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 A screen for downstream effectors of Neurogenin2 in the embryonic neocortex 

   

Pierre Mattar a, Olivier Britz b, Christine Johannes a, Marta Nieto c, Lin Ma a, Angela Rebeyka a, Natalia Klenin a, Franck Polleux d, François Guillemot b and Carol Schuurmans a,, 

   

 a University of Calgary, Calgary, Alberta, Canada T2N 4N1



 b Division of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK



 c Beth Israel Deaconess Medical Center, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA



 d Department of Pharmacology, University of North Carolina, Neuroscience Research Building, Chapel Hill, NC 27599-7250, USA

   Received 14 April 2004; revised 18 June 2004; accepted 22 June 2004. Available online 27 July 2004.    Abstract 

 Neurogenin ( Ngn ) 1 and Ngn2 encode basic-helix-loop-helix transcription factors expressed in the developing neocortex. Like other proneural genes, Ngns participate in the specification of neural fates and neuronal identities, but downstream effect...

   

 Keywords: neocortex development; Neurogenin ; downstream effectors; thalamocortical; subplate; neuronal specification; axonal targeting

   

 Abbreviations: CP, cortical plate; dig, digoxygenin; E, embryonic day; gz, germinal zone; IZ, intermediate zone; Ngn, Neurogenin; P, postnatal day; pp, preplate; sp, subplate; SVZ, subventricular zone; TCA, thalamocortical afferent; VZ, ventricular ...

    Article Outline Introduction Materials and methods Maintenance and genotyping of Ngn2 lacZ and Ngn1 mutant mice beta-galactosidase detection and cell sorting RNA extraction, cDNA synthesis, and subtraction Identification and isolation of full-len...

The neocortex is subdivided into more than 40 tangential areas and six radial layers, each characterized by unique neuronal morphologies, axonal projections, molecular identities, and functions ( Job and Tan, 2003 ). The striking degree of neuronal d...



 Ngn1 and Ngn2, which encode basic-helix-loop-helix (bHLH) transcription factors with proneural activity ( Fode et al., 1998, Fode et al., 2000 and Ma et al., 1998 ), contribute to the specification of a neuronal versus glial identity in cortical pro...



The changes in cell-fate exhibited following ectopic expression of Mash1 in Ngn1/2 expression domains are analogous to those obtained when the Drosophila proneural achaete-scute genes are ectopically expressed in precursors that normally express aton...



In order to understand better the genetic program(s) executed by the proneural gene Ngn2 in the neocortex, we performed a subtractive hybridization screen between wild-type and Ngn2 mutant telencephalons. We identified 46 genes expressed in the embry...

 Materials and methods Maintenance and genotyping of Ngn2 lacZ and Ngn1 mutant mice 

 Ngn2 lacZ +/-;Ngn1+/- single and double heterozygous intercrosses were set up to obtain heterozygous and homozygous single and double mutant embryos. Embryos were staged using the day of the vaginal plug as E0.5 and genotyping was performed by PCR a...

 beta-galactosidase detection and cell sorting 

E12.5 dorsal telencephalons were dissected from embryos obtained from Ngn2 lacZ +/- heterozygous intercrosses. Cortical cells were dissociated and stained with fluorescein digalactopyranoside (FDG; Sigma) and sorted by fluorescence-activated cell sor...

 RNA extraction, cDNA synthesis, and subtraction 

Cells (8000–10,000 lacZ+) were collected from each embryo and sorted directly into 200 muL of 4 M guanidinium isothiocyanate (EM Science). Approximately 1 mug of total RNA was extracted from the dorsal telencephalon of each embryo using a scaled-down...

 Identification and isolation of full-length cDNAs 

We sequenced 200 clones that came through our second round of subtractive screening of the wild-type minus Ngn2 mutant telencephalon library. Sequenced clones were identified using the BLAST algorithm (NCBI) to screen nucleotide and EST databanks. Th...

 RNA in situ hybridization 

RNA in situ hybridization was carried out on 10 mum cryostat sections as previously described ( Cau et al., 1997 ). Digoxygenin (dig)-labeled RNA probes were generated using T3, T7, or SP6 RNA polymerases and a dig-RNA-labeling mix (Roche). In additi...

 Immunohistochemistry, histology, and birthdating 

For histology, P0 brains were fixed in Bouin's fixative for 3 days, dehydrated in an ethanol–xylene series, embedded in paraffin, and 7 mum sections were cut and stained with hematoxylin–eosin as described previously ( Rhinn et al., 1998 ). For birth...

 Results Construction of a subtracted cDNA library enriched for neocortical genes dependent on Ngn2 function 

Early born preplate and deep-layer cortical plate neurons are misspecified in Ngn2 mutants, acquiring a GABAergic rather than glutamatergic identity ( Fode et al., 2000 and Schuurmans et al., 2004 ). To identify downstream components of the Ngn1/2 -d...

 Full-size image (163K) 

Fig. 1.Experimental design for subtracting expressed genes in Ngn2 lacZ +/- (”wild-type”) and Ngn2 lacZ -/- (”mutant”) cortical cells. (A) Frontal section of E13.5 telencephalon hybridized with a probe for Ngn2. (B) X-gal staining of frontal sections...

 View Within Article 

For each embryo, betagal (+) cortical cells were separated from betagal (-) cells by flow cytometry (FACS), yielding 8000–10,000 cells. RNA was extracted from betagal (+) cells and cDNA was synthesized and amplified by PCR. Two rounds of subtraction ...



To assess the efficiency of subtraction, pools of subtracted cDNA were radiolabeled by random priming and hybridized to Southern blots on which equivalent quantities of known genes had been transferred ( Fig. 1 C). The wild-type minus Ngn2 mutant sub...

 Sequence and expression analyses of telencephalic library clones 

We hypothesized that the wild-type minus Ngn2 mutant subtraction would yield genes important for the specification of a cortical, glutamatergic identity and thus focused on analyzing these clones. Subtracted cDNAs were cloned to generate a library; a...



To identify cDNA sequences, BLAST searches were performed. The majority (76%) of the genes identified were represented only once in the library, whereas 24% appeared more than once. Fifty-eight percent of the unique genes had been previously characte...



Expression patterns of the 46 genes presented in Supplementary Table 1 were examined by RNA in situ hybridization on sections of E13.5 telencephalon, and all were shown to be expressed in the dorsal telencephalon, their patterns overlapping at least ...

 Full-size image (197K) 

Fig. 2.Expression analysis of pan-neuronal markers in E13.5 Ngn2 and Ngn1;Ngn2 mutant cortices. Frontal sections of E13.5 wild-type (wt) telencephalon were hybridized in situ with dig-labeled RNA probes. Insets in A'–C' and D'–F' show higher magnific...

 View Within Article Full-size image (281K) 

Fig. 3.Expression analysis of regionalized clones in E13.5 Ngn2 and Ngn1;Ngn2 mutant cortices. Frontal sections of E13.5 wild-type (A, D, G, J, M, P, S, V, and Y), Ngn2 mutant (B, E, H, K, N, Q, T, W, and Z), and Ngn1;Ngn2 mutant (C, F, I, L, O, R, U...

 View Within Article 

Analysis of our subtracted clones revealed several expression patterns, including genes that were expressed in progenitors throughout the neural tube [i.e., pan-progenitor; e.g., G-protein coupled receptor EDG1, transcription factor Pegasus-like (dat...

 Expression analysis of pan-neuronal markers in Ngn2 and Ngn1;Ngn2 mutants 

We next set out to determine whether the genes we identified were misregulated in the Ngn2 single mutant cortex, as anticipated from the design of our screen. We first focused our analysis on the pan-neuronal genes, predicting that these genes would ...



We also examined Zfp10-like and Dcx expression in E13.5 Ngn1;Ngn2 double mutants. In striking contrast to the reduced expression of Zfp10-like and Dcx in Ngn2 single mutants, the pan-neuronal genes were up-regulated in the preplate of Ngn1;Ngn2 doubl...

 Expression analysis of regionalized markers in Ngn2 and Ngn1;Ngn2 mutants 

Early born preplate and deep-layer cortical plate neurons are misspecified in Ngn2 mutants, acquiring a GABAergic rather than a glutamatergic identity ( Fode et al., 2000 and Schuurmans et al., 2004 ). One of the goals of our subtractive screen was t...



We compared the expression profiles of our regionalized genes in E13.5 wild-type, Ngn2 single and Ngn1 ; Ngn2 double mutant cortices. As summarized in Table 1, 16/19 regionalized genes tested were clearly misregulated in Ngn2 and Ngn1;Ngn2 mutants by...

 Table 1. 

Alterations in expression patterns of regionalized genes isolated from subtractive screen in Ngn2 and Ngn1;Ngn2 mutants

      Gene name(s) Category Expression ( Ngn2 and Ngn1;Ngn2 mutants versus wild type)     Zac1 (Plagl1/LOT1) Transcription factor darr VZ, PP neurons   Id4 (IDB4) Transcription factor No change   Dach1 Transcription factor No change   Nuclear Factor ...

Summary of genes that displayed regionalized patterns of expression in the E13.5 neural tube. Description of changes in the expression patterns of these regionalized genes in Ngn2 and Ngn1;Ngn2 mutants at E13.5 is noted. VZ, ventricular zone; SVZ, su...

 View Within Article 

Of the regionalized genes expressed in cortical progenitors, Nlgn1, which is a postsynaptically localized protein in neurons ( Scheiffele et al., 2000 ), and srGAP3, an intracellular mediator of slit / robo signaling ( Wong et al., 2001 ), were clear...



The largest group of misregulated genes included those whose expression was disrupted in cortical preplate neurons. Included in this category were Bhlhb5 ( Figs. 3 G–I), which encodes a bHLH transcription factor; Mef2c ( Figs. 3 J–L), which encodes a...



Finally, we observed that several of our misregulated genes were aberrantly expressed in the SVZ. Genes disrupted in the Ngn1;Ngn2 mutant SVZ included Bhlhb5 ( Figs. 3 G–I), Unc5H4 ( Figs. 3 M–O), Dcc ( Figs. 3 P–R), EphA5 ( Figs. 3 S–U), and Sema3c ...

 Defects in the differentiation of Ngn2 mutant cortical plate and subplate neurons 

To investigate further Ngn1/2 -dependent defects in regionalized gene expression in postmitotic neurons, we analyzed Ngn2 single mutants at E15.5, a stage when deep-layer cortical plate neurons, which display aberrant molecular identities in Ngn2 and...

 Full-size image (152K) 

Fig. 4.Defects in gene expression in subplate and early born cortical plate neurons in E15.5 Ngn2 mutants. Expression analysis of sagittally sectioned E15.5 wild-type and Ngn2 mutant telencephalon. (A) Expression of Ngn2 in wild-type telencephalon. (...

 View Within Article 

As differentiation proceeds, cortical plate neurons migrate into the middle of the preplate, splitting this layer into an overlying marginal zone and underlying subplate layer. At E15.5, we noted that Mef2c was expressed in the subplate of wild-type ...

 Full-size image (274K) 

Fig. 5.Subplate neurons are generated but misspecified in Ngn2 mutants. (A) Expression of Ngn2 in sagittally sectioned perinatal (E18.5) wild-type telencephalon. (B) Higher magnification of germinal zone from (A). (C–F) P0 expression analysis reveale...

 View Within Article Aberrant thalamocortical and corticothalamic axonal trajectories in Ngn1/2 mutants 

Previous studies have demonstrated that the subplate is critical for TCA axonal pathfinding ( Ghosh and Shatz, 1993, McConnell et al., 1989, McConnell et al., 1994 and Super et al., 1998 ), suggesting that disruption and misspecification of this laye...



We examined the distribution of TCAs first by immunostaining with an anti-L1 antibody ( Figs. 6 A–F), which revealed an aberrant organization and fasciculation of thalamic axonal tracts that disrupted the cortical germinal zone in E18.5 Ngn2 mutants....

 Full-size image (181K) 

Fig. 6.Thalamocortical and corticofugal projections are perturbed in Ngn2 and Ngn1;Ngn2 mutants. (A–F) L1 immunostaining of E18.5 wild-type (A–C) and Ngn2 mutant (D–F) sagittal sections through the cortex revealed aberrant axonal bundles (arrowheads)...

 View Within Article Discussion 

The primary objective of this study was to gain new insights into the transcriptional program(s) executed downstream of the proneural transcription factor Ngn2. In this regard, our subtractive screen was successful; leading to the identification of t...

 Identification of differentiation cascades activated downstream of Ngn2 

Before this screen, we knew that Ngn1 and Ngn2 activities were absolutely required to induce a dorsal, glutamatergic-specific differentiation program in early born cortical neurons, but known components of the downstream pathway included only HLH (i....



Although genomic analyses for proneural-regulated genes have not been conducted in most neural lineages, analyses of expression profiles of candidate target genes in proneural mutants have led to the identification of several downstream genes that ty...



A second broad category of proneural-regulated genes includes markers of a mature neuronal phenotype, such as NCAM, neurofilament, and the ELAVL4 homologs ELAV and HuC/HuD ( Ferreiro et al., 1994, Park et al., 2003 and zur Lage et al., 2003 ). Here w...



Proneural transcription factors are known to be required to activate genes involved in cell–cell communication, such as rhomboid, scabrous, delta, c-RET, and the neuropeptide receptor NKD ( Heitzler et al., 1996, Lo et al., 1998, Okabe and Okano, 199...

 Full-size image (39K) 

Fig. 7.Model depicting Ngn2 -dependent genetic cascades in the cortex. Pax6 directly regulates Ngn2 (solid arrow; Scardigli et al., 2003 ), but whether Ngn2 directly regulates any of the targets identified in our screen remains to be determined (brok...

 View Within Article 

In summary, the wide range of Ngn1 - and Ngn2 -regulated genes uncovered in our screen suggests that these proneural transcription factors regulate a multitude of cellular processes ( Fig. 7 ). This is highly analogous to myogenic bHLH transcription ...

 Identification of a new biological function for Ngn2 in guiding TCAs trajectories in the neocortex 

We have shown here that subplate neurons, which normally provide attractive cues for TCAs, are disorganized in Ngn2 and Ngn1;Ngn2 mutants. Moreover, Ngn2 mutant subplate neurons are misspecified and fail to express genes like Mef2c, protocadherin 9, ...



The abnormal differentiation of the subplate layer in Ngn2 and Ngn1;Ngn2 mutants initially prompted us to examine the projection patterns of TCAs in mutant cortices given the known role of the subplate in guiding these axons. Indeed, we found that TC...



Another abnormal aspect of the TCA trajectories in Ngn2 and Ngn1;Ngn2 mutants was that the thalamic axons aberrantly projected towards and disrupted the cortical germinal zone. This phenotype is likely independent of the subplate defects given that c...



In summary, through the expression analysis of our subtractive clones, we have identified a number of genes that are regulated by Ngn1 and Ngn2 in the developing neocortex, including not only transcription factors, but also genes potentially involved...

    Acknowledgment 

We are very grateful to Helen Cooper, Isabel Hanson, Brian Hemmings, Hisashi Narimatsu, Eric Olson, Andreas Püschel, Albert Reynolds, Fritz Rathjens, Iljona Skerjanc, Marc Tessier-Lavigne, Pierre Vanderhaeghen, and Zheng-Ping Xu for kindly providing ...

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