know if the 1998 and 1997 engine are exactly the same? no one
seems to know the answer for sure.
3 Answers
manufacturers can change specifications through model years, so it may be different also on modern engines are controlled via electronics so make sure you get the ECU with the new engine.
a visual side by side check is the easiest way to tell if they are the same
I'm thinking about purchasing a 1998 dodge ram cummins turbo diesel, I already own two 1997's and love them. No one seems to know if dodge put the exact same engine in. They appear to be the same does anyone know for sure if dodge put the same in for these two years?
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text: I was a member of a gym called Zest and i was on a weekly contract, i signed up with them in 2004 and the company went into administration around 2007 or 2008 or around that time. So one day when i turned up they told me that the company was in administration but they allocated a new gym location in the same suburb for the current members. This location didn't have the name or any insignia of a business name. So i went to this location a few times but one day when i turned up there was nothing there and the doors were closed. So recently i accidentally had a look at my bank statement and i noticed that money was being withdrawn by a company called GoHealth which is another gym located in the same suburb but a few streets away. Their location is different to all previous locations mentioned above but in the same suburb. So i gave them a call and they told me that i was a member there on a 13$ fortnightly contract. Apparently what happened was, GoHealth took over Zest and took over all of their assets, including membership contracts from Zest, which according to them is completely legal since that was part of my original with Zest. When i spoke to GoHealth they had my wrong address since i changed my household, wrong email and wrong phone number because many years have passed and i didn't even know i was contracted with GoHealth so i guess even if they tried contacting me there was little chance they could. In the meantime i was a member of other health clubs but i never once went into GoHealth since i sincerely didn't know my membership was taken over my them. So they offered me some free time and a permission to cancel my membership but i was wondering if i could get my money refunded under Australian Consumer Law?


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Cincinnati Zoo
Lions, Tigers, Bears, Oh My!
Summer Vacation in Ohio
Our summer vacation began with wanting to visit Kings Island one of the best amusement parks in the world. the day before our trip to Kings Island we decide to check out he zoo. It was about 8:45 am when we arrived at zoo. We started out at the reptile house and we got a few picture but it was very crowded so we decided that we would move on to the next animal and we would come back to the reptile house later.
As we moved from one exhibit to the next we realized we had walked pass the zoo maps and it would be better if we marked off the areas visited as we passed them. Once we opened the map we were completely surprised at how much area we had to cover. We walked and walked the more animals we saw the more we wonder how many more animals could there be in one zoo. That is about the time we came upon the monkeys, I had never seen that many monkeys in one zoo. I have not visited that many zoos but I have visited the zoo in Atlanta Ga, and Nashville Tn. When you compared the Atlanta Zoo and Nashville Zoo to the Cincinnati Zoo you realize there is no comparison.
Zoo Animals
Dora the Explore in 4D
After viewing some of the animals we noticed a Dora the Explore show that was going to be showing at the zoo so we all walked over to catch the show. This was the first 4D experience our boys had ever had so they were really into it although my 13 year old tried to play it cool he too was excited at the show. Not only was it nice to see an animated 4D show it was also a good chance to cool off. There were times that water was sprinkled into the audience as a splash back effect from the show. Our seats vibrated and moved from time to time and that was cool also. My four year old was excited at the bubbles Dora was blowing into the audience. After the show he looked up at his dad and said, "that was tso cool dad you are most awesome dad ever.
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Good hotel for solo travelers. Great location. Popular among business travelers.
Guest Review Highlights
- Great location
- Quite clean
- Room cleanliness is acceptable
- Room cleanliness is acceptable
- Room needs to be redecorated
- Bathroom could be optimized
- WiFi system needs upgrading
- Tasty breakfast
- Service could be improved
- Service should be more professional


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heritage. About Us. FAI - Villa dei Vescovi, Luvigliano Di
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Derrick Rose rips up his ACL on a non-contact play. Hours later, the Knicks' Iman Shumpert tears his ACL on another non-contact play. Two major injuries that happen just after a lockout-induced, compressed NBA schedule where a lot of guy missed …
also served as a much-needed boost following Sergio Ramos' red card 11 minutes earlier. Umut Bulut pulled Galatasaray level a minute after Bale's opener before Real clinched a comfortable victory courtesy of second-half goals from Alvaro Arbeloa, Angel Di ...
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text: Home > Sports Why Pinay got USD 8,000 from Pacquiao ABS-CBN News Posted at Apr 08 2014 08:39 PM | Updated as of Apr 09 2014 04:40 AM Share Save Facebook Twitter GPlus LinkedIn Pinterest MANILA, Philippines – Manny Pacquiao gave away thousands of dollars to his team members who won in their own version of "The Biggest Loser" before they left for Las Vegas for his rematch against Timothy Bradley. The winners included Marites Diamond, who got USD 8,000 for losing eight percent of her body fat in their 2-week contest. The US-based Pinay is the same woman who kept up with Pacquiao during his morning jogs in Los Angeles, according to Gerry Ramos on Spin.ph. "Nagulat si Manny, akala niya 'di makakatagal lalo na nung paakyat ng Griffith Park. Pero natawa siya kasi nung pababa na, nakita niya kasunod pa rin niya tsaka ni Bobby [Pacquiao]," Buboy Fernandez, a co-trainer of Pacquiao, told Ramos. Diamond said it wasn't easy shedding away the excess pounds. "I've been into physical fitness pero not something like this," she said. "Ang hirap pala." Former beauty queen and actress Anjanette Abayari also joined the contest. She, too, was able to lose eight percent of her bodyweight. "Today I achieved my goal Precision Fitness losing 8% of my body weight in 14 days... I held my breath as Manny Pacquiao personally watched our weight on the scale. And was so thrilled when I made it," Abayari said. Abayari, who was crowned Binibining Pilipinas-Universe in 1991, is best known for playing sexy roles in the 1990s. She portrayed the iconic Pinay heroine "Darna" in 1994. Share Save Facebook Share on Twitter GPlus LinkedIn Pinterest Read More: boxing Manny Pacquiao Pacman Features biggest loser marites diamond /life/02/22/17/rb-chanco-iiwanan-ko-si-miss-france-para-kay-maam-charo/entertainment/02/22/17/panoorin-robi-domingo-brokenhearted-nga-ba/business/02/22/17/agrinurtures-big-chill-to-boost-expansion-in-china/overseas/02/22/17/us-powerball-lottery-jackpot-snowballs-past-400-million/entertainment/02/22/17/janella-salvador-to-hold-digital-concert


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Reviews, evaluates, designs, implements and maintains company database[s]. Identifies data sources, constructs data decomposition diagrams, provides data flow diagrams and documents the process. Writes codes for database access, modifications, and constructions including stored procedures. Familiar with a variety of the field's concepts, practices, and procedures. Leads and directs the work of others.
Key Activities:
-Data management and analysis, including daily data updates, review, and evaluation; query and stored procedures revisions; data scrubbing; code modifications and constructions.
-Design, implementation, and enhancement of operational progress reports; as well as review/quality inspection of daily and adhoc reports.
-Support of outbound call campaigns through design, implementation, and maintenance of database tables used with responses gathered via front end web-based application.
Other Activities:
-Request for Changes submittal and presentation to the Change Control Board.
-Service request monitoring, scheduling, updating.
-Data flow diagrams, technical requirements and practice, procedure, process documentation.


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Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx−/Y mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.
Mutations in the Aristaless-related homeobox gene (ARX) are a common cause of X-linked intellectual disability. Over 40 different mutations have been identified in ARX resulting in a spectrum of disorders that vary from mild intellectual disability with no brain malformations to severe brain malformations such as lissencephaly and hydranencephaly (Gecz et al. 2006). Currently the pathogenesis of these ARX-related disorders are poorly defined.
During mouse cerebral cortical development Arx is expressed in the ventricular zone (VZ) (Colasante et al. 2008; Colombo et al. 2004; Friocourt et al. 2006). Arx deficient mice die shortly after birth with a thin and disorganized neocortex in addition to other brain abnormalities (Kitamura et al. 2002). The neocortical alterations appear to be the result of decreased pallial progenitor cell proliferation (Friocourt et al. 2008; Kitamura et al. 2002). However, it remains unclear how Arx regulates cortical progenitor proliferation, cell specification, and layer formation in the neocortex.
To define the endogenous function of Arx in the cortical VZ, Arx-floxed mice were mated to Emx1-Cre mice, removing Arx specifically in the dorsal telencephalon. The number and proliferation rate of progenitor cells, their cell cycle length, and final cortical laminar fate were then assayed. Our data show that Arx regulates the expansion of both radial glial cells (RGC) and intermediate progenitor cells (IPC), with a more pronounced effect on the latter. The decrease in proliferation in the Arx cKO mice resulted in a loss of neurons specifically in the upper layers of the neocortex. This is consistent with the hypothesis that IPC derived neurons contribute to all cortical layers, but predominately upper layers (Nieto et al. 2004; Sessa et al. 2008; Tarabykin et al. 2001; Zimmer et al. 2004).
We also identified a cohort of genes whose expression is consistently altered in Arx KO mice cortices when compared with wild-type mice. Among them, Cdkn1c, encoding for CDKN1C, was found to be a direct target and its role in the identified IPC phenotype was analyzed. As a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CDKi), CDKN1C antagonizes cell cycle progression by inhibiting G1/S transition (Sherr and Roberts 1999). Interestingly, CDKN1C, whose expression is normally detected in the cortical SVZ, was overexpressed in both the VZ and SVZ of Arx KO cortices. Thus, ARX appears to regulate cortical progenitor pool expansion by repressing the premature expression of Cdkn1c in the cerebral cortex.
Arx conditional knock out mice (Arxfl/fl) (Fulp et al. 2008) and Arx knock out mice (Arx−/y) (Collombat et al. 2003) were maintained on a C57Bl/6 background. Timed-pregnant mice were considered embryonic day 0.5 (E0.5) on the morning of the confirmation of the vaginal plug. To inactivate Arx in the dorsal telencephalon during development Arxfl/fl mice were crossed with Emx1-Cre animals (Jin et al. 2000). Genotyping of Arx conditional knock out and germline knock out mice was performed as described (Collombat et al. 2003; Fulp et al. 2008; Jin et al. 2000). Sex assessment for control embryos was performed by Sry PCR (SryFw 5′-CAGAAATGAACTACTGCATCCC-3′ and SryRev 5′-AACTTGTGCCTCTCACCACG-3′).
Tbr2::GFP transgenic mice (Kwon and Hadjantonakis 2007) were crossed with Arxfl/y;Emx1-Cre animals. Mice were maintained at the Children's Hospital of Philadelphia and at the San Raffaele Scientific Institute lab animal facilities. All experiments were approved by the Institutional Animal Care and Use Committees of the Children's Hospital of Philadelphia and of San Raffaele Scientific Institute.
BrdU Administration for Cell Cycle Dynamic Analysis
BrdU was administered (50 mg/kg, i.p.) to pregnant mice 1 h before embryo harvesting to assess the S-phase labeling index (LI). A cumulative BrdU-labeling protocol was followed to determine the growth fraction (GF) (fraction of the cell population that is proliferative), to determine the time to reach the GF (Tc−Ts), and, subsequently, to estimate the length of the cell cycle (Tc) and the duration of S-phase (Ts) for both radial glia and intermediate progenitors (Takahashi et al. 1995). Pregnant mice were injected with BrdU (50 mg/kg, i.p.) starting at E13.5, with injections every 3 h and with survival times of 0.5 h. Thus, a total of 6 time points were examined: 0.5, 3.5, 6.5, 9.5, 12.5, and 15.5 h. For cell cycle exit experiment, BrdU (50 mg/kg, i.p.) was injected in a pregnant female at E13.5 and 24 h later embryos were harvested and processed for analysis.
Histological Techniques
Nissl Staining
Sections were stained in a 0.1% cresyl violet solution for 5–10 min and then, rinsed quickly in distilled water. Thereafter, sections were dehydrated in 100% ethanol (2 × 5 min), dried and coverslipped with Eukitt (Electron Microscopy Science).
RNA In Situ Hybridizations
ISH on frozen sections was performed as previously described by (Schaeren-Wiemers and Gerfin-Moser 1993) with the modifications previously reported (Colombo et al. 2007). Cdkn1c in situ probe was a kind gift of L. Muzio (Muzio et al. 2002).
Whole heads (E11.5 and E12.5) or brains were dissected from embryonic and P1 mice and fixed overnight in 4% PFA at 4 °C. P14 and P30 mice were perfused with 4% PFA, then the brains were removed and fixed in 4% PFA overnight at 4 °C. Fixed brains were frozen and 10-μm-thick coronal sections were obtained. Antigen retrieval was performed in citric acid-based Antigen Unmasking Solution (Vector Laboratories) autoclaved at 105 °C for 10 min; for BrdU staining slides were also treated with 2N HCl for 20 min. No antigen retrieval was performed for ARX and TBR2. Sections were then blocked for 1 h at room temperature with 10% normal goat serum (Gibco) and 0.1% triton in PBS. Primary antibodies against ARX (rabbit, gift provided by Professor Kunio Kitamura, 1:500), KI67 (rabbit, Immunological Sciences, 1:300 and mouse, BD Pharmingen, 1:200), BrdU (rat, Accurate Chemical and Scientific, 1:200), PH3 (rabbit, Chemicon, 1:100), TBR2 (rabbit, Abcam, 1:200 and Chemicon, 1:300), PAX6 (mouse, Developmental Studies Hybridoma Bank, 1:1000), CUX1 (rabbit, Santa Cruz Biotechnology, 1:50), SATB2 (mouse, Bio Matrix Research, 1:200), CTIP2 (rat, Abcam and Beckton-Dickinson 1:300), GFP (chicken, 1:2000), Caspase3 (rabbit, Cell Signaling, 1:200), and TBR1 (rabbit, Abcam, 1:25 and Chemicon, 1:400) were diluted in 10% normal goat serum and incubated on slides overnight at 4 °C. Secondary antibodies were conjugates of Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 (1:500, Invitrogen), biotinylated goat anti-mouse and anti-rabbit (Dako, 1:100). They were diluted in 10% normal goat serum and incubated on slides for 2 h at room temperature. Biotinylated secondary antibodies were subsequently incubated with strepdavidin-Cy3 (Jackson ImmunoResearch, 1:300) in PBS. Slides were then counterstained with DAPI (Invitrogen, 1:1000), washed and mounted in Fluorescent Mounting Medium (DakoCytomation).
For the ARX/TBR2 double labeling, sections were blocked in 10% normal donkey serum (Jackson ImmunoResearch) with 0.3% triton in TBS (Tris buffered saline pH 7.4, USB Cleveland, OH, USA; the buffer used throughout the procedure) for 30 min at room temperature. Anti-TBR2 antibody (Abcam, 1:200) in 10% donkey serum next was applied for 2 h at room temperature followed by washing with TBS and then incubation with goat anti-rabbit monoclonal Fab fragments (Jackson ImmunoResearch,, 1:25) in 10% donkey serum for 30 min at room temperature. They were then washed and secondary Alexa 594 donkey anti-goat 1:200 in 10% donkey serum in TBS was applied for 30 min at room temperature. For the second primary antibody, the sections were again washed and blocked with the goat anti-rabbit monoclonal Fab fragments 1:25 in 10% donkey serum. After washing, the anti-ARX antibody was applied overnight at 4 °C in 10% donkey serum. Finally, the slides were washed and the secondary Alexa 488 donkey anti rabbit was applied at 1:200 in 10% donkey serum for 30 min at room temperature. The slides went through on final wash, were counterstained with DAPI and mounted.
Quantifications and Statistical Analysis
All quantifications on P14 cryostat brain sections were performed using a 200-μm section of the dorsomedial cortex at the midhemispheric level from pia to white matter. For quantifications on E11.5 and P1 brain sections, cell counts were derived from 25% of the neocortex. The area of interest was derived by measuring the linear distance along the pial and ventricular surfaces of the entire lateral cortex, dividing those into quarters, and then using the total area, from pia to ventricle, in the third quartile from the dorsal midline. Quantifications on E14.5 brain sections were performed using a 150-μm section of the dorsomedial and lateral cortex. At least 3 serial sections from 3 different animals for each genotype were photographed using a Leica DMR fluorescent microscope. Images were imported into Photoshop CS3 and double-positive cells were overlaid manually by color-coded dots in new layers. The number of labeled cells (dots) was calculated using the Record measurements of Photoshop CS3 and imported into Excel 2008. The percentage of labeled cells per region per section was calculated for each brain, and the final mean percentage calculated across all brains. Results were expressed as mean value ±SD and were tested for statistical significance by the 2-tailed, unpaired Student's t test (P < 0.05 level of significance).
RNA Isolation, Processing, and Microarray Analysis
The experiment was performed as previously described (Colasante et al. 2009). Briefly, 3 pregnant Arx heterozygous dams crossed with C57Bl/6 males were sacrificed at E14.5. A total of 5 wt and 5 Arx KO embryos were harvested and after brain isolation the cerebral cortices were separated from the ventral telencephalon. Total RNA was extracted from the cortices using the Qiagen RNA micro kit (Qiagen, Valencia, CA, USA). cRNAs were generated and hybridized on a total of 10 different MOE430v2 Affymetrix DNA chips according to the Affymetrix protocol. The chips were scanned with an Affymetrix scanner to generate digitized image data files. The data were deposited in the NCBI Gene Expression Omnibus (Edgar et al. 2002) and are accessible through GEO Series (# GSE12956).
Luciferase Reporter Assays
P19 cells (105) were seeded and transfected 24 h after plating, with 400 ng of each of the following constructs in different combinations: pT81 luciferase reporter plasmids, control plasmid pCAG-iresGFP, pCAGArx-iresGFP, pCDNA3-Tle1, and 80 ng of pRL-TK-Renilla luciferase plasmid DNA (Promega) using Lipofectamine PLUS™ Reagent (Roche Diagnostics, Alameda, CA). Forty-eight hours post-transfection, cell lysis, and measurement of firefly and Renilla luciferase activity was performed using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. The firefly luciferase activity was normalized according to the corresponding Renilla luciferase activity, and luciferase activity was reported as mean relative to pCAG-iresGFP/luciferase transfection. The 2 Cdkn1c −549/−68 and −1490/−720 enhancer regions were PCR amplified from E14.5 mouse genomic DNA using the following primers: HindIII-Cdkn1c-F1 5′-CCCAAGCTTGCGCGCGGGCCTCCTCAC-3′; BamHI-Cdkn1c-R1 5′-CGCGGATCCCCAGGACCAGGACCCAGCTG-3′; HindIII-Cdkn1c-F2 5′-CCCAAGCTTGAGATCTAAAAGATCTGTAG-3′, and BamHI-Cdkn1c-R2 CGCGGATCCTAGTGAATGAGGGGTTCATG-3′, respectively. The amplified products were cut with HindIII and BamHI and cloned into the BamHI and HindIII sites of pT81-TK-Luciferase, which contains the thymidine kinase minimal promoter upstream of luciferase. The (−549/−68) enhancer region carrying mutagenized ARX binding site was generated by site-directed mutagenesis with the following primers 5′-CTGAAAATACTACAGGATGCTCCTCGTGAGGAG-3′ and 5′-CTCCTCACGAGGAGCATCCTGTAGTATTTTCAG-3′ using the QuikChange II site-directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions.
Chromatin Immunoprecipitation
E14.5 mouse embryonic cortices were isolated and single-cell suspension was derived by enzymatic treatment. Cells were crosslinked with 1% formaldehyde for 10 min and chromatin prepared essentially as described previously (Colasante et al. 2008; Ferrai et al. 2007). The chromatin aliquots (1 mL) were incubated overnight with 1 μg of anti-GFP (Chemicon) as mock and anti-ARX(C-14) (Santa Cruz). PCR primers used are the following: ChIPEbf3-F, 5′-GTCTATAAGTACAATGGTGACAC-3′; ChIPEbf3-R, 5′-CTCCATCAAGATCCTTCTC-3′ (amplification product, 240 bp); ChIPCdkn1c-F, 5′-GCGCCCCTTTATACGCGCTG 3′; ChIPCdkn1c-R, 5′-TCACGTTACCGCCCGCAGAG-3′ (amplification product 200 bp); ChIPCdkn1a-F 5′-GCTGTCAAAACGACCTTGAATG-3′; ChIPCdkn1a-R: 5′-GGAAGGACTAACTCTTTTCCAG-3′ (amplification product 220 bp); ChIPCdkn1b-F 5′-CCCTGATAAGAGCGGTCAGTC-3′; ChIPCdkn1b-R 5′-TTACGGAGCTTCGGTGGCTAG-3′ (amplification product 250 bp). PCR products were analyzed on 2% agarose gels in TBE buffer.
Reduced Cerebral Cortex and Olfactory Bulbs after Deletion of Arx from the Dorsal Telencephalon
The germline loss of Arx in mice results in a thinner and disorganized cortex that was attributed to a primary defect in cell proliferation (Kitamura et al. 2002). To further define the role of Arx in cortical progenitor cell proliferation, we conditionally deleted Arx from the E9.5 dorsal telencephalon by crossing our Arxfl/+ mice to Emx1-Cre mice (Jin et al. 2000) (Fig. 1A,B). In Arxfl/y; Emx1-Cre mice (here after Arx cKO), no ARX protein was observed in the VZ and SVZ of the cortex by E11.5 and throughout the rest of development (Fig. 1A,B and data not shown). Morphological analyses performed at E14.5 revealed that, at this developmental stage, alterations of the gross morphology of Arx cKO brains are already evident. The cerebral hemispheres are reduced in size and the olfactory bulbs are smaller and characterized by an abnormally wide interspace separating them (Fig. 1C,D). Nissl staining on E14.5 coronal sections highlighted the diminution of cortical thickness affecting Arx cKO cerebral cortex in its whole medio-lateral extension (E14.5 relative volume (rv) Arx cKO/ctrl = 0.86 ± 0.03; n = 3, P < 0.005 (Fig. 1E,F,O). Arx−/y mice die perinatally (Collombat et al. 2003; Kitamura et al. 2002), whereas crosses between Arxfl/+ female and Emx1-Cre male mice recovered all expected genotypes in Mendelian ratios (data not shown). Morphological assessments at P30 of Arx cKO and relative control brains confirmed the analysis made at E14.5. Indeed, the cerebral hemispheres and olfactory bulbs in Arx cKO mice are reduced in size, whereas the cerebellum and the brain stem do not appear to be affected (Fig. 1I,J). In addition, P30 Nissl-stained coronal sections highlight an overall reduction of the cerebral cortex, most evident at the level of pyriform cortex and the hippocampus (rv P30 = 0.84 ± 0.05; n = 3, P < 0.005) (Fig. 1K,L,O).
In the Arx cKO, the laminar structure cannot be clearly delineated (Fig. 1M,N) and the corpus callosum is absent in Arx cKO brains (Fig. 1P,Q); this feature, together with the microcephaly, strongly resembles the morphologic abnormalities observed in patients with the XLAG syndrome (Kitamura et al. 2002).
Our data indicate the loss of Arx in cortical progenitors recapitulates the morphologic defects observed with the germline loss of Arx. These data support the model by which the reduced brain size observed in Arx KO mice and children with loss of function ARX mutations, is largely or completely due to a loss of Arx in cortical progenitor cells.
Loss of Arx in the Pallial Progenitor Cells Reduces the Proliferation Rate of Cortical Progenitor Cells
Previous data suggested a decrease in cortical progenitor cell proliferation as the basis for the reduced cerebral cortical thickness in Arx KO mice (Kitamura et al. 2002). At E11.5 in cortical development, there are similar numbers of progenitor cells in wild-type and Arx cKO mice cortices as measured by the number of KI67-positive cells (ctrl: 392 ± 33.5; cKO: 368 ± 45.5, n = 3, P = 0.579: cells counted in 25% of the cortex from ventricular to pial surface) (data not shown). This suggests normal numbers of cortical progenitor cells exist early in development when the progenitor cells are being produced. However, when the same analysis was performed at E12.5, we observed a reduction in the percentage of proliferating progenitor cells (% KI67+/Hoechst, ctrl: 96.65 ± 2.90; cKO: 87.49% ± 5.58, n = 6, P = 0.016, cells counted in a 150-μm bin from the ventricular to the pial surface) (Supplementary Fig. 1C,D′,G), and an increase in ßIII-tubulin-positive young neurons in Arx cKO compared with control (% TUBB3+/Hoechst, ctrl: 10.88 ± 2.96; cKO: 20.75 ± 3.39, n = 6, P = 0.003) (Supplementary Fig. 1A,B′,H).
At E14.5, the cortical plate (CP) is already thinner (Fig. 1E,F) and the reduction in the total number of proliferative cells is evident in Arx cKO cortices (Fig. 2A,C) (ctrl: 259 ± 21.8; cKO: 202.5 ± 16.3; n = 6, P < 0.001, cells counted in a 150-μm bin from the ventricular to the pial surface). Moreover, both the types of cortical progenitor cells, RGCs and IPCs, are diminished in Arx cKO cortices at this embryonic stage. Indeed, we observed a reduced PAX6-labeling intensity in Arx cKO mice (Fig. 2D′,E′) in addition to a 27% reduction in the number of PAX6+ cells (ctrl: 323.6 ± 13.3; cKO: 237.5 ± 26.8; n = 3, P < 0.001; Fig. 2J) as well as a 20% reduction of TBR2+ cells (ctrl: 223.6 ± 16.4; cKO: 179.3 ± 10.8; n = 3, P < 0.001) (Fig. 2F′,G′).
In order to exclude an increase in cell-death contributing to the reduced cortical thickness in Arx cKO cortices, the apoptotic marker Caspase3 was analyzed at different embryonic stages (E13.5, E15.5, and E17.5). We found no difference between the Arx cKO and control cortices (Supplementary Fig. 2).
To determine if the reduction in proliferation was affecting both types of cortical progenitor cells (RGCs and IPCs) in the same way, their rate of BrdU incorporation was measured in control and Arx cKO cerebral cortices after a 1-h BrdU pulse. As expected, general reduction in BrdU incorporation (∼24%) (arrowheads in Fig. 2F″), was found in Arx cKO cortices with respect to controls (ctrl: 142.3 ± 18.2; cKO: 109.0 ± 10.2; n = 3, P < 0.001 and Fig. 2J). Interestingly, double labeling with PAX6 and BrdU revealed little differences in the PAX6+/BrdU+ cell number between Arx cKO and control (ctrl: 125.6 ± 19; cKO: 103.7 ± 7.17; in both cases about 40% of the total PAX6+ cells; n = 3, P = 0.012) (Fig. 2D′″,E′″,J). Conversely, double labeling with TBR2 and BrdU revealed a reduction of dual-labeled cells (ctrl: 52.9 ± 12.9, about 25% of the total TBR2+; cKO: 17.5 ± 2.9, about 10% of the total TBR2+; n = 3, P < 0.001) (arrowheads in Fig. 2F′″,G′″,J). Consistent with these data, staining with the M-phase marker phosphohistone 3 (PH3) revealed an overall reduction of positive cells in the Arx cKO cerebral cortex with a greater loss in the basal area (IPCs) than the apical area (RGCs) (apical, ctrl: 122.0 ± 5.6, cKO: 107.5 ± 3.5; basal, ctrl: 39.0 ± 4.8; cKO: 11.5 ± 2.12 n = 3, P < 0.001, Fig. 2H,I,H′,I′,J).
Taken together, these data indicate that at E14.5 both progenitor populations (RGCs and IPCs) are reduced in Arx cKO cerebral cortices and their proliferation is affected. However, BrdU incorporation rates and PH3 staining indicate that there is a disproportionate deficit in the proliferation of IPCs compared with RGCs in Arx cKO mice.
Cortical Progenitors Show a Normal Cell Cycle Length, but IPC Mitoses Are Significantly Reduced in Arx cKO Mice
To further understand the mechanism by which loss of Arx resulted in fewer proliferating cells, we tested 2 nonmutually exclusive hypotheses: that the cell cycle changes are related to lengthening of the cell cycle or to precocious exit of cortical progenitors from the cell cycle.
An increased cell cycle length during the neurogenic period would result in fewer cellular divisions, fewer progenitor cells and the observed reduction in BrdU incorporation. To examine neocortical cell cycle dynamics in the absence of Arx, the length of the cell cycle phases was estimated between E13.5 and E14.5 in wild-type and Arx cKO mice using a cumulative BrdU pulse-labeling paradigm (Takahashi et al. 1995). BrdU was injected intraperitoneally in gravid females every 3 h up to 6 time points with survival time being 0.5 h after the last injection. At least 2 brains of each genotype were assayed at each time point for BrdU labeling to evaluate the cell cycle parameters in the VZ precursors, where VZ was defined by 2 criteria: the region under the area with high density of TBR2+ cells and where cells with vertically oriented nuclei were present. Moreover, TBR2/BrdU double staining was performed in order to gain information regarding the BrdU incorporation kinetic of IPCs. Representative cortical fields of wild-type and Arx cKO BrdU- and TBR2-labeled coronal sections are shown for 5 out of the 6 time points (Fig. 3A–J).
The GF in the VZ—defined as the maximum percentage of VZ progenitors entering the S-phase on the total of nuclei in the VZ—did not change between the 2 genotypes, and in both cases, almost 100% of VZ cells were BrdU+ at the 9.5 h time point (Fig. 3G,H). The LI of VZ (BrdU+cells/total cell number) at each time point was plotted against the corresponding time after BrdU injection, and, from the slope of the interpolating lines, the cell cycle parameters Tc and Ts were calculated (Fig. 3N,P). Interestingly, no significant difference was found in both parameters between control and Arx cKO brains (ctrl: tc = 14.5 h and Ts = 4 h; cKO: tc = 14.1 h and Ts = 4.1 h).
A similar analysis of the cell cycle kinetic was performed in the SVZ. First, the GF at E13.5 was evaluated as the percentage of proliferating IPC (KI67+ TBR2+) divided by the total number of IPCs (TBR2+) (Fig. 3K,L′). Interestingly, while ∼75% of IPCs in the control is proliferating, only 45% of Arx cKO IPCs enter any cell cycle phase (% KI67+ TBR2+/TBR2, ctrl: 74.30 ± 1.99; cKO: 45.17 ± 2.64, n = 4, P < 0.0004, Fig. 3M). A similar reduction in the SVZ GF of Arx cKO was noticed also later in cortical development (Supplementary Fig. 3; E17.5% KI67+ TBR2+/TBR2, ctrl: 16.57 ± 2.49; cKO: 7.58 ± 0.26). These data confirmed and completed the previous observations relative only to S and M phases of the cell cycle.
Then, LI in the SVZ was calculated as the ratio between the TBR2+ cells incorporating BrdU on the total of TBR2+ proliferating cells (BrdU+ TBR2+/Ki67+ TBR2+) and plotted against the corresponding time after BrdU injection and interpolating lines generated for both control and Arx cKO (Fig. 3A″–J″,O). The slope of the Arx cKO interpolating line resulted slightly higher; indeed, cKO IPCs seem to reach the value of GF = 1, a little before the control (Tc-Ts, control: 7.8 h; cKO: 6.3 h). As a consequence, Tc and Ts is slightly shorter in cKO IPCs with respect to control (Fig. 3P). The ratio between Ts and Tc is not significantly altered (Ts/Tc, ctrl: 0.25; cKO: 0.24), suggesting that the relative length of the different cell cycle phases is constant.
Interestingly, the same conclusions were reached through a different approach. We measured the BrdU LI (0.5 h pulse) in SVZ precursors (Ki67+) at E14.5 (Arnold et al. 2008), finding significant decrease in the number of proliferating Ki67+ cells/100 μm2 area in cKO SVZ with respect to control (ctrl: 19.83 ± 6.04 and cKO: 3.83 ± 1.94; n = 4, P < 0.005; Fig. 4A–C), comparable to the reduction in BrdU+ (Supplementary Fig. 4) and PH3+ cells in cKO SVZ (Fig. 2J). Consequently, the ratio between the number of S-phase cells (BrdU+) and the total of those proliferating (Ki67+) show no significant difference (ctrl: 0.44 ± 0.02 and cKO: 0.40 ± 0.10; n = 4, P = 0.34; Supplementary Fig. 4C). This last set of data again indicates that Ts/Tc ratio of Arx cKO SVZ precursors is comparable to control.
Consequently, the reduced BrdU incorporation observed in cumulative labeling experiment of TBR2+ cells can be attributed to a consistent reduction in the proliferation ability of Arx cKO SVZ progenitors and not to an alteration of their cell cycle length.
Precocious Cell Cycle Exit in Arx cKO Cortical Progenitors
Since only about half of the pool of IPCs is able to proliferate in Arx cKO cortex, we predicted precocious cell cycle exit of these cells. Thus, we assessed the fraction of cells exiting the cell cycle 24 h after a BrdU pulse (between E13.5 and E14.5). Cells remaining in the cycle are BrdU+/Ki67+, while those that have stopped cycling exhibit BrdU labeling only (Chenn and Walsh 2002). At E13.5, an increase of 18% in the cells leaving the cycle is observed in Arx cKO progenitors (leaving fraction in ctrl: 0.49 ± 0.06; in cKO: 0.58 ± 0.05; n = 8 for each, P < 0.0005; Fig. 4A″,B″,C) suggesting precocious neurogenesis. Consistent with that, more cells with undiluted BrdU exist in the Arx cKO when compared with control (arrowheads, Fig. 4A′,B′). Although it is not possible to distinguish the contribution of RG cells and IPCs to the fraction of precociously differentiating cells, it is likely that most of those cells are IPCs that may exit cell cycle without any round of amplification.
In order to evaluate the effects of IPC reduced proliferation in Arx cKO, we analyzed GFP expression in E15.5 Arx cKO and relative control mice crossed with Tbr2::GFP transgenic animals (Fig. 4D,E′), where GFP is under the control of the Tbr2 endogenous regulatory sequences (Kwon and Hadjantonakis 2007). Due to the stability of the GFP protein in vivo, GFP fluorescence in Tbr2::GFP mice can be used as a lineage tracer of the IPC progeny (Sessa et al. 2008). We noticed an overall decrease in GFP+ cells in Arx cKO SVZ and CP with respect to control consistent with a reduction in the amplification of IPCs in the cKO (GFP+ cells/150 um area unit, ctrl: SVZ, 55.7 ± 5.13; IZ, 80.00 ± 16.7; CP, 180.33 ± 6.51; total, 316.00 ± 15.39; cKO: SVZ, 27.70 ± 7.37; IZ, 63.33 ± 26.63; CP, 83.00 ± 2.64; total 181.70 ± 21.01; n = 4 P < 0.005 (Fig. 4F).
Given the more pronounced effect of Arx ablation on IPCs than on RGCs, we sought to assess if Arx is also expressed in IPCs in addition to RGCs (Friocourt et al. 2006). We performed immunofluorescence for ARX and TBR2 in wild-type cortex at E14.5 and showed that ARX colocalizes with TBR2 in many cells of the VZ and SVZ (Supplementary Fig. 5).
Arx cKO Mice Have a Reduced Number of Upper Cortical Layer Neurons with Normal Number of Deeper Layer Neurons
In order to determine if the reduced proliferation of cortical progenitors has an effect on cortical lamination, we evaluated the cortical layers using molecular markers at different developmental stages. The expression of TBR1, which labels neurons in the subplate, layer VI, and few in layer V (Molyneaux et al. 2007), was evaluated in control Arx cKO mice at E16.5. In Arx cKO, the number of TBR1+ cells in layers V–VI was slightly decreased with respect to control (TBR1, ctrl layers V–VI = 98.2 ± 6.5; cKO layers V–VI = 82.0 ± 7.8) (Fig. 5A,B). Interestingly, more neurons were found in Arx cKO subplate, still detectable at this stage (TBR1, ctrl: SP = 36.5 ± 4.5; cKO: SP = 60.0 ± 3.5) (arrowheads in Fig. 5A,B).
A similar difference, although more dramatic, was observed staining for CTIP2, which labels predominately layer V, but also layer VI neurons (Molyneaux et al. 2007). CTIP2+ cells are already well organized in control layers V–VI, with some positive cells localized in the subplate, whereas in the Arx cKO, they appear less compact and completely disorganized (arrowheads in Fig. 5C,D). However, the total number of CTIP2+ cells does not change between Arx cKO and control (CTIP2, ctrl: 166.9 ± 4.2 and cKO: 155.7 ± 5.7) (Fig. 5U). To assess upper layer stratification, we used P1 and P14 animals. At P1 Arx cKO mice had a decreased number of cells labeled with SATB2 in layer II/III in the Arx cKO cortices with respect to controls (SATB2 ctrl: layer II/II = 131.4 ± 18.5 and SATB2 cKO: layer II/III = 96.9 ± 12.6; n = 5, P = 0.0126) (arrowheads in Fig. 5I,J), but not in layer IV/V (SATB2 ctrl: layer IV/V = 84.0 ± 6.8 and SATB2 cKO: layer IV/V = 77.9 ± 7.6; n = 5, P = 0.547) (Fig. 5I,J). In addition, the number of cells labeled with CUX1, a marker of layers II–IV, is also decreased in the Arx cKO cortices (CUX1 ctrl: layers II–IV = 125.9 ± 7.0 and CUX1 cKO: layers II–IV = 95.9 ± 7.8; n = 5, P = 0.0327) (arrowheads in Fig. 5K,L). However, no significant difference between the Arx cKO and control cortices was detected for layers V and VI markers (CTIP2 ctrl: total = 97.5 ± 7.5 and CTIP2 cKO: total = 93.2 ± 9.6; n = 3, P = 0.587; TBR1 ctrl: total = 116.9 ± 4.9 and TBR1 cKO: total = 92.1 ± 14.2; n = 3, P = 0.132) (arrowheads in Fig. 5E–H).
At P14, a time in which laminar organization is complete, a decrease in the number of SATB2- and CUX1-positive cells persisted in layer II/III in the Arx cKO cortices when compared with control cortices (SATB2 ctrl: layer II/II = 29.5 ± 2.4, layer IV = 41.4 ± 3.8 and SATB2 cKO: layer II/III = 21.2 ± 3.9, layer IV = 24.5 ± 3.0; n = 4, P = 0.0286; CUX1 ctrl: layers II–IV = 30.5 ± 2.1 and CUX1 cKO: layers II–IV = 24.9 ± 3.5; n = 4, P = 0.0482) (arrowheads in Fig. 5Q–T). However, similar numbers of CTIP2-positive and TBR1-positive cells are present in the Arx cKO cortices as compared to control (CTIP2 ctrl: total = 35.5 ± 3.7 and CTIP2 cKO: total = 29.2 ± 1.7, n = 3, P = 0.272; TBR1 ctrl: total = 32.7 ± 2.1 and TBR1 cKO: total = 32.5 ± 2.5, n = 3, P = 0.406) (Fig. 5M–P).
In summary, we find a reduction of upper layer (II–IV) neurons with no change in deeper layer (V–VI) neurons in Arx cKO mice.
Identification of Genes Differentially Expressed in Arx KO Versus Wild-Type Cerebral Cortex by Transcriptome Analysis
To gain insight into the Arx-dependent mechanism responsible for the reduced proliferation in Arx cKO cortical progenitor cells observed at E14.5, we performed a gene expression profile analysis. Since all the proliferation defects observed in the Arx cKO cortices were also detected in Arx KO mouse model (data not shown), we decided to perform this experiment in Arx KO dorsal telencephalon. E14.5 embryonic stage was selected for analysis, as the proliferation defect was clearly detectable. Brains were harvested, and the cerebral cortices were carefully dissected and processed for gene expression profiling (Fig. 6). RNA was separately extracted from the isolated tissues and used to hybridize Affymetrix microarrays (MOE430v2) containing 45 101 probe sets. Cortical tissues from Arx KO (n = 5) and control (n = 5) embryos were independently processed on 10 different gene-chips in order to minimize individual biological differences. Datasets obtained were then used to identify differentially expressed transcripts between Arx KO and wild-type dorsal telencephali (Fig. 6A–I).
Using a log2 (fold-change ≥ 1 and a FDR ≤ 0.05) linear model, the analysis revealed that a total of 147 probe sets corresponding to 86 different Entrez Gene Identifiers presented significantly different expressions between Arx KO and wild-type cortices (Fig. 6A–I).
Among the full set of identified genes, 3 (DDX3Y, KDM5D, and EIF2S3Y) (Fig. 6I) were considered as Arx-independently misregulated. In fact, they correspond to Y-linked expressing units and their upregulation in Arx KO tissues is indicative of a sex-biased harvest of the embryos. This was expected because all Arx KO embryos were males (Arx−/Y), while wild-type embryos displayed an equal probability to be males or females. The remaining 83 differentially expressed genes were grouped according to the known or predicted protein function they coded for: transcription factors (Fig. 6A); receptors (Fig. 6B); intracellular regulatory proteins (Fig. 6C); structural proteins (Fig. 6D); extracellular regulatory proteins (Fig. 6E); extracellular junctions and adhesion molecules (Fig. 6F); carriers and transporters (Fig. 6G); extracellular matrix proteins (Fig. 6H).
To ensure the reliability of our analysis, a subset of the misregulated genes were selected and analyzed in control and KO tissues using quantitative real-time RT-PCRs (qPCRs). In this experiment, only RNA extracted from male control embryos was used, in order to avoid any sex-biased gene expression between males and females. In all cases analyzed, qPCR results confirmed previous gene-array analyses both for the upregulated genes RARb, Id1, Id3, Tub6, Olig2, and Cdkn1c as well as the downregulated Pax6 gene (Fig. 6J, all normalized to ß-actin). However, the magnitude of the changes in expression levels detected by qPCRs was, in some cases, significantly higher (up to 2.5 fold) when compared with the microarray analyses (RARb, Olig2, Cdkn1c), while, in other instances, the results were comparable (Id1, Tub6, Pax6). Interestingly, the expression of Tbr2 was decreased in Arx KO compared with control (20% reduction) in agreement with our immunohistochemistry data, despite the microarray analysis did not show a significant alteration.
Cdkn1c is Overexpressed in Arx KO Cerebral Cortex
Among the genes found to be misexpressed in Arx KO cerebral cortices, we noticed the upregulation of Cdkn1c. Cdkn1c encodes for CDKN1C (also known as p57/KIP2), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs). Together with the 2 other members of this family, CDKN1A and CDKN1B, it exerts a basilar role in the regulation of cell cycle progression of cortical progenitors at level of G1/S transition by inhibiting the cyclin/CDK complexes (Sherr and Roberts 1999). Recently, Mairet-Coello and coworkers showed that CDKN1C overexpression in the cerebral cortex elicits precursor cell cycle exit and promotes a transition from proliferation to neuronal differentiation, while the opposite effects occur in CDKN1C-deficient precursors. Indeed, they reported that, in mutant mice, proliferation of RGC and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs (Mairet-Coello et al. 2012).
As this function is particularly consistent with the phenotype observed in Arx cKO brains and might underlie the reduction in the cerebral cortex volume, we focused further analyses on this gene.
In order to ascertain if Cdkn1c misregulation plays a role in Arx KO tissues, we first assessed its expression by in situ hybridization throughout embryonic development (E12.5–E18.5) in wild-type and Arx KO mouse brain sections (Fig. 7A,B,D,E and Supplementary Fig. 6). The expression of Arx was evaluated on adjacent wild-type brain sections (Fig. 7C,F and Supplementary Fig. 6). At E12.5, Cdkn1c transcript was barely detectable in the SVZ of control cortices whereas it was significantly overexpressed in both VZ and SVZ of Arx KO (Fig. 7A,B, arrowheads A′,B′). Interestingly, the upregulation of Cdkn1c seemed to follow a gradient in Arx KO cortices, higher lateral lower medial (Fig. 7B). Similarly, at E14.5, Cdkn1c is still expressed in the SVZ in control tissues (Fig. 7D, arrowheads D′) and overexpression persists in Arx KO cortices (Fig. 7E, arrowheads E′). In contrast to E12.5, Cdkn1c expression clearly followed a higher medial-lower lateral gradient in both control and KO cortices (Fig. 7D,E). Beginning on E16.5, Cdkn1c transcript was additionally detected in CP cells of both control and KO brains and, at E18.5, the level of Cdkn1c upregulation in Arx KO cortices was significantly diminished, inversely correlating with the Arx expression pattern at the same developmental stages (Supplementary Fig. 6). Similar results were also observed in Arx cKO cortices.
Unexpectedly, although Arx is strongly expressed in the ganglionic eminences, in particular at E12.5, E14.5, and E16.5 (Fig. 7C,F and Supplementary Fig. 6), its ablation in Arx KO mice does not induce Cdkn1c upregulation in this compartment (Fig. 7B,E and Supplementary Fig. 6).
In conclusion, our expression analysis of Cdkn1c in wild-type and Arx KO cerebral cortices throughout the neurogenic period, showed that this gene is upregulated in Arx KO cortical VZ and SVZ, with a pattern that parallels Arx expression in control tissues. This correlation raises the possibility that Arx depletion directly promotes Cdkn1c upregulation in cKO tissues.
Cdkn1c is a Direct Transcriptional Target of ARX
To determine if Cdkn1c is a direct transcriptional target of ARX, a 5-kb genomic region upstream to the murine Cdkn1c gene locus was screened for the presence of putative ARX binding sites. Previous studies identified palindromic sequences TAAT/ATTA spaced by 3 nucleotides (5′-ATTANNNTAAT-3′) as favored binding sites for paired homeodomain proteins (Wilson et al. 1993). Indeed, our search highlighted the presence of a unique palindromic sequence (5′-ATTAGCATAAT-3′) located between bases −71/−81 upstream of the Cdkn1c transcription start site (named +1) (Fig. 7G). As binding sites for transcriptional regulators are usually evolutionary conserved, we performed a multiple alignment of the aforementioned genomic region among different species, finding a high level of conservation in the ARX putative binding site in mouse, rat, human, and orangutan, except for the 3 spacing nucleotides that were not conserved in orangutan (Fig. 7G). A genomic region of about500 bp (−549/−68) containing the putative ARX binding site and another of about 750 bp (−1490/−720 containing some TA repeats, were separately cloned, upstream to a minimal promoter, in a construct containing the Luciferase gene and tested for transcriptional reporter activity (Fig. 7H). The 500 bp (−549/−68)-Luc and 750 bp (−1490/−720)-Luc constructs were separately cotransfected with control plasmid pCAG-IRESGFP or pCAGArx-IRESGFP in P19 cells and the effect of ARX expression on the basal transcriptional activity of the 2 putative regulative sequences was evaluated. ARX cotransfection reduced the basal transcriptional activity of the 500-bp regulative region by 30% (fold increase/decrease in transcriptional activity of pCAG-ArxIRESGFP coinfected with respect to pCAG-IRESGFP coinfected: 0.67 ± 0.04; n = 3, P < 0.005). However, higher reduction was detected when the corepressor Tle1 (McKenzie et al. 2007) was cotransfected in association with ARX (0.42 ± 0.06, n = 3, P < 0.005). Surprisingly, ARX and in particular TLE1 alone are able to repress luciferase transcription; this is due to the fact that both proteins are already expressed at low levels in P19 cells (data not shown). Conversely, when pCAG-ArxIRESGFP was cotransfected with the Luciferase construct regulated by 750 bp enhancer, only a slight reduction in the basal transcriptional activity was assessed (fold increase/decrease in transcriptional activity of pCAG-ArxIRESGFP coinfected with respect to pCAG-IRESGFP coinfected (transcriptional activity; 0.94 ± 0.01, n = 3, P < 0.05), even in combination with TLE1 (Fig. 7H). When the binding site 5′-ATTAGCATAAT-3′ was mutagenized to 5′-AGGAGCATCCT-3′, no significant reduction in Luciferase expression was appreciated in cotransfection experiments (Fig. 7H).
These results indicate that ARX exerts a specific transcriptional repression activity on this 500 bp (−549/−68) genomic region; in order to assess if this repression is direct or mediated by other factors expressed in P19 cells, we performed chromatin immunoprecipitation experiments. Anti-ARX antibody was used to immunoprecipitate chromatin isolated from E14.5 telencephali; then, PCR amplification reactions, with primers designed to span the 500 bp (−549/−68) region upstream to Cdkn1c gene, allowed us to evaluate an enrichment of that region in the ARX-immunoprecipitated chromatin with respect to IgG only and anti-GFP immunoprecipitated chromatin (Fig. 7I). Amplifications with primers spanning Ebf3 regulatory region were also performed and used as a positive control, as Ebf3 was previously showed to be a direct target of ARX in the ventral pallium (Fulp et al. 2008). We found enrichment of the Cdkn1c (and Ebf3) regulatory regions when pulled down with an anti-ARX antibody but not with the anti-GFP or no antibody controls. No enrichment in ARX-immunoprecipitated chromatin samples was detected amplifying for Cdkn1a and Cdkn1b regulative regions (Fig. 7I), as expected by the absence of any alteration of the 2 gene transcripts in Arx KO cortices. Taken together, these results suggest that ARX is a direct repressor of Cdkn1c transcription, and it likely exerts this role by direct interaction with the palindromic binding site 5′-ATTAGCATAAT-3′ located in the 500 bp enhancer region.
Loss of ARX/Arx in both humans and mice results in a reduced size of the cerebral hemispheres. In this study, by crossing Arxfl/y mice with Emx1-Cre mice, we have confirmed that the reduced brain size is largely if not completely due to the loss of Arx in cortical progenitor cells. Indeed, in E14.5 Arx cKO, we observed a reduction in the number of cortical progenitor cells, both PAX6+ and TBR2+, that might be the result of the precocious RG progenitor differentiation observed at E12.5 in Arx cKO.
However, this cannot be the only underlying mechanism. In fact, the proliferation rate of cortical progenitor cells is also affected in Arx cKO and our data suggest that the IPC pool is more effected than the RGC compartment: most of S-phase progenitors in E14.5 Arx cKO cortices are PAX6+ cells and only a little percentage is TBR2+. Consistent with this, basal mitoses are dramatically reduced. Interestingly, the determination of the GF at E13.5 allowed us to understand that, whereas all the cKO RG progenitors were able to proliferate at that stage, only 45% of TBR2+ cells in Arx cKO could enter the cell cycle (vs. the 75% of the control). These observations support the hypothesis that the drastic effect on the IPC proliferation is not only due to a premature differentiation among VZ progenitors but essentially to a direct effect of ARX ablation on IPCs. Consistent with this, we found ARX expression in many TBR2+ cells of the VZ/SVZ.
The reduction of cortical progenitor cells led to an alteration of the cortical layering: a significant loss of cells from the superficial layers of Arx cKO cerebral cortex with relative preservation of the deeper layers. Our observations in this genetic model complement those found after an acute reduction of ARX through the in utero electroporation of an Arx shRNA in midgestation where an increased number of progenitors exiting the cell cycle and prematurely adopting the neuronal fate were found (Friocourt et al. 2008).
We also performed a gene expression profile analysis in wild-type and Arx KO cerebral cortices that identified 83 misregulated genes. Interestingly, this analysis found very little overlap with the results of the same analysis that we previously performed in the ganglionic eminences (Colasante et al. 2009; Fulp et al. 2008). These data reinforce the assertion that ARX exerts region-specific roles in the ventral telencephalon and in the cerebral cortex through the activation/repression of different transcriptional programs (Nasrallah et al. 2012). In agreement with the reduction of GABAergic interneurons migrating to the cerebral cortex in Arx KO brains (Colombo et al. 2007; Kitamura et al. 2002), markers of specific interneuron subclasses (NPY, Somatostatin and also LHX6) were downregulated in KOs. However, among the several misregulated genes, we focused our attention on Cdkn1c, as we considered it the most interesting ARX putative target, whose upregulation could account for the phenotype observed in Arx cKO cortices (Mairet-Coello et al. 2012).
Our characterization of the Cdkn1c expression pattern during normal corticogenesis found Cdkn1c to be expressed in the SVZ in the first phases of neurogenesis (E12.5–E14.5). At later stages it is also detected in the CP. Conversely, in Arx cKO, it is strongly upregulated in the VZ and SVZ starting at E12.5. The expression of Cdkn1c during corticogenesis has been debated for a long time. While early reports indicated that Cdkn1c mRNA is localized in the VZ, SVZ, and CP of embryonic rat cortex (Campagne and Gill 1998), CDKN1C protein has been localized in the CP, with faint expression in the IZ and no signal in the VZ/SVZ (Itoh et al. 2007; Nguyen et al. 2006). A few recent studies have helped clarify this issue (Mairet-Coello et al. 2012; Nguyen et al. 2006; Tury et al. 2011, 2012). Using an antibody against CDKN1C, they found it is expressed mainly in differentiating postmitotic neurons, but also it colocalizes with TBR2+ cells in SVZ and with some PAX6+ in VZ. These data are fully consistent with our data on Cdkn1c expression obtained by in situ hybridizations.
Recently, Tury et al. (2011) showed that CDKN1C overexpression in E14.5–15.5 rat embryos elicits precursor cell cycle exit and promotes transition from proliferation to neuronal differentiation. Comparing the neurogenic and astrogliogenic effects of CDKN1B and CDKN1C, they discovered that CDKN1C overexpression promotes more neuronal differentiation than CDKN1B. The specific neurogenic effect of CDKN1C is particularly interesting in relation to its expression in IPCs, which, distinct from RGCs, generate only neurons. However, they do not clearly analyze the specific effect of CDKN1C overexpression on the 2 cortical progenitor cell populations. In their follow-up work, Mairet-Coello et al. (2012), analyze the phenotype of Cdkn1c mutant mice, finding that proliferation of both RGCs and IPCs is increased, expanding both precursors with greater impact on IPCs.
In this context, our report is the first to describe the effect of CDKN1C overexpression on cortical progenitor cell proliferation, integrating the analysis previously performed. Interestingly, our data also support the hypothesis that CDKN1C is more relevant for IPC than RGC cell cycle exit, although CDKN1C is overexpressed in both cortical VZ and SVZ, the most dramatic effect is observed on IPC and not on RGC proliferation.
We propose a model for the ARX-dependent control of cortical progenitor cell proliferation (Fig. 8). ARX expression in the VZ and partially in the SVZ is able to repress Cdkn1c premature expression, allowing proliferation of RG cells and expansion of IPCs. When Arx is normally downregulated, the consequent upregulation of CDKN1C in the SVZ favors IP cell cycle exit. In contrast, in Arx KO cortex, CDKN1C expression is high in the VZ resulting in a mild proliferation effect on RG cells. More dramatic is the impact on TBR2+ IPCs; these progenitors have already such a high level of CDKN1C that directly exit the cell cycle without significant cell division.
Certainly, the presence of other alterations or compensatory mechanisms cannot be excluded in Arx cKO cortices. For example, the microarray analysis highlighted the upregulation of Id1 and Id2 genes in Arx KO cortices. It is known that their targeted disruption in mice results in premature withdrawal of progenitor cells from the cell cycle and expression of neural-specific differentiation markers (Lyden et al. 1999). Thus, it is likely that their overexpression in Arx cKO cortices might compensate for the Cdkn1c upregulation, mitigating the full phenotypic expression. Among the compensatory mechanisms, we must also consider the slight acceleration of cell cycle duration in IPCs observed in Arx cKO contributing to the observed phenotype.
We also demonstrated that Cdkn1c is a direct transcriptional target of ARX. The genomic region 5′ to the murine Cdkn1c gene locus was screened for the presence of the ARX putative binding site and a unique palindromic sequence (5′-ATTAGCATAAT-3′) was located a few bases upstream of the Cdkn1c transcription start site. Luciferase assays allowed us to demonstrate that ARX, together with the corepressor TLE1, is able to repress transcription through binding at the identified 500 bp enhancer region. Furthermore, chromatin immunoprecipation revealed that ARX directly binds in this 500 bp Cdkn1c enhancer region.
Surprisingly, the ARX binding site that we identified in this study is different from the one described in our previous work (Fulp et al. 2008). In that work, we analyzed the transcription factor binding sites significantly enriched in the regulatory regions of genes whose expression increased within Arx KO subpallium, we identified the sequence TAATTA. In a recent study using the ChIP-chip methodology in ARX transfected N2a cells almost 50% of Arx-immunoprecipitated sequences N2a cells were unequivocally enriched for TAATTA binding site in comparison to controls (Quille et al. 2011). In this same study, only 20% of the genes ARX-immunoprecipitated in E15.5 mouse embryonic brains were found to have this binding site; furthermore, any other motif enriched in ARX-bound sequences by comparison to control sequences, could not be identified (Quille et al. 2011). These results suggest that whereas overexpressed ARX seems to be recruited primarily to TAATTA, in more physiological conditions such as in embryonic brain, ARX is recruited to other less common motifs or needs other factors for DNA binding. Based on these data, the ATTAGCATAAT site we identified might be one of the in vivo binding sites to which ARX is recruited. Surprisingly, the ChIP on chip analysis performed by Quillé and coworkers revealed that ARX binds the Cdkn1a (p21/Cip1) promoter but not Cdkn1c; however, this might be due to limitations of the ChIP-chip technique. Indeed, ChIP-seq experiments might allow further delineation of ARX direct transcriptional target genes.
In summary, our data indicate ARX plays a unique role in the progenitor cells of the cerebral cortex when compared with its role in the ventral forebrain. By genetically eliminating Arx from pallial progenitor cells, we find a reduction in the number of RGC due to a premature exit from the cell cycle and significant skipping of the IPC amplification stage of corticogenesis. This defect results in the predicted deficit in upper layer neurons of the cerebral cortex. We further elucidated the pathogenesis of this defect by identifying the cell cycle regulator CDKN1C as a downstream target of ARX. Our study extends our understanding of the aberrant molecular network responsible for the reduced proliferation observed in Arx cKO forebrains; indeed, this is a critical point for understanding the pathogenic mechanisms underlying these devastating neurologic disorders and in devising genetic or cellular therapeutic strategies to ameliorate Arx mutation-induced pathological phenotypes.
Supplementary Material
Supplementary Material can be found at http://www.cercor.oxfordjournals.org/ online.
This work was supported by following grants: Telethon, the NIH NS46616 and HD26979 and the Neurodevelopmental Disabilities Training Grant T32NS007413.
We thank K. Kitamura for the anti-ARX antibody and L. Muzio for helpful discussion. The anti-PAX6 antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, USA. Finally, we also thank George Clement for his assistance with animal husbandry and genotyping. Conflict of Interest: None declared.