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Colloquium Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox7-gfp knock-in mice Jerome Aubert*t, Marios P. Stavridis*t, Susan Tweedie*, Michelle O'Reilly*, Klemens Vierlingert, Meng Li*, Peter Ghazalt, Tom Pratt, John 0. Mason, Douglas Royt, and Austin Smithson *Institute for Stem Cell Research, University of Edinburgh, King's Buildings, West Mains Road, EH9 3JQ Edinburgh, Scotland; tThe Scottish Centre for Genomic Technology and Informatics, University of Edinburgh, Chancellor's Building, 49 Little France Crescent, EH16 4SB Edinburoh, Scotland; and Division of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, EH8 9XD Edinburgh, Scotland The transcription factor Sox1 is the earliest and most specific known marker for mammalian neural progenitors. During fetal development, Sox1 is expressed by proliferating progenitor cells throughout the central nervous system and in no tissue but the lens. We generated a reporter mouse line in which egfp is inserted into the Sox1 locus. Sox1GFP animals faithfully recapitulate the expression of the endogenous gene. We have used the GFP reporter to purify neuroepithelial cells by fluorescence-activated cell sorting from embryonic day 10.5 embryos. RNAs prepared from Sox16FP+ and Sox1GFP- embryo cells were then used to perform a pilot screen of subtracted cDNAs prepared from differentiating embryonic stem cells and arrayed on a glass chip. Fifteen unique differentially expressed genes were identified, all previously as- sociated with fetal or adult neural tissue. Whole mount in situ hybridization against two genes of previously unknown embry- onic expression, Lrrn1 and Musashi2, confirmed the selectivity of this screen for early neuroectodermal markers. Neural stem cells are promising candidates for the development of cellular and genetic therapies for neurodegenerative disor- ders such as Parkinson's disease and Huntington's disease (1), and for creation of in vitro drug discovery and toxicological screens (24. However, the biomedical application of neural stem cells will require the generation of large homogenous populations of these cells in vitro. One source of neural stem cells is embryonic stem (ES) cells (34. ES cells are derived from the inner cell mass of the preimplantation blastocyst-stage embryo and can be propagated indefinitely in an undifferentiated, pluripotent state (44. The for- mation of multicellular aggregates called embryoid bodies permits the commitment of pluripotent ES cells into multiple cellular lineages in vitro (5), mimicking aspects of cellular differentiation in early embryos (6~. This provides a powerful system for the discovery of genes induced early during development and for functional validation of candidate genes (74. A favored protocol for the commitment of ES into neural lineage is the treatment of embryoid bodies with all-trans retinoic acid (~-104. After induction and outgrowth onto an adhesive substratum, up to 50% of cells express the neural precursor markers Soxl and Sox2 and can generate neurons and glia (114. Key advances in defining the optimal conditions for generating and propagating neural stem cells are likely to come from a proper understanding of the molecular mechanisms controlling the fate decisions of pluripotent cells and of fetal and ES cell-derived neural precursors. Here we describe a refined approach to identify genes induced during neural specification and/or maintained in neural progenitor cells in vivo and in vitro. Transgenic mice (SoXlGFP) were generated in which the en- hanced GFP (e~p) reporter is inserted into the Soxl gene via gene targeting (12~. Soxl is the earliest specific marker of neural 11836-11841 1 PNAS 1 September 30, 2003 1 vol. 100 1 suppl. 1 precursors in the mouse embryo (134. It is present in prolifer- ating neural precursors from the neural plate stage onwards. The only other site of expression in the mid-gestation fetus is the lens (Fig. L43. Exit from mitosis and neuronal or glial differentiation is accompanied by down-regulation of Soxl (144. The GFP knock-in allows the visualization of Soxl expression in these animals by fluorescence microscopy, and the purification of Soxl -positive cells by f luorescence-activated cell sorting (FACS). This purification allows preparation of RNAs for highly selective differential screening of microarrays. We have tested this approach by application to a custom microarray of a subtractive cDNA population prepared from retinoic acid- induced embryoid bodies. Materials and Methods Gene Targeting. A phage genomic DNA library from 129/Ola strain mouse was screened with a 2-kb probe containing the Soxl ORF (generously provided by Larysa Pevny). From a resulting phage containing 12 kb of Soxl genomic sequence, 5.5- and 2.5-kb fragments flanking the Soxl ORF were taken as 5' and 3' homology arms to prepare a targeting vector. The gene for e~p was fused in-frame into the second of three consecutive ATGs at the Soxl translation initiation site (15, 16) via PCR. The fusion product, linked via an internal ribosome entry site (IRKS) (17) to the gene encoding puromycin acetyltransferase (pac), was cloned between the homology arms. A cytomegalovirus promot- er-driven hygromycinR-thymidine kinase dual selection cassette (18) flanked by loxP sites was inserted downstream of the GFPirespac cassette (Fig. 1A). After electroporation in E14Tg2a ES cells and selection in hygromycin, three targeted clones were identified by Southern analysis with flanking 5' and 3' probes, and unique integration was confirmed for two of these by using an egfp probe. Transient transfection with a Cre recombinase expression vector was used to remove the selection cassette. Clones that had undergone excision were selected for in the presence of ganciclovir and screened by Southern analysis (Fig. 1B). One such clone (46C) was injected into blastocysts and This paper results from the Arthur M. Sackier Colioquium of the Nationai Academy of Sciences, "Regenerative Medicine," held October 18-22, 2002, at the Arnoid and Mabei Beckman Center of the National Academies of Science and Engineering in irvine, CA. Abbreviations: ES, embryonic stem; FACS, fluorescence-activated ceil sorting; En, embry- onic day n. ~J.A. and M.P.S. contributed equally to this work. ~A.S. is a scientific adviser to Stem Cell Sciences, Ltd., and holds nonvoting equity in the company. Stem Cell Sciences funds research in the laboratory and has patents granted and pending on technology used in this article. IlTo whom correspondence should be addressed. E-mail: austin.smith@ed.ac.uk. 2003 by The National Academy of Sciences of the USA www.pnas.org/cgi/doi/10.1 073/pnas.1 734197100

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B C 14 46 ~ cat ~ Sox1 ORf it/ ~ l`= ~ 8~t _ | . Genomic locus ~ i;;= ~ Aft\ ~ EcoRI: 12Kb X ~71ntemay ~ Lox\ ~=~ Targeting vector egfiP pllC C~V try-= En Xbal Xbal Score I 1 ~ 1 Targeted locus On deal: 9Kb ~~ ~ ~ ~ EcoRI: SKb CO 14 46 53 46C + Cry ~ ~ ~~> _ .~ ~ ~ 1 2Kb L_' ,_ 46C locus ~-~ ~ 6.6Kb _ _ ~ 5Kb TV 14 46 S3 cot 1~~ _ ~ 9Kb Fig. 1. Targeting Sox1 and Sox1GFP expression. (A) In situ hybridization of an E10.5 embryo with a Sox1 riboprobe, showing expression restricted to and throughout the neuraxis. (B) Schematic showing the design of the targeting vector and the screening strategy for identification of correctly targeted clones. (C) Southern blots showing the correct targeting events and the excision of the cytomegalovirus (CMV) HyTK cassette after transient transfection with a Cre expression plasmid. (Top) 5' probe. (Middle) Internal EGFP probe. (Bottom) 3' probe. Clone 14 is correctly targeted but has multiple integrations; clones 46 and 53 are correctly targeted, single integration clones. Clone 46C is a derivative of clone 46 after Cre-mediated deletion of the CMV Hy-TK cassette. Control is DNA from the parental E14Tg2a ES cells. (D) Sox1GFP expression in embryos and adult animals. (] Sox1GFP embryo at E9.5 showing expression throughout the length of the neural tube. (i/) Dorsal anterior view of a Sox1GFP embryo at E11.5 showing exclusion of GFP fluorescence from the midline, indicating no expression in roofplate and floorplate. (ii/) Coronal section through the head of an E12.5 embryo showing Sox1GFP expression in the ventricular and subventricular zone and the lens. (iv) Section through an adult SoxlGFP mouse brain, showing Sox1GFP-expressing cells in the subgranular layer of the hippocampus. (Inset) Higher magnification of boxed area, showing a GFP-expressing cell in the granular layer. Tissue Preparation. For analysis of SoxlGFP expression, heterozy- gous Sox1GFP males were crossed with wild-type females. Midday after vaginal plug was considered as embryonic day 0.5 (E0.54. Females were killed by cervical dislocation, and the embryos were dissected free of the uterus, washed in PBS, and observed under a fluorescence microscope. For cryosectioning, embryos were fixed in 4% paraformaldehyde (PFA), cryoprotected in 30% sucrose in PBS, and embedded in OCT compound before cryosectioning at 10-12 ,um. Adult (4-7 weeks old) heterozy- gous brains were dissected out and fixed in 4% PEA before embedding in 2% agarose in PBS and sectioning at 50 ,um by using a vibratome. Sections were counterstained with propidium iodide and analyzed by confocal microscopy. passed through the germ line of chimeras to generate the RT-PCR.To eliminate contaminating genomic DNA, 1 ,ugof total Soxl GFP mouse line. Mice were maintained on a mixed 129xMF1 RNA was treated with 1 unit of DNase I (GIBCO/BRL) for 15 background by breeding of heterozygotes to outbred MF1 mice. min at 25C. DNase I was inactivated with 25 mM EDTA (pH 8.0, GIBCO/BRL) at 65C for 10 min and chilled on ice. First strand random-primed cDNA was synthesized by using Super- script II Preamplification System (GIBCO/BRL) as described by the supplier. The cDNA was analyzed by PCR amplification using individual primer pairs for specific marker genes. The PCR cycling sequence used was 94.0C for 3 min. followed by 20-35 cycles of 94.0C for 30 s, 58.0-60.0C for 30 s and 72.0C for 1 min. This was followed by a final extension time of 7 min. All PCR samples were analyzed by electrophoresis on No agarose gels stained with ethidium bromide. FACS Purification and RNA Preparation. E10.5 Soxl-GFP-positive embryos were dissected free of extraembryonic membranes, digested in 0.1% trypsin, and resuspended in cold 10% FCS in PBS. The cells were sorted by flow cytometry to give two cell populations, Soxl-GFP-positive (SoxlGFP+) cells and Soxl- GFP-negative (SoxlGFP-) cells. The sample was kept cold at all times to minimize RNA degradation and cell death during sorting. Viable cells were gated by their forward and side scatter characteristics, and gates were set to sort positive and negative cell populations. Total RNA was extracted from both cell populations by using the RNeasy Minikit (Qiagen, Valencia, CA), according to the manufacturer's instructions. RNA yield was determined by measuring absorbance at 260 nm. RNA quality was assessed by electrophoresis of 1 ,~g of RNA on a standard 1.2% formalde- hyde agarose gel. Aubert et al. Microarray and Sequence Analysis. A subtracted library enriched for genes expressed during retinoic acid-induced neural com- mitment of ES cells (7) was spread out on LB plates containing ampicillin and 5-bromo-4-chloro-3-indolyl ,l3-D-galactoside (X- gal). A total of 384 white bacterial colonies were randomly picked and cultured in 96-well plates. One microliter of each bacterial culture was amplified by using the Advantage cDNA PCR kit (CLONTECH) and PCR primers that are homologous to the flanking regions of the cDNA insert (30-35 cycles: 30 s at 95C, followed by 3 min at 68C). PCR fragments were analyzed by electrophoresis on 2% agarose gel. The average insert size was between 200 and 800 bp. PCR products were printed onto poly~lysine)-coated glass slides by using an Affymetrix (Santa Clara, CA) 417 arrayer. Each cDNA insert was spotted in triplicate. A collection of marker genes (see Fig. 2B) whose expression was predetermined by RT-PCR analysis was included to act as control for the specificity of neural expression in the sorted Soxl+/- RNA populations. Twenty micrograms of Soxl+ or Soxl- total RNA obtained from disaggregated embryos was reverse transcribed with Su- PNAS | September 30, 2003 I vol. 100 | suppl. ~ | 11837

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A 489692 357194 sly o 244796 122398 it. i ~ i.: - P~ ..... . art L2''::::::f:,:: '.:' ~ ~ ''.2''~''-:.:'."'''''''.",'' ~~'~'~'~'~'~'~'~'~''''~'' N:: .. ..... 6 Hi I. I I ~ - ~ ..~:..:.,.:..i..,~.,.,.,.,.~.. ~~ it.: ;. . Neslin _ Patch sFRP2 R1 R2 ~ _' 0 10' 1o2 103 Sox1-GFP FAX \, ~0* ~0* P311 Riken cDNA 2810027019 ShcC Soxl O Bmp4 p-aclin Fig. 2. FACS purification of neural precursors. (A) FACS profile showing cell sorting of the Sox1 + neural precursor population from the Sox1- cell popu- lation from E10.5 mouse embryos. (B) Positive (R2, 30.35%) and negative (R1, 44.02%) populations were sorted and analyzed by RT-PCR for marker gene expression. perscript II (GIBCO/BRL) in the presence of Cy3 dCTP after priming with poly~dT) according to methods described by Brown et al. (http://brownlab.stanford.edu). Cy3-labeled Soxl+ and Soxl- cDNA was hybridized (19, 20) to separate microarrays. Table 1. Genes identified by microarray analysis Clone Fold Increase P5C3 2.40 P2H5 2.40 P2C5 1.98 P3B7 P5C1 1 1.96 P5H8 P3C1 P3B1 P2E5 P5G9 P5D1 1 P5D5 P5H2 P5A9 P2E7 Scanning was performed with an Affymetr~x 428 scanner, and the program QUANTARRAY (Perkin-Elmer) was used for image analysis. Multiple scans were taken to define the optimal dy- namic range of signal for subsequent analysis. The background was first subtracted for each respective probe element on the array. The median value for the triplicate probe elements representing each gene or insert was then defined. A scaling factor was applied to the arrays representing normalization to the 75th percentile of the global signal distribution. Ratios of expression were then calculated by using these normalized median values. A ratio value (Soxl+/Soxl-) of 1.5 or greater was used for the selection of neural specific expression. Clones encoding 15 up-regulated transcripts (Table 1) were sequenced, and the corresponding genes were identified by BLASTN searches of nonredundant (nr), dbEST, and mouse genomic databases. Protein domains were identified by using SMART. In Situ Hybridization. Subtracted library clones were used to generate antisense RNA probes labeled with digoxygenin-UTP. Automated in situ detection was carried out on E8.5, E10.5, and E11.5 outbred mouse embryos by using an InsituPro machine (Abimed, Langenfeld, Germany). Results and Discussion Expression of Sox1GFP. In undifferentiated ES cells in vitro there is no detectable activity of the SoXlGFP allele but expression is specifically activated on induction of neural differentiation as described elsewhere (12, 14~. In viva after germ-line transmis- sion, the GFP reporter is faithfully expressed in the nervous system and lens, with no apparent ectopic expression. SoxlGFP fluorescence is first detected around E8.5 throughout the neural plate and headfolds (ref. 12 and data not shown). This is slightly GenBank accession no. 1.96 CB968098 1.96 CB9681 07 1.95 CB968099 1.94 CB968097 1.81 CB968094 1.76 CB9681 05 1.69 CB9681 03 1.65 CB9681 04 1.63 CB9681 06 1.60 CB9681 00 1.51 CB968095 Gene symbol (synonyms), name Expression CB968102 Nhlh2 (HenZ, Nsc1-2, NSCL2), nescient helix-loop-helix 2 CB968096 Mm.1 56164 CB968093 Khdrbs3 (SLM-2, Et/e, T-STAR), KH domain containing, RNA binding, signal transduction associated 3 Slc2a1 (Glut-1), solute carrier family 2 (facilitated glucose transporter), member 1 CB968101 sFRP2 (Sdf5), secreted frizzled-related sequence protein 2 Lrrn1 (NLRR-1), leucine-rich repeat protein 1, neuronal Sox4, SRY-box-containing gene 4 Zic1, zinc finger protein of the cerebellum 1 Vim, vimentin Rtn1, reticulon 1 Msi2h (msi2), musashi homolog 2* Sox11, SRY-box-containing gene 11* Qk, Quaking Hrmt113, HMT1 hnRNP methyltransferase-like 3* Tuba 1 (Talpha 1), tubulin or1 Dev NS and PN cerebellum Dev and mature NS, epididymis Ad B and S muscle/testes E10 NT, OV, heart and gut E neuroepithelium Dev CNS, DRG and cartilage Dev CNS, thymus, BM E cerebellum and dorsal 1/2 of NT Radial glia in Dev hindbrain Dev and mature NS Dev and mature NS, ubiquitous by NB Dev NS, sites of Ep-Mes interactions Myelin-forming cells Dev and mature NS NPCs Domains HLH KH TM CRD LRR/TM HMG ZnF CC TM RRM HMG KH Data are fold increase in expression, accession number of each clone sequence, gene symbols/names, characterized expression pattern, and protein domain information (given where known). Ad, adult; B. brain; BM, bone marrow; CC, coiled coil; CRD, cysteine-rich domain; Dev, developing; DRG, dorsal root ganglia; E, embryo; Ep, epithelial; HLH, helix-loop-helix; HMG, high-mobility group; KH, K homology RNA-binding domain; LRR, leucine-rich repeat; M, muscle; Mes, mesenchymal; NB, Northern blot; N PCs, neural precursor cells; NS, nervous system; NT, neural tube; OV, optic vesicle; PN, postnatal; RRM, RNA recognition motif; S. skeletal; TM, transmembrane; ZnF, zinc finger. *The predicted transcripts from these genes are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank databases under the accession numbers TPA: BK001349, BK001483, and BK001484. 11838 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 1734197100 ~ ~ ~ G Aubert et a/. ,.

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later than the reported onset of expression of Soxl mRNA and protein (13, 14), presumably because of the time required for correct folding and accumulation of GFP to detectable levels. At E9.5 SoxlGFP is expressed along the entire neuraxis but in no other tissue (Fig. lDi). At mid-gestation, SoxlGFP is maintained throughout the brain and the neural tube but is excluded from the roofplate and floorplate (Fig. lDii). At this stage, SoxlGFP expression also becomes evident in the lens where Soxl has been shown to regulate the ~y-crystallin genes and to be necessary for lens fiber cell elongation (214. The distribution of SoxlGFP is in agreement with the published expression of Soxl mRNA and protein (14~. In later stages of embryonic development, SoxlGFP is excluded from most differentiated neurons and glia but is maintained in the proliferative ventricular zone and in the lens (Fig. lDiii). In the brains of adult animals SoxlGFP is prominently ex- pressed in the subgranular layer of the dentate gyrus (Fig. lDiv). Numerous GFP-expressing cells are seen in the inner subgranu- lar layer, the area where adult neural progenitors have been shown to reside (22~. Neurons born in this area subsequently migrate through the granular layer of the dentate gyrus. At higher magnification, smaller numbers of GFP-positive cells can be observed in the granular layer in SoxlGFP mice. Heterozygous SoxlGFP animals are viable and apparently healthy with no obvious phenotype. Homozygous null SoxlGFP mice have small eyes with opaque lenses and suffer from spontaneous sei- zures, as described for Soxl mutants (21~. FACS Purification and RNA Probe Generation. E10.5 SoXlGFP em- bryos were trypsinised, and the resultant pool of cells was sorted by flow cytometry based on GFP expression. Cell sorting yielded a SoxlGFP+ neural precursor population (gate R1) and a SoxlGFP- control population (gate R2) (Fig. 2A). RNA was extracted from the Soxl+ and Soxl- cells. To confirm the identity of the two RNA populations, cDNA was produced by reverse transcription and analyzed by RT-PCR. We examined the presence in both populations of a number of known genes whose expression pattern is both spatially and temporally de- fined during development (Fig. 2B). As expected, Soxl mRNA was restricted to SoxlGFP+ cells. Genes known to be restricted to neural progenitors populations such as Ngn2 and Pax6 (23) displayed a similar restriction to the GFP+ population. Pax7, Nestin, and sFRP2 all show strong expression in the GFP+ population but are also represented in the GFP- fraction. This is in agreement with a predominant expression of these genes in the developing CNS with additional expression in the somitic mesoderm (Nestin and Pax7) and in the mesonephros (sFRP2) (23-25~. RT-PCR analysis revealed weak expression of the ShcC gene in the Sox1 + cell population. ShcC is an adapter protein that is predominantly expressed in mature neurons (26, 273. As neurogenesis in the neural tube begins at E9.5, the expression seen here could be indicative of a small number of early neurons present in the Soxl+ cell population. This could arise from perdurance of GFP after differentiation and Soxl down-regulation. RNAs for bone morphogenic protein 4 (BMP4), a marker for early mesodermal differentiation (28), and SoxlO, a key regu- lator in the differentiation of peripheral glial cells, with high expression in neural crest cells and cells of the melanocyte lineage, were preferentially expressed in SoxlGFP- cells. To- gether, these results confirm the efficient separation of neural and nonneural cell populations by flow cytometry. P311 and Riken cDNA2810027019 represent two genes that are induced in retinoic acid treated embryoid bodies (74. These genes show similar expression levels in Soxl+ and Soxl- cell popula- tions. This result is consistent with the expression profile revealed by in situ hybridization analyses. Both mRNAs are abundant in the neural tube, but P311 is also found in the somites and apical Aubert et a/. ectodermal ridge (7) and Riken cDNA 2810027019 mRNA is present in migrating neural crest cells, the apical ectodermal ridge, and in condensing mesenchymal cells (data not shown). These two examples illustrate a limitation in the selectivity of screens based on total cell populations from retinoic acid-induced differentiating embryoid bodies (see below). Microarray Analysis. We have previously generated a subtracted library enriched for genes induced during retinoic acid-induced differentiation of ES cells (74. Analysis by differential filter hybridization indicated substantial enrichment for genes of interest. Of 480 clones, 138 (29%) were preferentially expressed during retinoic acid-mediated differentiation. These corre- sponded to 96 unique genes, 40% of which showed enriched expression in the developing or adult CNS (7~. However, most of these clones also showed appreciable expression in nonneural tissues of the developing fetus as exemplified by P311 and Riken cDNA 2810027019. We reasoned that a more stringent screen of the SSH library would be to perform the differential hybrid- ization with SoxlGFP-purified cell populations from embryos rather than whole cell populations from embryoid bodies. To test this idea, we generated a cDNA microarray from 384 randomly picked clones from the SSH library and carried out a differential hybridization screen with RNAs generated from the SoxlGFP embryos. Also included on the array were a number of the marker genes used for characterization of the Soxl GFP RNA isolated from embryos (Fig. 2~. The Soxl + and Soxl - RNA samples were labeled and hybridized to the array. The majority of the marker genes shown to be preferentially expressed in Soxl+ RNA by RT-PCR analysis (e.g., Soxl, Ngn2, Nestin, and Pax6; Fig. 2B) showed expression fold changes of >1.5 when analyzed by microarray analysis. The expression of SoxlO, P311, and the motor neuron precursor marker Isletl was not elevated in the Soxl+ popula- tion, consistent with additional sites of expression in ventral mesoderm and endoderm (29~. The expression of these marker genes determined by the microarray screen is in a broad agree- ment with the RT-PCR profiles of the sorted RNA populations and known expression within and outwith the embryonic CNS. Clone Identification. Fifteen clones that demonstrated the highest differential Soxl+/Soxl- expression ratios (>1.5) were taken for sequencing. Fourteen sequences correspond to known genes, all of which had previously been associated with either devel- oping or adult CNS (Table 1~. Clones PSD11 (Msi2h) and P5D5 (Soxl l ~ do not match known genes directly but are derived from extended 3' UTR regions that lie downstream of the current gene annotation. In each case, the sequence can be linked to the identified gene via a contiguous assembly of expressed se- quences. The discovery of additional 3' UTR sequences is consistent with the fact that a Musashi2 (Msi2h) 3' UTR probe detects a 7.1-kb transcript and suggests that the original 2.3-kb Msi2h cDNA sequence (AB056103) is incomplete (30~. We also noted that the first 513 base pairs of the original Msi2h cDNA sequence do not align with other Msi2h ESTs or the human MSI2 sequence. In fact, this presumed 5' UTR sequence has a 100% match with a genomic sequence on mouse chromosome 1 suggesting that the original cDNA is a hybrid of two clones. Msi2h is thought to play a role in the maintenance and prolif- eration of CNS precursor cells (30, 31~. The relationship between clone P5D5 and Soxl l was detected by combining sequences from two unigene clusters Mm.41704 and Mm.254253 into a single contiguous sequence of ~7 kb. This relatively long predicted transcript is consistent with human SOX11, which has a 3' UTR of >8 kb (32~. Interestingly, the screen identified a second SRY-box-containing gene, Sox4, which is expressed in the differentiating subventricular zone progenitors during neurogenesis (33) and has a role during B cell PNAS | September 30, 2003 | vol. 100 | suppl. ~ | 11839

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differentiation and heart development (34). Sox4 and Soxll :~ ~ ~ ~ IRK encode class C Sox proteins, are closely related at the sequence ~~ ~~ ~~ ~ ~ ~ ~ level, and have similar expression pattern. It has been proposed ~ ~~-~ ~ ~~ ~ i`) that they may be functionally redundant during CNS develop- ~ DID 1~1~t '6~ Hrmtll3 (clone P5A9) has not been described in mouse, but is ~ ~ ~~ ~~ ~~ ~~ ~~ ~ ~ Be ~~]~ ~ ~; presumed to be the ortho ague of human hnRNP me by rans- ferase- ke 3 HRMTIL3~-based high identity (>99%) and conserved synteny. The gene encoding P2H5 remains to be identified; this sequence matches a unigene cluster containing ESTs that are derived exclusively from neural tissues and have moderate similarity to Neural Wiskott-Aldrich syndrome pro- tein (N-WASP). The remaining up-regulated genes were identified as being expressed predominantly in developing or adult nervous system. The identification of Reticulon 1, an endoplasmic reticulum protein of unknown function, in this screen is consistent with the fact that this protein is localized in neuroepithelial progenitors at the pallio-subpallial boundary of the developing telencepha- lon (354. The gene for the intermediate filament protein, vimentin, which is expressed in radial glia (36), was up-regulated in the Soxl+ fraction. Radial glia are thought to serve as precursor cells in the developing forebrain (374. The neuronal precursor cell marker tubulin on (38) was also enriched in Soxl + cells. Nescient helix-loop-helix 2 (~Nhlh2) is a basic helix-loop-helix transcription factor that is reported to be transiently expressed in subependymal cells throughout the CNS at mid-gestation and also transiently in the postnatal cerebellum (39, 404. Expression in the Soxl + fraction could reflect the perdurance of GFP or an earlier onset of Nhlh2 expression than previously described. sFRP2, encoding the Wnt antagonist secreted frizzled-related protein-2, is expressed in the embryonic neuroepithelium (25, 414. This gene was also isolated in the previous screen of this library and demonstrated to promote neural differentiation of ES cells (74. Detection of sFRP2 in the microarray screen demonstrates the potential of this approach for identification of functionally significant players in neural differentiation. The KH domain containing, RNA binding, signal transduc- tion-associated 3 (Khdrbs3) gene is a predominantly nuclear RNA-binding protein which heterodimerizes with Sam68 (68- kDa Src substrate associated during mitosis). Khdrbs3 expression has been observed in adult brain and also skeletal muscle (424. Its embryonic expression and functional role have yet to be defined. Expression of Lrrnl (leucine-rich repeat protein 1, neuronal) has been reported in the CNS at E11.5 by Northern-blot, and whole mount in situ hybridization on E13.5 revealed a predom- inant expression in the developing nervous system (434. Al- though its role remains unknown, the ERR domain is proposed to function in cell adhesion and has been implicated in a variety of events in neural development. In Situ Hybridization. We used whole mount in situ hybridization to examine the embryonic expression of two genes emerging from the microarray screen, Lrrnl and Msi2h. Both have been suggested to play significant roles in neural development (Fig. 3~. At E8.5, Msi2h hybridization is readily detected in the hindbrain and the otic vesicle, and is not evident in any other tissue. Lrrnl mRNA is detectable along the entire antero-posterior axis of the neuroectoderm, with additional faint expression in somites. At E10.5, Msi2h expression is maintained in the hindbrain and otic vesicle, but also extends along the neural tube. Hybridization is also apparent in dorsal root ganglia and limb bud (Fig. 3D). Lrrnl mRNA on E10.5 is present in the ventral-most neural tube as well as the hindbrain and the telencephalic vesicle, and is also prominent in the somites. 11840 1 www.pnas.org/cgi/doi/10.1 073/pnas.17341 97100 ;~ ~^ Fig. 3. Expression of Lrrn1 and Musashi2 mRNAs in mouse embryos. (A) Lateral view of E8.5 embryo showing Msi2h hybridization in the hindbrain and the otic vesicle. (B and C) Lateral and dorsal view, respectively, of an E10.5 embryo showing Msi2h hybridization in the neural tube, hindbrain, and otic vesicles. (D) Transverse section at E11.5 showing specific Msi2h hybridization in the ventral half of the neural tube and the dorsal root ganglia. (E) Lateral view of an E8.5 embryo showing Lrrn1 hybridization in the neural tube and weakly in the somites. (F and G) Lateral and dorsal view, respectively, of an E10.5 embryo showing Lrrn1 hybridization in the telencephalic vesicle, the hindbrain, the otic vesicle, and the somites. (H) Transverse section at E10.5 shows Lrrn1 hybridization in the ventral part of the neural tube and the som ites. Conclusion In this study, we have shown that Soxl-~p knock-in mice allow reliable visualization and purification of pan-neural progenitor cells from mid-gestation mouse embryos. Importantly, Soxl is expressed in neuroepithelial cells throughout the entire neuraxis, labeling all categories of regionally specified neural precursor. The particular advantage of Soxl over the other well established pan-neural marker nestin is that there is no detectable expres- sion outwith the CNS during early to mid-fetal development apart from in the well defined structure of the lens. Examination in whole mount embryos shows that the SoxlGFP reporter reproduces faithfully the expression of Soxl. Interestingly, pre- liminary analyses of adult brains have highlighted expression in the subgranular layer of the dentate gyrus (Fig. 1), a region for which there is now overwhelming evidence of the persistence of neural precursor cells (22, 444. Thus, SoxlGFP may be a useful marker of adult neural precursors. Further studies are required to test this directly. In the present study, we used FACS to separate Soxl-GFP- positive and -negative populations from whole E10.5 mouse embryos. Analysis of a panel of markers by RT-PCR yielded expression data consistent with the substantial elimination of nonneural cells from the GFP-positive population and con- versely the absence of neural precursors from the GFP-negative population. We then carried out a pilot microarray screen with the aim of identifying genes specifically expressed both during neural com- mitment of ES cells and in neural progenitor cells in vivo. From 384 arrayed SSH clones, we identified 15 unique clones showing preferential expression in the GFP-positive cell population. Of these, 11 represent known genes previously reported as ex- pressed in embryonic and/or adult neural tissues, particularly in the brain. The remaining genes were ESTs, each of which has originated from libraries derived from neural tissues. We have been able to identify three of the ESTs as corresponding to Musashi2, Soxll, and Hrmtll3. Musashi2 has previously been described as ubiquitously expressed based on Northern analyses Aubert et a/.

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~ al r~ ~ i ~ of adult tissues (30~. However, our in situ hybridization data show that this gene is preferentially expressed in neural tissue in the fetus. It is noteworthy that marker genes with expression in the developing nervous system but substantial additional nonneural expression (i.e., Soxl O. Isletl, P311, and Riken cDNA 2810027019) were not significantly enriched in the Soxl + population. Furthermore, several clones were found to be present at higher levels in the Soxl - RNA population (data not shown). These clones most likely correspond to transcripts expressed in nonneural tissues induced by retinoic acid treatment of embryoid bodies. The fold enrichment value is not an absolute measure of differential expression, and in many cases may be a considerable underestimate of the selectivity of expression caused by the heterogeneity of the Soxl+ population. 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