Martinez-De Luna RI , Ku RY , Lyou Y , Zuber ME .

P40 OD010997 NIH HHS , R01 EY015748 NEI NIH HHS , R01 EY017964 NEI NIH HHS , R01EY015748 NEI NIH HHS , R01EY017964 NEI NIH HHS

	Fig. 1. Maturin predicted protein sequence, conservation and expression during Xenopus laevis embryonic development. (A) Maturin protein sequence alignment. Predicted amino acid sequence of X. laevis Maturin aligned with putative orthologs from other vertebrates. Periods indicate amino acid identity, while gaps (dashes) were inserted to optimize the alignment. The 29-residue Maturin Motif is indicated by a red over-line. Aspartic (D) and glutamic (E) amino acids in the acidic domain are labeled with asterisks. The four positions at which nonconservative changes of highly acidic amino acids have taken place are indicated with a caret (^). (B) Amino acid identity among vertebrate Maturins. Percent identity shared with X. laevis Maturin is shown. Genbank accession numbers are: X. laevis BC045253, H. sapiens NM_152793 (C7orf41), M. musculus BC042507 (2410066E13Rik), G. gallus XM_003640720.1, D. rerio NM_001144806. (C) Detection of maturin and pax6 transcripts using RT-PCR. Gene specific primers were used to detect maturin and pax6 transcripts in total RNA isolated from eggs (E) and whole embryos of the indicated developmental stage. Histone h4 was used as a loading control. (D–K) Maturin expression in developing embryos. Whole mount in situ hybridization was used to detect Xenopus laevis maturin expression. Developmental stage is indicated above each panel. (E) Stage 9 embryo cut open with a razor following whole mount in situ hybridization. An, animal; Veg, vegetal; b, blastocoel; A, anterior; P, posterior; np, neural plate; ef, eye field; nc, neural crest; D, dorsal; V, ventral; sc, spinal cord; ov, optic vesicle; e, eye. Scale bars = 400μm.
	Fig. 2. Maturin expression during neural differentiation. Expression of maturin (A–K), differentiation (A′–K′) and proliferation markers (A″–K″) in frog (A–D″), zebrafish (E–H″) and mouse (I–K″) neural tissues. Expression of maturin (A–K) and tubb2b (A′–D′) was determined using in situ hybridization. Differentiating neurons were identified by GFP immunolabeling to detect elav3l:GFP transgene expression in zebrafish (green in E′–H′) and by acTUBB3b (orange in I′–K′) immunolabeling in mouse. Proliferating neuroblasts were identified by either BrdU in Xenopus (pink in A″–D″) or PCNA immunolabeling in zebrafish and mouse (orange in E″– H″ and green in I″– K″). Nuclei were stained blue with DAPI (A″–D″). Inset in panel D is magnified view of region encompassing the dorsal CMZ. MZ, marginal zone; VZ, ventricular zone; LP, lens placode; gc, ganglion cells; *, retinal periphery; L, lens; G, ganglion cell layer; I, inner plexiform layer; P, photoreceptor layer; NE, neuroepithelium; CP, cortical plate; IZ, intermediate zone; DCL, differentiated cell layer; NBL, neuroblastic layer. Scale bars, 50 μm.
	Fig. 3. Maturin knockdown results in neural plate expansion. (A–F″) Design and test of Maturin morpholino activity. (A) Sequence alignment of a and b homeologs of X. laevis maturin showing relative position of the Maturin morpholino MatMO2 (red overline). (B) Schematic of the Mat target-YFP reporter construct used to test morpholino activity. (C) Western blots were used to detect the expression of YFP and β-actin (loading control) in extracts prepared from embryos injected with the indicated morpholino, and cRNA coding for YFP or Mat target-YFP. (D–F″) Brightfield (D, E and F), mCherry fluorescent (D′, E′ and F′) and YFP fluorescent (D″, E″ and F″) images of stage 15 embryos unilaterally injected with cRNA for mCherry and Mat target-YFP alone (D – D″), with CoMO (E–E″), or MatMO2 (F–F″). (G– M) Neural plate expansion following Maturin knockdown. The extent of neural plate expansion was determined by comparing the distance between the embryonic midline (white dashed line) and outer edge of the neural ridge on the control (uninjected) and injected side of embryos injected with CoMO (G–I) or MatMO2 (J–L). In situ hybridization for sox2 (H, K) and rax (I, L) was used to more precisely quantitate the extent of neural plate expansion (M). Graph shows the size of the sox2 and rax expression domains in the uninjected and injected side of either CoMO or MatMO2 embryos. Error bars show the s.e.m. Asterisks indicate P-values calculated using a one-way ANOVA analysis (ns P>0.05; *** P<0.0001). Right side (viewer’s perspective) of all embryos is the injected side. Scale bars, 400μm.
	Fig. 4. Maturin knockdown increases neural cell number without reducing cell death or inducing neural cell fate. (A–C) Neural plate cell density in MatMO2-injected embryos. (A) Diagram illustrating the predicted effect on cell density if a change in cell size is responsible for neural plate expansion. (B) The number of nuclei (Blue - DAPI) in a 100 μm by 200 μm region on either side of the midline of embryos unilaterally injected with MatMO2 and GFP cRNA (green) was determined. Inset shows how the embryos were positioned for sectioning. (C) Average number of nuclei observed in 0.02 mm2 area. (D, E) Neural plate cell number in MatMO2-injected embryos. (D) Neural plate sections were stained for sox2 expression (red) and DAPI (blue). Dashed line indicates embryonic midline, and the lateral extent of sox2 expression is indicated by the arrowheads. Inset shows how the embryos were positioned for sectioning. (E) Total number of cells (nuclei) within the sox2 expression domain. (F–H) TUNEL staining was used to detect cell death in CoMO- (F) and MatMO2- injected embryos (G). Insets in F and G show the location of the 0.138 mm2 area on the control and injected side of each embryo scored to generate results (H). (I) RT-PCR for ncam1, tubb2b, actc1 (actin, alpha cardiac muscle 1) and H4 on animal cap explants from embryos injected into both blastomere at the 2-cell stage with CoMO, MatMO2, noggin or neurog2. Actc1 was used to confirm neural induction resulting from nog and neurog2 injection was direct (not via mesoderm induction) (Hemmati-Brivanlou and Melton, 1994). H4 was used as a loading control. Controls included RNA isolated from whole embryos and processed without (WE-RT) and with (WE) reverse transcriptase, as well as uninjected animal cap explants. Similar results were obtained from two independent experiments and when explants were cultured to the equivalent of stg 22. (J–L) Sox2 in situ hybridization on Martinez-De Luna et al. Page 21 embryos injected into one ventral blastomere at the 8-cell stage with noggin (J), CoMO (K) or MatMO2 (L). The injected side is on the right (viewer’s perspective). In the graphs the error bars show the s.e.m. P-values obtained using Student’s t-test: ns,not significant; *P=0.0101; ***P<0.0001. Scale bars, (B)=100 μm; (F and J)=400 μm.
	Fig. 5. Maturin knockdown inhibits differentiation of neural progenitors. (A–C) Whole mount in situ hybridization for tubb2b following Maturin knockdown. tubb2b expression in CoMO injected (A), uninjected (B), or MatMO2 injected embryos (C). A similar effect on tubb2b expression was also observed with MatMO1 (Fig. S1G). (D–F) Proliferation in Morpholinoinjected embryos. Proliferating cells were labeled using pHH3 whole mount immunostaining in CoMO (D) and MatMO2 injected embryos (E). Insets in D and E show the 0.138 mm2 area on the control and injected side of each embryo in which pHH3-positive cells were counted to generate the results (F). (G–I) Whole mount in situ hybridization for tubb2b after maturin overexpression. tubb2b expression in β-Gal (G) and maturin cRNA (H) injected embryos. In all panels of this figure, the injected side is to the right (viewer’s perspective). The s.e.m. is shown. P-values obtained using Student’s t-test: **P=0.0023. Scale bars, 400μm.
	Fig. 6. Maturin transcription is induced by, and Maturin function is required for the activity of, the proneural transcription factors. (A–D′) Anterior (A–D) and lateral (A′–D′) views of maturin expression in embryos misexpressing YFP-only (A, A′), neurog2 (B, B′), neurod1 (C, C′), and ebf3 (D, D′), respectively. Arrows show areas normally lacking maturin (A, A′), express ectopic maturin when proneural transcription factors are misexpressed (B–D′). (E–M) One blastomere of 2-cell staged embryos were injected with the indicated proneural transcription factor alone (E, H and K), with control (F, I and L) or Maturin (G, J and M) morpholinos. The effects on tubb2b expression were detected by whole mount in situ hybridization at stage 15. Right side (viewer’s perspective) is the injected side. Scale bar, 400μm.
	Fig. 7. Maturin and Pak3 synergy. (A–K) Embryos were injected unilaterally into one blastomere at the 2-cell stage with the indicated morpholino(s) and/or cRNA(s). At stage 15 in situ hybridization was used to determine the effect on tubb2b expression. (L, M) Graphs illustrate the percent of embryos with additional tubb2b-positive cells in the lateral stripe, when injected with sub-maximal amounts of maturin, myrPak3, pak3 or the indicated combination of cRNAs. Scale bars, 400μm. Injected side in all embryos is right side (viewer’s perspective).
	Fig. 8. Proposed gene network illustrating the most likely position of Maturin in the proneural pathway. Maturin and Pak3 function together downstream of the proneural transcription factors to promote differentiation of the primary neurons. Our results are consistent with models in which Maturin and Pak3 form a complex (A), or function independently (B). In both models, Maturin and Pak3 are both required for normal primary neurogenesis.
	mturn (maturin, neural progenitor differentiation regulator homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, horizontal view, animal up.
	mturn (maturin, neural progenitor differentiation regulator homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
	mturn (maturin, neural progenitor differentiation regulator homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
	mturn (maturin, neural progenitor differentiation regulator homolog) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior right, dorsal up.

Agathocleous, From progenitors to differentiated cells in the vertebrate retina. 2009, Pubmed

Agathocleous, From progenitors to differentiated cells in the vertebrate retina. 2009, Pubmed
Andreazzoli, Xrx1 controls proliferation and neurogenesis in Xenopus anterior neural plate. 2003, Pubmed , Xenbase
Arias-Romero, A tale of two Paks. 2008, Pubmed
Bokoch, Biology of the p21-activated kinases. 2003, Pubmed
Chalmers, Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation. 2002, Pubmed , Xenbase
Chalmers, Oriented cell divisions asymmetrically segregate aPKC and generate cell fate diversity in the early Xenopus embryo. 2003, Pubmed , Xenbase
Chitnis, Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. 1995, Pubmed , Xenbase
Dehay, Cell-cycle control and cortical development. 2007, Pubmed
Donovan, Regulation of proliferation during central nervous system development. 2005, Pubmed
Dyer, Regulating proliferation during retinal development. 2001, Pubmed
Galisteo, The adaptor protein Nck links receptor tyrosine kinases with the serine-threonine kinase Pak1. 1996, Pubmed
Good, The sequence of a nervous system-specific, class II beta-tubulin gene from Xenopus laevis. 1989, Pubmed , Xenbase
Guillemot, Cell fate specification in the mammalian telencephalon. 2007, Pubmed
Gururaj, p21-activated kinase signaling in breast cancer. 2005, Pubmed
Hartenstein, Early neurogenesis in Xenopus: the spatio-temporal pattern of proliferation and cell lineages in the embryonic spinal cord. 1989, Pubmed , Xenbase
Hemmati-Brivanlou, Inhibition of activin receptor signaling promotes neuralization in Xenopus. 1994, Pubmed , Xenbase
Hensey, Programmed cell death during Xenopus development: a spatio-temporal analysis. 1998, Pubmed , Xenbase
Hindley, Co-ordination of cell cycle and differentiation in the developing nervous system. 2012, Pubmed
Hing, Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila. 1999, Pubmed
Holt, Cellular determination in the Xenopus retina is independent of lineage and birth date. 1988, Pubmed , Xenbase
Kimmel, Stages of embryonic development of the zebrafish. 1995, Pubmed , Xenbase
King, Phosphorylation site specificity of the Pak-mediated regulation of Raf-1 and cooperativity with Src. 2001, Pubmed
Klisch, Mxi1 is essential for neurogenesis in Xenopus and acts by bridging the pan-neural and proneural genes. 2006, Pubmed , Xenbase
Kreis, PAK signalling in neuronal physiology. 2009, Pubmed
Lee, Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. 1995, Pubmed , Xenbase
Lu, Mechanism of activation of Pak1 kinase by membrane localization. 1999, Pubmed
Ma, Identification of neurogenin, a vertebrate neuronal determination gene. 1996, Pubmed , Xenbase
Marchler-Bauer, CDD: a Conserved Domain Database for the functional annotation of proteins. 2011, Pubmed
Mathers, The Rx homeobox gene is essential for vertebrate eye development. 1997, Pubmed , Xenbase
Mizuseki, Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. 1998, Pubmed , Xenbase
Oschwald, Localization of a nervous system-specific class II beta-tubulin gene in Xenopus laevis embryos by whole-mount in situ hybridization. 1991, Pubmed , Xenbase
Pagni, MyHits: improvements to an interactive resource for analyzing protein sequences. 2007, Pubmed
Park, Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. 2000, Pubmed
Petersen, SignalP 4.0: discriminating signal peptides from transmembrane regions. 2011, Pubmed
Pozzoli, Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus. 2001, Pubmed , Xenbase
Quevillon, InterProScan: protein domains identifier. 2005, Pubmed
Regad, The neural progenitor-specifying activity of FoxG1 is antagonistically regulated by CKI and FGF. 2007, Pubmed , Xenbase
Rennefahrt, Specificity profiling of Pak kinases allows identification of novel phosphorylation sites. 2007, Pubmed
Saka, Spatial and temporal patterns of cell division during early Xenopus embryogenesis. 2001, Pubmed , Xenbase
Salomoni, Cell cycle control of mammalian neural stem cells: putting a speed limit on G1. 2010, Pubmed
Sigrist, PROSITE, a protein domain database for functional characterization and annotation. 2010, Pubmed
Souopgui, XPak3 promotes cell cycle withdrawal during primary neurogenesis in Xenopus laevis. 2002, Pubmed , Xenbase
Viczian, XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina. 2003, Pubmed , Xenbase
Viczian, Expression of Xenopus laevis Lhx2 during eye development and evidence for divergent expression among vertebrates. 2006, Pubmed , Xenbase
Viczian, Generation of functional eyes from pluripotent cells. 2009, Pubmed , Xenbase
Zuber, Giant eyes in Xenopus laevis by overexpression of XOptx2. 1999, Pubmed , Xenbase
Zuber, Specification of the vertebrate eye by a network of eye field transcription factors. 2003, Pubmed , Xenbase