Hulstrand AM , Houston DW .

R01 GM083999 NIGMS NIH HHS , R01GM083999 NIGMS NIH HHS

	Fig. 1. Cep4l is vegetally localized in oocytes and expressed in the developing mesoderm and neural crest during development. (A) Diagram of Cep4l protein domains. (B) RT-PCR showing expression of cep4l throughout development compared with the housekeeping gene odc. Stages as determined by Nieuwkoop and Faber. ((C)–(H)) Whole mount in situ hybridization of cep4l expression in oocytes and embryos. (C) Stage VI oocyte, lateral view. Early (D) and late (E) gastrulae, vegetal views. Early (F) and late (G) neurula. (H) Tailbud. Bp, blastopore ((I)–(L)) embryo sections following in situ hybridization. (I) Stage 15 neurula. ((J) and (K)) Stage 25 tailbud. (L) Stage 31 tailbud. All views transverse sections, dorsal to the top.
	Fig. 2. Cep4l misexpression induces ectopic neurogenesis. ((A)–(H)) Cep4l overexpression phenotype. Two-cell embryos were injected with 500 pg cep4l mRNA and incubated to the indicated stage. ((A) and (D)) Stage 12, animal view. ((B) and (E)) Stage 17, dorsal view, anterior to the left. ((C) and (F)) Stage 26, lateral view, anterior to the left. ((G)–(H)) Cep4l and human CDC42EP4 overexpression induces ectopic neurons. Tubb2b expression in (G) uninjected (un), (H) cep4l-injected (500 pg), (I) CDC42EP4-injected (500 pg) embryos. ((J) and (K)) Tubb2b-stained stage 15 embryos were transverse sectioned, dorsal to the top left, ectopic neurons in the deep layer in (K) are indicated with arrows. ((L) and (M)) Zn-12 antibody-stained stage 24 embryos indicate ectopic neurons in cep4l-injected embryos ((M) and (M′)) compared with control ((L) and (L′)). Lateral views, dorsal to the top, ((L′) and (M′)) close-up images of ((L) and (M)) outlined by the boxed regions.
	Fig. 3. Cep4l induces specifically primary sensory neurons. ((A)–(H)) Cep4l overexpression induces ectopic lateral neurons. ((A)–(D)) Unilateral β-gal alone, 50 pg (red). ((E)–(H)) β-gal+250 pg cep4l. Neuronal markers are pan-neuronal tubb2b ((A) and (E)), Rohon–Beard sensory runx1 ((B) and (F)), sensory and motor islet1 ((C) and (G)), interneuron pax2 ((D) and (H)). Brackets in ((E)–(G)) indicate unilaterally expanded lateral neuron region in cep4l-injected embryos. (M) Quantification of the mean difference in marker positive neuron populations, injected–uninjected sides. Error bars indicate 95% CI.
	Fig. 4. Cep4l is required for neurogenesis. (A) Cep4l morpholino design and cep4l constructs. (B) Cep4l MO blocks translation of cep4l mRNA. Immunoblotting against HA in embryo lysates with the indicated constructs and MO doses. ☐-catenin was used as a loading control. ((C)–(T)) Cep4l depletion inhibits endogenous neurogenesis. ((C) and (D)) Phenotypes of controls and embryos injected with 40 ng Cep4l MO. Lateral views, anterior to the left. ((E)–(F)) Tubb2b expression in Cep4l MO-injected embryos. Dorsal (top) and lateral (bottom) views, anterior to the left. ((G)–(L)) Dorsal and ventral Cep4l depletion differentially affects neurogenesis. (G) Diagram of lineage targeting strategy. Tubb2b ((H), (I), (K)–(M)) or islet1 ((J) and (N)) expression in controls ((H), (I) and (J)), and in embryos injected with 8 ng Cep4l MO into the right dorsal animal (DA) (K) or ventral animal (VA) ((L), (M) and (N)) cells. Arrow in (K) marks depleted brain and placode neurons. Brackets in ((M) and (N)) mark depleted sensory neurons, small arrows mark unaffected areas. ((O)–(V)) cep4l rescue injections. Embryos were injected at two cells with 50 pg β-gal mRNA alone ((O) and (S)) or with a sub-phenotypic dose (125 pg) of cep4l ((R) and (V)), and subsequently at eight cells with 8 ng Cep4l MO into the right VA blastomere ((P), (T), (Q) and (U)). Bracket in ((P) and (T)) marks depleted sensory neurons. Panels ((S)–(V)) are closeup images of those above. Arrowheads in ((Q) and (U)) marks rescued sensory neurons. m.n., motorneurons, s.n., sensory neurons.
	Fig. 5. Cep4l binds Cdc42 through the CRIB domain to induce neurons. (A) Co-immunoprecipitation of in vitro translated Cep4l-HA with active His-tagged Cdc42 and Rac1. ((B)–(F)) The Cdc42 binding (CRIB) domain is required for induction of ectopic neurons by Cep4l. (B) Immunoblot of embryos injected with cep4l or cep4l H31A,H34A CRIB domain mutant. (C) Co-immunoprecipitation of Cep4l-HA or mutant Cep4l H31A,H34A-HA with FLAG-Cdc42 in embryo lysates. Tubb2b expression at stage 18, uninjected control (D), cep4l (E), cep4l H31A,H34A (500 pg each) (F). ((G)–(L)) Activated Cdc42 functionally interacts with Cep4l. Tubb2b expression in embryos unilaterally injected with 125 pg cep4l ((J)–(L)) and 50 pg cdc42 ((H) and (K)) or cdc42-G12V ((I) and (L)). Arrow indicates ectopic neurons on the injected side in (L).
	Fig. 6. Cdc42 is required for neurogenesis. (A) Cdc42MO depletes Cdc42 protein. Immunoblot of GFP-tagged Cdc42 in embryos injected with 500 pg cdc42-GFP alone or with 60 ng cdc42 MO. ((B)–(E)) Cdc42 is required for neurogenesis. ((B) and (C)) Tubb2b expression in controls (B) and in embryos unilaterally injected with 60 ng cdc42 MO (C). ((D) and (E)) Quantification of tubb2b+ lateral neurons in control and cdc42 MO-injected embryos. (D) Mean number of neurons in uninjected and injected sides and (E) mean difference injected–uninjected sides. Error bars indicate 95% CI, ⁎⁎=p-value<0.001.
	Fig. 7. Cep4l and Fgf8a expand neurons and inhibit non-neuronal deep cell fates. ((A)–(E)) Expression of neuronal tubb2b (A), neural plate sox2 (B), neural crest sox9 (C), and superficial epidermal xk81a1 ((D) and (E) in embryos injected with 200 pg β-gal and 250 pg cep4l. ((F)–(I)) Cep4l misexpression in animal caps. Tubb2b expression in controls ((F) and (G)) or cep4l-injected ((H) and (I)) embryos and explants. ((K)–(P)) Expression of epidermal deep layer multiciliated cell marker crcc ((K)–(M)) and ionocyte marker foxi1 ((N)–(P)) in controls ((K) and (N)), cep4l-injected ((L) and (O)), and fgf8a-injected embryos ((M) and (P)). Insets in (N)–(P) show expression of tubb2b.
	Fig. 8. Cep4l interacts with and is required for Fgf8a in neuronal induction. ((A)–(H)) Cep4l functionally interacts with Fgf8a. Tubb2b expression in embryos overexpressing ((A)–(D)) or depleted of either Cep4l or Fgf8a ((E)–(F)). (A) BSA treated control, (B) cep4l-injected (125 pg), (C) FGF8a protein treated (0.5 ng), and (D) both cep4l and FGF8a-injected embryos. (E) Uninjected control, (F) cep4l MO-injected (8 ng VA), (G) fgf8a MO-injected (10 ng ventral blastomeres at 4 cell stage), and (H) both cep4l and fgf8a MOs. ((I)–(L)) Cep4l depletion blocks Fgf8a-induced ectopic neurons. Two-cell embryos injected unilaterally with 100 pg β-gal mRNA alone (I) or with 125 pg cep4l (J), 125 pg fgf8a ((K) and (L)) mRNA and subsequently with 8 ng cep4l MO in the right VA blastomere ((I), (J) and (L)).
	Fig. 9. Fgf8a acts independently of MAPK activation and regulates association of Cep4l with Cdc42. (A) Immunoblotting against diphospho-ERK in animal caps, isolated 60 min post dissection. Embyros were injected with 500 pg of the indicated RNAs. WE=whole embryo, stage 9; An=uninjected animal caps. ((B)–(E)) Immunostaining against dp-ERK in control BSA treated (B), cep4l-injected (500 pg) (C), FGF8a (1.5 ng) treated (D), FGF8b (1.5 ng) treated (E) gastrulae. Arrow indicates activated ERK staining. Animal views. Insets: vegetal views showing internal control staining of dp-ERK in the marginal zone. ((F) and (H)) Immunoblots of FLAG immune complexes from embryos co-injected at two cells with 500 pg cep4l-HA and FLAG-cdc42 RNA. (F) Co-immunoprecipitation of Cep4l-HA with FLAG-Cdc42 in embryos injected at stage 9 with BSA or 1 ng human FGF8a or Fgf8b protein into the blastocoel. (G) Relative quantification of HA pulldown normalized to FLAG pulldown and lysate levels of expression, using ImageJ. (H) Co-immunoprecipitation of Cep4l-HA with FLAG-Cdc42 in embryos co-injected at four cells with Fgf8a MO.
	Supplementary Fig. 2. Cep4l depletion affects neuronal, not neural or neural crest fate (A-D) Cep4l depletion does not inhibit neural or neural crest fates. Eight-cell embryos were injected with 8 ng Cep4l MO into the right VA blastomere and stained using in situ hybridization at stage 15. Neural pax6 (A, B) and neural crest snai1 markers (C, D). Dorsal views, anterior to the top.
	Supplementary Fig. 3. Cep4l functions independently of cell division (A-F) Cep4l does not depend on cell proliferation to induce ectopic neurons. Control (A, D) or cep4l-injected embryos (B, C; E, F) were cultured in normal MMR (A-C) or hydroxyurea-aphidicolin (HUA) (D-F) from stage 10.5 and stained for tubb2b at stage 20. Lateral views, anterior to the left. (E-F) Immunostaining for mitotic cells in untreated and HUA-treated embryos with anti-phospho-histone H3 (C, F). (G) Quantitative RT-PCR analysis of tubb2b, pax6, nrp1, neurod and neurog2 expression in cep4l-injected (500 pg) and noggin-injected (200 pg) animal caps. WE, whole embryo.
	Supplementary Fig. 3. Cep4l functions independently of cell division (A-F) Cep4l does not depend on cell proliferation to induce ectopic neurons. Control (A, D) or cep4l-injected embryos (B, C; E, F) were cultured in normal MMR (A-C) or hydroxyurea-aphidicolin (HUA) (D-F) from stage 10.5 and stained for tubb2b at stage 20. Lateral views, anterior to the left. (E-F) Immunostaining for mitotic cells in untreated and HUA-treated embryos with anti-phospho-histone H3 (C, F). (G) Quantitative RT-PCR analysis of tubb2b, pax6, nrp1, neurod and neurog2 expression in cep4l-injected (500 pg) and noggin-injected (200 pg) animal caps. WE, whole embryo.
	Supplementary Fig. 5. Cep4l and Cdc42 depletion block Fgf8a neurogenic activity (A-G) Two-cell embryos were unilaterally injected with 50 pg beta-gal (A-C) alone or (D-F) with 500pg fgf8a mRNA, and subsequently with (B,E) 8 ng cep4l MO or (C,F) 60 ng cdc42 MO VA-targeted, and stained for runx1 using in situ hybridization. Red stain indicates gal expression. Dorsal views, anterior to the top. (G) Quantification of runx1 sensory neurons in each injection condition. Data points indicate mean difference between Injected - Uninjected sides, error bars indicate 95% CI.
	Supplementary Fig. 6. Cep4l depletion does not block Fgf8a neural/neural crest patterning (A-L) Two-cell embryos were unilaterally injected with 50 pg beta-gal alone (A-F) or with 500pg fgf8a mRNA (G-L), and subsequently with 8 ng cep4l MO VA targeted (D-F, J-L), and stained for sox2 (A, D, G, J), hoxB9 and krox20 (B, E, H, K) and snai1 (C, F, I, L). Red stain indicates beta-gal expression. Anterior views, dorsal to the top.
	Supplementary Fig. 7. Low doses of Fgf8b do not phenocopy Fgf8a Blastula stage embryos were injected with BSA (A, B), 1.5 ng FGF8a (C, D), 1.5 ng FGF8b (E, F), 0.3 ng FGF8b (Fgf8b 1:50) (G, H), or 1.5 ng FGF17b (I, J) protein into the blastocoel and assessed for phenotype (top row) and in situ hybridization for tubb2b at stage 18 (bottom row). Dorsal views, anterior to the top.

Ash, Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae. 2003, Pubmed

Ash, Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae. 2003, Pubmed
Bos, GEFs and GAPs: critical elements in the control of small G proteins. 2007, Pubmed
Briscoe, Specification of neuronal fates in the ventral neural tube. 2001, Pubmed
Burbelo, A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. 1995, Pubmed
Cappello, The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface. 2006, Pubmed
Chalmers, Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation. 2002, Pubmed , Xenbase
Chitnis, Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. 1995, Pubmed , Xenbase
Cornell, Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate. 2000, Pubmed
Crossley, The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. 1995, Pubmed
Cuykendall, Identification of germ plasm-associated transcripts by microarray analysis of Xenopus vegetal cortex RNA. 2010, Pubmed , Xenbase
Dailey, Mechanisms underlying differential responses to FGF signaling. 2005, Pubmed
Fletcher, FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus. 2006, Pubmed , Xenbase
Guo, Fgf8b-containing spliceforms, but not Fgf8a, are essential for Fgf8 function during development of the midbrain and cerebellum. 2010, Pubmed
Hardcastle, FGF-8 stimulates neuronal differentiation through FGFR-4a and interferes with mesoderm induction in Xenopus embryos. 2000, Pubmed , Xenbase
Harris, Neuronal determination without cell division in Xenopus embryos. 1991, Pubmed , Xenbase
Hartenstein, Early neurogenesis in Xenopus: the spatio-temporal pattern of proliferation and cell lineages in the embryonic spinal cord. 1989, Pubmed , Xenbase
Hayes, Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development. 2007, Pubmed , Xenbase
Hirsch, A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes. 2001, Pubmed
Hong, Fgf8a induces neural crest indirectly through the activation of Wnt8 in the paraxial mesoderm. 2008, Pubmed , Xenbase
Houston, Maternal Xenopus Zic2 negatively regulates Nodal-related gene expression during anteroposterior patterning. 2005, Pubmed , Xenbase
Joberty, Borg proteins control septin organization and are negatively regulated by Cdc42. 2001, Pubmed
Joberty, The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins. 1999, Pubmed
Keller, The cellular basis of the convergence and extension of the Xenopus neural plate. 1992, Pubmed , Xenbase
Kerr, Maternal Tgif1 regulates nodal gene expression in Xenopus. 2008, Pubmed , Xenbase
Lamborghini, Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation. 1980, Pubmed , Xenbase
Lee, Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. 1997, Pubmed
Lee, Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. 1995, Pubmed , Xenbase
Liu, FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate. 1999, Pubmed
MacArthur, FGF-8 isoforms differ in NIH3T3 cell transforming potential. 1995, Pubmed
Mancilla, Neural crest formation in Xenopus laevis: mechanisms of Xslug induction. 1996, Pubmed , Xenbase
Metcalfe, Primary neurons that express the L2/HNK-1 carbohydrate during early development in the zebrafish. 1990, Pubmed
Moody, Quantitative lineage analysis of the origin of frog primary motor and sensory neurons from cleavage stage blastomeres. 1989, Pubmed , Xenbase
Moury, The origins of neural crest cells in the axolotl. 1990, Pubmed
Olsen, Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain. 2006, Pubmed
Ossipova, PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC. 2007, Pubmed , Xenbase
Park, Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus. 2008, Pubmed , Xenbase
Park, Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus. 2012, Pubmed , Xenbase
Pera, Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. 2003, Pubmed , Xenbase
Rana, Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides. 2006, Pubmed , Xenbase
Ribisi, Ras-mediated FGF signaling is required for the formation of posterior but not anterior neural tissue in Xenopus laevis. 2000, Pubmed , Xenbase
Rossi, Transcriptional control of Rohon-Beard sensory neuron development at the neural plate border. 2009, Pubmed
Rossi, Rohon-Beard sensory neurons are induced by BMP4 expressing non-neural ectoderm in Xenopus laevis. 2008, Pubmed , Xenbase
Rual, Towards a proteome-scale map of the human protein-protein interaction network. 2005, Pubmed
Sabherwal, The apicobasal polarity kinase aPKC functions as a nuclear determinant and regulates cell proliferation and fate during Xenopus primary neurogenesis. 2009, Pubmed , Xenbase
Sato, The Fgf8 signal causes cerebellar differentiation by activating the Ras-ERK signaling pathway. 2004, Pubmed
Sato, Inductive signal and tissue responsiveness defining the tectum and the cerebellum. 2001, Pubmed
Schneider, Differential role of Axin RGS domain function in Wnt signaling during anteroposterior patterning and maternal axis formation. 2012, Pubmed , Xenbase
Selleck, Origins of the avian neural crest: the role of neural plate-epidermal interactions. 1995, Pubmed
Sinha, Cellular signaling for activation of Rho GTPase Cdc42. 2008, Pubmed
Stubbs, Radial intercalation of ciliated cells during Xenopus skin development. 2006, Pubmed , Xenbase
Thompson, Delineation of the Cdc42/Rac-binding domain of p21-activated kinase. 1998, Pubmed
Turner, Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. 1994, Pubmed , Xenbase