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.

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