Yan B , Neilson KM , Moody SA .

NS23158 NINDS NIH HHS , R01 NS023158-15 NINDS NIH HHS , R01 NS023158-15S1 NINDS NIH HHS , R01 NS023158-16 NINDS NIH HHS , R01 NS023158-17 NINDS NIH HHS , R01 NS023158-18 NINDS NIH HHS , R01 NS023158-19A1 NINDS NIH HHS , R01 NS023158-20 NINDS NIH HHS , R01 NS023158-20S1 NINDS NIH HHS , R01 NS023158-20S2 NINDS NIH HHS , R01 NS023158-21 NINDS NIH HHS , R01 NS023158-22 NINDS NIH HHS , R01 NS023158 NINDS NIH HHS

	Fig. 2. FoxD5 is required for the normal expression of NE genes. (A) Myc-tagged FoxD5 (foxD5-MT) is highly expressed in both the cytoplasm and nucleus (arrowheads); this expression is greatly reduced in the presence of foxD5-MOs (foxMOs). Myc-tagged N-terminal deleted FoxD5 (δN-foxD5) is strongly expressed even in the presence of foxMOs. (B) Control MO (cMO) does not alter the expression of sox2 on the injected side () of the embryo (st14, dorsal view, anterior is down). (C) Co-injection of δNfoxD5 mRNA prevents the foxMOs reduction of sox2 expression (cf. E) on the injected side () of the embryo (st15, same view as in B). (D) The percentage of embryos exhibiting the phenotypes shown in E (reduced expression above x-axis; expanded expression below x-axis) after injection of foxD5-MOs (analyzed at four different stages), cMO (analyzed at st14) or foxD5-MOs + δNfoxD5 mRNA (analyzed at st14). These data indicate that the effects of foxD5-MOs are specific and can be rescued in a high percentage of cases by δNfoxD5. Sample sizes are indicated above each bar. (E) Arrows denote patches of reduced gene expression at the site of foxD5-MOs injection for each NE gene at gastrulation (st10.5–12) and neural plate (st13–15) stages. For zic1 and zic3, foxD5-MOs expand their expression domains at neural plate stages (lines denote width of neural plate on injected sides). For gem, sox2, zic2, zic3, Xiro1 and Xiro2: dorsal views with anterior to the bottom; for sox3, anterior view with dorsal to the top; for sox11, soxD, and zic1: gastrula stages are dorsal views (as for gem) and neural plate stages are anterior views (as for sox3); for Xiro3: st10.5 is vegetal view with dorsal to the top and st13 is dorsal view (as for Xiro1, 2).
	Fig. 3. foxD5 mRNA injected into D1.1 lineages affects all 11 NE genes. (A) foxD5-expressing cells (red nuclei) showed increased expression of gem at both gastrulation (left) and neural plate (right) stages. For gastrula, small arrow denotes blastopore lip and inset shows cells at a higher magnification. Dorsal views with anterior to the bottom. sox2 (B) and sox3 (C) were weakly reduced (small arrows) compared to adjacent NE cells () at gastrula stages (dorsal views with anterior to the bottom). This effect waned by neural plate stages, when their expression domains were expanded on the injected side (black lines); anterior views with dorsal to the top. (D) foxD5-expressing cells showed reduced sox11 expression (small arrows) compared to adjacent NE cells () at early gastrula (dorsal view with anterior to the bottom), but later (by st12) showed highly increased expression in both the neural plate (above white arrows) and in the adjacent ectoderm (red asterisk). Black bar indicates the expanded sox11 domain on the injected side. Anterior view with dorsal to the top. (E) soxD expression was unaffected at early gastrulation (dorsal view with anterior to the bottom, and top inset), began to be reduced (small arrows) compared to adjacent NE cells () by stage 12 (bottom inset). The reduced phenotype was maintained at neural plate (anterior-lateral view) stages; higher magnification shows reduced soxD in foxD5-expressing cells (red nuclei) compared to adjacent NE cells (). For zic2 (G), foxD5-expressing cells (dark blue) showed increased expression at both gastrula (bracket and inset) and neural plate stages (arrow depicts ectopic expression along the midline). Both are dorsal views with anterior to the bottom. zic1 (F) and zic3 (H) showed reduced expression in foxD5-expressing cells (red nuclei) compared to adjacent NE cells () at gastrulation. Gaps in their expression (large arrows) coinciding with foxD5-expressing cells (red nuclei) also were observed at neural plate stages. Xiro1 (I), Xiro2 (J) and Xiro3 (K) were repressed (arrows) at both gastrulation (not shown) and neural plate stages. For A–K, the percentage of embryos affected and sample sizes are indicated. (L) These results were confirmed for several of the NE genes by RT-PCR comparing uninjected (Un) and foxD5-injected ACs. H4 is internal control.
	Fig. 4. foxD5 ectopically induces a subset of NE genes and represses the BMP pathway. (A) Left side: foxD5-expressing cells (arrows, red nuclei) express zic2 (blue cytoplasm) in the st11 ventral epidermis, but do not express sox2; inset shows higher magnification of sox2 to demonstrate lack of blue cytoplasm surrounding red nuclei. Asterisks denote endogenous gene expression on the dorsal sides of the embryos. Right side: high magnifications show strong induction () of gem, zic2 and sox11 in the st11 ventral epidermis; the red nuclei are partially masked by the blue signal in the cytoplasm. A few cells (arrows) weakly express sox3. (B) The percentage of embryos showing ectopic ventral induction of each NE gene (collected at st10.5–13). Sample sizes are above each bar. (C) foxD5 expression in the ventral epidermis at gastrulation (st10–11) reduces the expression of two epidermal genes downstream of BMP signaling. AP2 is expressed throughout the control ectoderm (left: animal view of whole embryo; right: higher magnification). foxD5-expressing cells (red nuclei) show reduced AP2 expression. Asterisk denotes adjacent unaffected cells. Epidermis-specific keratin (epi-keratin) is expressed throughout the ventral ectoderm (left: ventral view with animal hemisphere to the top of whole embryo; right: higher magnification). foxD5-expressing cells (red nuclei) show reduced epi-keratin expression. Asterisk denotes adjacent unaffected cells. The frequencies of the phenotypes and sample sizes are given. (D) Injection of foxD5 mRNA into a neural progenitor cell-autonomously induces szl (arrow) in the neural plate (st13, anterior view). The frequency of the phenotype and sample size are given. (E) Injection of foxD5 mRNA cell-autonomously reduces the number of cells displaying nuclear phospho-SMAD1/5/8 staining (brown nuclei) in the st11 ventral epidermis. Top panel illustrates staining in ventral epidermis of uninjected control embryo. Bottom panel illustrates staining in a region of foxD5-expressing cells stained blue by expression of the cytoplasmic βGal lineage marker (asterisk) and in an adjacent region (right side) that does not contain foxD5-expressing cells. Graph on right indicates the mean number of cells displaying nuclear phospho-SMAD1/5/8 staining in foxD5-expressing ventral epidermis clones compared to the uninjected side (control) of the same embryos (n = 16 per group). Bars indicate s.e.m. and indicates p < 0.0001 (paired t-test).
	Fig. 6. foxD5 regulates transcription by both activation and repression. (A) The percentage of embryos showing either increased (above x-axis) or decreased (below x-axis) expression after injection of wt foxD5, an activating construct (foxD5VP) or a repressing construct (EnRfoxD5); embryos were collected at st10.5–11 (gem, sox2, sox3, sox11, zic1–3) or st12 (soxD, Xiro1–3). Sample sizes for wt foxD5 are in Fig. 3, and for other constructs are above each bar. (B) foxD5VP strongly induced gem in st11 ventral epidermis, whereas EnRfoxD5 induced only a few cells (arrows in both low and high magnification pictures). foxD5VP induced zic2 expression in the st12 neural plate midline (bracket), distant from the endogenous expression () along the lateral neural plate border, whereas EnRfoxD5 induced only a few patches of cells (arrows in both low and high magnification pictures). (C) EnRfoxD5 reduced zic1 expression in the st11 NE (arrow).
	Fig. 7. When foxD5-hGR was activated by Dex in the presence of Chx (+ Chx/+ Dex), the cells expressed high levels of gem, sox11 or zic2 (arrows), indicating that protein synthesis is not required. This induction did not occur in the absence of Dex (+ Chx/− Dex) (arrows, red nuclei). Dex treatment alone (− Chx/+ Dex) induced each gene (arrows) either above their endogenous levels of gene expression (, gem) or outside the normal endogenous domains of gene expression (, sox11, zic2). The frequencies of the phenotypes and sample sizes are indicated below each example. Embryos were fixed when siblings reached st12.
	Fig. 8. In combination, gem, sox11 and zic2 account for the foxD5 effects. (A) The percentage of embryos in which NE genes showed increased (above x-axis) or decreased (below x-axis) expression after injection of wt foxD5, n-gem, sox11 or zic2 (analyzed at st10–11). Sample sizes for n-gem, sox11 and zic2 are above each bar. (B) The percentage of embryos showing the st14 foxD5-MOs phenotype (cf. Fig. 2) after injection of foxD5-MOs alone or in combination with n-gem, sox11 or zic2 mRNAs. Asterisks denote significant differences (i.e., rescue of the phenotype) compared to foxD5-MOs alone (p < 0.05, Chi-squared test). Sample sizes for rescue experiments are above each bar. (C) Injection of n-gem mRNA induced midline expression (arrows) of both sox11 and zic2 in the st13 neural plate (dorsal views, anterior to the bottom). The asterisks on the zic2 sample denote endogenous expression domains; the sox11 sample was developed only until the induced expression was detected, so endogenous levels are not visible. (D) Co-expression of n-gem rescues the foxD5-MOs reduction in zic2 on the injected side (arrow, anterior view at st12, dorsal to the top) but does not rescue the effect on sox2 (arrow, dorsal view at st11, anterior to top). cf. Fig. 2E.
	Fig. 10. Effects of NE genes on foxD5 expression at gastrulation (st10.5 unless otherwise noted). (A) Cells (red nuclei) expressing n-gem do not alter foxD5 expression (blue); black arrow points to blastopore lip. Cells expressing (B) sox11 or (F) zic2 show weak repression of foxD5 (clear area surrounding red nuclei noted by small red arrows) in a low percentage of embryos; cells expressing zic1 usually do not show altered foxD5 expression (E), but in a few cases (12%) there were phenotypes similar to that shown for zic2 (F). Cells expressing (C) sox2 or (D) sox3 show weak repression of foxD5 (small red arrows) in a majority of embryos. Cells expressing (G) zic3, (H) soxD, (I) Xiro1 or (L) Xiro3 show strong repression of foxD5 (clear area surrounding red nuclei). Cells expressing Xiro2 rarely showed repression at stage 10.5 (J) but repression is strong by stage 12 (K). Only sox3 and Xiro3 induce ectopic foxD5 expression (arrows) in the ventral epidermis (inserts, blue cytoplasm surrounding the red nuclei). The frequencies of the phenotypes and sample sizes are indicated in each panel.

Aruga, Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation. 2002, Pubmed

Aruga, Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation. 2002, Pubmed
Bani-Yaghoub, Role of Sox2 in the development of the mouse neocortex. 2006, Pubmed
Bellefroid, Xiro3 encodes a Xenopus homolog of the Drosophila Iroquois genes and functions in neural specification. 1998, Pubmed , Xenbase
Bergsland, The establishment of neuronal properties is controlled by Sox4 and Sox11. 2006, Pubmed
Brewster, Gli/Zic factors pattern the neural plate by defining domains of cell differentiation. 1998, Pubmed , Xenbase
Bylund, Vertebrate neurogenesis is counteracted by Sox1-3 activity. 2003, Pubmed
Carlsson, Forkhead transcription factors: key players in development and metabolism. 2002, Pubmed
de Graaf, Hormone-inducible expression of secreted factors in zebrafish embryos. 1998, Pubmed
de la Calle-Mustienes, Xiro homeoproteins coordinate cell cycle exit and primary neuron formation by upregulating neuronal-fate repressors and downregulating the cell-cycle inhibitor XGadd45-gamma. 2002, Pubmed , Xenbase
De Robertis, Dorsal-ventral patterning and neural induction in Xenopus embryos. 2004, Pubmed , Xenbase
Ellis, SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. 2004, Pubmed
Faure, Endogenous patterns of TGFbeta superfamily signaling during early Xenopus development. 2000, Pubmed , Xenbase
Fetka, Neuroectodermal specification and regionalization of the Spemann organizer in Xenopus. 2000, Pubmed , Xenbase
Glavic, Xiro-1 controls mesoderm patterning by repressing bmp-4 expression in the Spemann organizer. 2001, Pubmed , Xenbase
Gomez-Skarmeta, Araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes. 1996, Pubmed
Gómez-Skarmeta, The Wnt-activated Xiro1 gene encodes a repressor that is essential for neural development and downregulates Bmp4. 2001, Pubmed , Xenbase
Gómez-Skarmeta, Xiro, a Xenopus homolog of the Drosophila Iroquois complex genes, controls development at the neural plate. 1998, Pubmed , Xenbase
Graham, SOX2 functions to maintain neural progenitor identity. 2003, Pubmed
Hiraoka, XLS13A and XLS13B: SRY-related genes of Xenopus laevis. 1997, Pubmed , Xenbase
Hollenberg, Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination. 1993, Pubmed
Huang, Asymmetrical blastomere origin and spatial domains of dopamine and neuropeptide Y amacrine subtypes in Xenopus tadpole retina. 1995, Pubmed , Xenbase
Huang, Noggin signaling from Xenopus animal blastomere lineages promotes a neural fate in neighboring vegetal blastomere lineages. 2007, Pubmed , Xenbase
Hutcheson, The bHLH factors Xath5 and XNeuroD can upregulate the expression of XBrn3d, a POU-homeodomain transcription factor. 2001, Pubmed , Xenbase
Hyodo-Miura, Involvement of NLK and Sox11 in neural induction in Xenopus development. 2002, Pubmed , Xenbase
Inoue, Zic1 and Zic3 regulate medial forebrain development through expansion of neuronal progenitors. 2007, Pubmed
Kaestner, The mouse fkh-2 gene. Implications for notochord, foregut, and midbrain regionalization. 1995, Pubmed
Kato, Neuralization of the Xenopus embryo by inhibition of p300/ CREB-binding protein function. 1999, Pubmed , Xenbase
Katoh, Human FOX gene family (Review). 2004, Pubmed
Kishi, Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm. 2000, Pubmed , Xenbase
Kolm, Efficient hormone-inducible protein function in Xenopus laevis. 1995, Pubmed , Xenbase
Kroll, Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation. 1998, Pubmed , Xenbase
Kroll, Geminin in embryonic development: coordinating transcription and the cell cycle during differentiation. 2007, Pubmed , Xenbase
Kuo, Opl: a zinc finger protein that regulates neural determination and patterning in Xenopus. 1998, Pubmed , Xenbase
Kuroda, Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus. 2004, Pubmed , Xenbase
Kurth, Establishment of mesodermal gene expression patterns in early Xenopus embryos: the role of repression. 2005, Pubmed , Xenbase
Lee, Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. 2006, Pubmed , Xenbase
Levine, Proposal of a model of mammalian neural induction. 2007, Pubmed
Levine, Gene regulatory networks for development. 2005, Pubmed
Li, Generation of purified neural precursors from embryonic stem cells by lineage selection. 1998, Pubmed
Luo, The cell-cycle regulator geminin inhibits Hox function through direct and polycomb-mediated interactions. 2004, Pubmed
Mattioni, Regulation of protein activities by fusion to steroid binding domains. 1994, Pubmed
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
Mizuseki, SoxD: an essential mediator of induction of anterior neural tissues in Xenopus embryos. 1998, Pubmed , Xenbase
Moody, Fates of the blastomeres of the 16-cell stage Xenopus embryo. 1987, Pubmed , Xenbase
Moody, Cell lineage analysis in Xenopus embryos. 2000, Pubmed , Xenbase
Moody, Neural induction, neural fate stabilization, and neural stem cells. 2002, Pubmed
Nakata, Xenopus Zic3, a primary regulator both in neural and neural crest development. 1997, Pubmed , Xenbase
Nakata, Xenopus Zic family and its role in neural and neural crest development. 1998, Pubmed , Xenbase
Niehrs, Mesodermal patterning by a gradient of the vertebrate homeobox gene goosecoid. 1994, Pubmed , Xenbase
Papanayotou, A mechanism regulating the onset of Sox2 expression in the embryonic neural plate. 2008, Pubmed
Penzel, Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis. 1997, Pubmed , Xenbase
Pitulescu, The regulation of embryonic patterning and DNA replication by geminin. 2005, Pubmed
Rogers, Sox3 expression is maintained by FGF signaling and restricted to the neural plate by Vent proteins in the Xenopus embryo. 2008, Pubmed , Xenbase
Rogers, Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives. 2009, Pubmed , Xenbase
Rozen, Primer3 on the WWW for general users and for biologist programmers. 2000, Pubmed
Sasai, Identifying the missing links: genes that connect neural induction and primary neurogenesis in vertebrate embryos. 1998, Pubmed
Seo, Geminin's double life: chromatin connections that regulate transcription at the transition from proliferation to differentiation. 2006, Pubmed
Seo, Geminin regulates neuronal differentiation by antagonizing Brg1 activity. 2005, Pubmed , Xenbase
Snape, Transcription factor AP-2 is tissue-specific in Xenopus and is closely related or identical to keratin transcription factor 1 (KTF-1). 1991, Pubmed , Xenbase
Sölter, Characterization of a subfamily of related winged helix genes, XFD-12/12'/12" (XFLIP), during Xenopus embryogenesis. 1999, Pubmed , Xenbase
Spella, Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle. 2007, Pubmed
Sullivan, foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent on the C-terminal domain. 2001, Pubmed , Xenbase
Taylor, Tcf- and Vent-binding sites regulate neural-specific geminin expression in the gastrula embryo. 2006, Pubmed , Xenbase
Tuteja, SnapShot: forkhead transcription factors I. 2007, Pubmed
Uwanogho, Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. 1995, Pubmed
Wang, Sox3 expression identifies neural progenitors in persistent neonatal and adult mouse forebrain germinative zones. 2006, Pubmed
Wegner, From stem cells to neurons and glia: a Soxist's view of neural development. 2005, Pubmed
Wegner, From head to toes: the multiple facets of Sox proteins. 1999, Pubmed
Wijchers, In control of biology: of mice, men and Foxes. 2006, Pubmed
Yaklichkin, Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators. 2007, Pubmed
Yang, Beta-catenin/Tcf-regulated transcription prior to the midblastula transition. 2002, Pubmed , Xenbase
Zappone, Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells. 2000, Pubmed