Peyrot SM , Wallingford JB , Harland RM .

GM 42341 NIGMS NIH HHS , R01 GM042341-26 NIGMS NIH HHS , R01-GM074104-01 NIGMS NIH HHS , R01 GM042341 NIGMS NIH HHS , R01 GM074104 NIGMS NIH HHS

Xla Wt + Cyclopamine (fig.3.e)
Xla Wt + Cyclopamine (fig.5.h)
Xla Wt + Cyclopamine (fig.6.g)
Xla Wt + Cyclopamine (fig.S2.h)
Xla Wt + Cyclopamine (fig.S3. b)
Xla Wt + Cyclopamine (fig.S3. b^1)
Xla Wt + Cyclopamine (fig.S3. b^1^1)
Xla Wt + Cyclopamine (fig.S4.b)
Xla Wt + Cyclopamine (fig.S4.e)
Xla Wt + shh (fig.3.c)
Xla Wt + shh (fig.5.i)
Xla Wt + shh (fig.6.i)
Xla Wt + shh (fig.S2.d)
Xla Wt + shh (fig.S3.e)
Xla Wt + shh (fig.S3.e')
Xla Wt + shh (fig.S3.e^1)
Xla Wt + shh MO (fig.3.b)
Xla Wt + shh MO (fig.3.e)
Xla Wt + shh MO (fig.5.h)
Xla Wt + shh MO (fig.6.c)
Xla Wt + shh MO (fig.S2.e, i)
Xla Wt + shh MO (fig.S3.c)
Xla Wt + shh MO (fig.S3.c')
Xla Wt + shh MO (fig.S3.c^1)
Xla Wt + shh MO (fig.S3.d)
Xla Wt + shh MO (fig.S3.d')
Xla Wt + shh MO (fig.S3.d^1)
Xla Wt + {ca}notch1 (fig.1.c)
Xla Wt + {ca}notch1 (fig.1.f)
Xla Wt + {ca}notch1 (fig.1.i)
Xla Wt + {ca}notch1 (fig.1.i, l)
Xla Wt + {ca}notch1 (fig.1.l)
Xla Wt + {ca}notch1 (fig.1.o)
Xla Wt + {ca}notch1 (fig.1.o)
Xla Wt + {ca}notch1 (fig.2.c, c^1)
Xla Wt + {ca}notch1 (fig.2.d, d^1)
Xla Wt + {ca}notch1 (fig.2.f)
Xla Wt + {ca}notch1 (fig.2.g, h)
Xla Wt + {ca}notch1 (fig.2.k, l)
Xla Wt + {ca}notch1 (fig.4.i)
Xla Wt + {ca}notch1 (fig.4.i)
Xla Wt + {ca}notch1 (fig.6.b, f)
Xla Wt + {ca}notch1 (fig.7.i, i^1)
Xla Wt + {ca}notch1 (fig.S1.b, c, e, f)
Xla Wt + {ca}notch1 (fig.S1.b, e)
Xla Wt + {ca}notch1 (fig.S1.c, f)
Xla Wt + {dn}rbpj (fig.1.b)
Xla Wt + {dn}rbpj (fig.1.h)
Xla Wt + {dn}rbpj (fig.1.k)
Xla Wt + {dn}rbpj (fig.1.n)
Xla Wt + {dn}rbpj (fig.1.n)
Xla Wt + {dn}rbpj (fig.2.b, b^1)
Xla Wt + {dn}rbpj (fig.6.j)
Xla Wt + {dn}rbpj (fig.7.h, h^1)

	Fig. 2. Perturbation of Notch signaling has variable effects on Shh expression. Shh expression in neurula (st.16, A–H) and tadpole (st.35, I–L) embryos upon blockade (B, F, J) and activation (C, D, E–H, K, L) of Notch signaling. Shh is expressed in the floor plate and notochord (A, A', I, I'). Blockade of Notch signaling led to a slight reduction in the floor plate domain of Shh in 31% of embryos (B, B'), but many embryos showed normal expression (J', 69%). Activated Notch signaling led to increased expression throughout the midline (C, C'), though the floor plate domain was normal (F, K'), in 27% of embryos. In a significant proportion of embryos (69%, p < 0.05), activated Notch led to a decrease in Shh expression throughout the midline (H, L') or in the floor plate domain (G). In NICD tadpoles, Shh expression was decreased and/or patchy in the spinal cord (K, L). The variable response of Shh to Notch activation is not correlated with amount or distribution of NICD protein (E–H, αnti-myc staining for NICD-myc in brown). NICD in the lateral neural plate does not cause ectopic Shh expression (E), while NICD in the FP domain can result in Shh expression (F), or repression (G, H). (A'–D') Transverse sections of (A–D); faint notochord expression of Shh is not visible in thin sections. (E–H) αnti-myc staining (brown) was performed on transversely bisected embryos after ISH for Shh (purple). The notochord is outlined in red and the ventral boundary of the neural plate in blue. (I–L) Lateral view of tailbud stage embryos. (I'–L') Transverse vibratome sections through middle of spinal cord at tailbud stages.
	Fig.3. Shh is not a major regulator of dorsal midline fates. Shh signaling was blocked by injection of Shh MO or by cyclopamine treatment (“−Shh” B, E, G, J, M, P) and activated by injection of Shh mRNA (“+Shh,” C, H, K, N, Q). Netrin expression in the floor plate was decreased in 42% of − Shh embryos (B, N = 387) and slightly upregulated in 44% of + Shh embryos (C, N = 139). Floor plate expression of Shh was slightly narrower in 29% of embryos with blockade of Shh signaling (E inset, N = 194). Perturbation of Shh signaling had no effect on notochord (AxPC, F–H and Tor70, I–Q) or hypochord (O–Q) or floor plate markers F-spondin and FoxA2 in the spinal cord of tadpoles (I–N). (A–H) Dorsal views, anterior up, with transverse bisection in inset (A–E). (G–O) Transverse vibratome sections through middle of spinal cord at tailbud stages.
	Fig.4. Ectopic Shh does not affect notochord formation and Notch signaling acts after specification to repress the notochord fate. Shh mRNA overexpression does not repress notochord specification at stage 10, assayed by chd (B) and Xbra (E), or notochord formation during gastrulation (Xbra, H). Activated Notch signaling does not affect notochord specification (C, F), but potently represses subsequent notochord formation (I). Pink is lacZ staining of co-injected lineage tracer.
	Fig.5. Lateral floor plate is regulated by Shh signaling. Nkx2.2 is a marker of the LFP in zebrafish and chick. At onset in frog (stage 22), it is expressed in two stripes in the ventral neural tube (C) lateral to Shh (A) and Netrin (B). At tadpole stages, Shh (D), Netrin (E), and Nkx2.2 (F) clearly mark distinct domains. LFP formation requires Shh signaling (H) and Shh overexpression can induce ectopic Nkx2.2 throughout the brain and upregulation in the spinal cord (I) at stage 22. (A–F) Transverse vibratome sections following ISH and Tor70 staining of the notochord (brown, D–F). (G–I) Dorsal views, anterior up.
	Fig.6. Shh and Notch signaling act in parallel to specify floor plate. Epistasis (A–L) and enhancement (M–P) experiments to determine the contribution of Notch and Shh signaling to floor plate formation. Embryos were injected and/or treated with reagents listed, fixed at stage 20–22, bisected and stained for co-injected lineage tracer (pink), then processed for Netrin ISH. Activated Notch signaling upregulates Netrin (B, F), while blockade of Shh signaling slightly reduces Netrin (C, G). Activation of Notch and blockade of Shh in the same embryo results in a Notch phenotype (D, H). Shh overexpression leads to a slight increase in FP (I), and blockade of Notch signaling leads to a decrease (J). Activation of Shh and blockade of Notch in the same embryo results in a Notch phenotype (K). Blockade of Notch signaling downstream of NICD with SDBM is able to rescue FP and notochord development to normal (L). Blockade (M, O) or activation (P) of both Notch and Shh signaling in the same embryo gives more severe phenotypes than perturbing either pathway alone.
	Fig.7. Notch signaling does not regulate gastrula midline markers or 1° MFP. Shh and plvs are markers of 1° MFP ( Fig. S6). Blockade (B, E, H) and activation (C, F, I) of Notch signaling does not affect gastrula stage expression of these markers (A–F). Expression of plvs upon perturbation of Notch signaling (G–I) looks much like that of Shh (see Fig. 2), with FP expression only mildly affected (G'–I'). (G'–I') Transverse bisections of G–I.
	Fig. 1. Notch signaling promotes formation of floor plate and hypochord and represses notochord. Notch signaling was repressed by injection of mRNA encoding Su(H) DNA-binding mutant (SDBM, B, E, H, K, N) and activated by Notch intracellular domain mRNA (NICD, C, F, I, L, O). Floor plate development was assayed by expression of Netrin (A–C), F-spondin (G–I), and FoxA2 (J–L). Notochord formation was assayed by Axial protocadherin (AxPC, D–F) and Tor70 staining (brown, G–O). Tor70 staining has high variability and background, and thus should not be considered quantitative. Hypochord development was assayed by F-spondin (G–I, staining ventral to notochord) and VEGF (M–O). (A–F) Dorsal views, anterior up. (G–O) Transverse vibratome sections through middle of spinal cord at tailbud stages.
	Fig. S1. Notch-expressing cells do not contribute to notochord. NICD-myc mRNA was injected with (B, C) or without (E, F) β-galactosidase mRNA as a lineage tracer into both dorsal blastomeres to target the midline. Embryos were fixed, bisected transversely, stained for lacZ (pink, A–C), and processed by ISH for AxPC expression in the notochord (purple, A–F). This injection technique efficiently targets the midline (A), with labeled cells in FP, notochord, dorsal endoderm and sometimes medial somite. Embryos with activated Notch have reduced (B, E) or absent (C, F) notochord. Co-injected lineage tracer never labels any notochord that forms (B). Staining for NICD protein (αnti-myc, brown in E, F) shows that NICD+ cells never give rise to the notochord.
	Fig. S2. Shh MO efficiently and specifically blocks Shh function. (A) Schematic of Shh transcript with exons (blue), introns (Vs), and UTRs (gray) shown. “Shh spliceMO” (red) was designed to overlap the first splice donor site. RT-PCR primers (black) were designed flanking the splice junction to assay for correct splicing. (B) RT-PCR of embryos injected with Shh spliceMO shows dose-dependent reduction of Shh splicing. EF1α serves as a loading control. (C–J) ISH for Hh pathway target genes Patched1/2. Shh mRNA upregulates Ptc (D), while Shh MO reduces Ptc (E). Reduction by Shh MO is specific, as it can be rescued by co-injection of Shh mRNA (F). In a separate round of experiments, cyclopamine treatment shows a similar amount of Ptc reduction (H) to Shh MO (I). Combination of these reagents does not enhance the phenotype (J), indicating that maximal possible knockdown of Shh function is achieved by either Shh MO or cyclopamine alone.
	Fig. S3. Loss of Shh signaling and Hh signaling cause similar developmental defects. (A) Control embryos at stage 42, lateral (A), dorsal, (A') and ventral (A") views. (B) Cylopamine treatment blocks all Hh signaling, resulting in reduced melanocytes, edema, and kinked tail (B), closely spaced eyes (B’), and gut-looping defects (B"). Injection of MOs designed to the translation start site (“Shh ATGMO,” C–C") and splice donor site (“Shh spliceMO,” D–D") show similar phenotypes. (E) Injection of Shh mRNA (pink, lacZ staining) results in excess melanocytes (D, arrow), ventralization of the eye (E, E'), and gut looping defects (E").
	Fig. S4. Perturbation of S/Hh signaling only affects the floor plate of the brain. Lateral views of tailbud stage control (A, D), cylopamine-treated (B, E), and Shh mRNA injected (C, F) embryos, processed by ISH for F-spondin (A–C) and FoxA2 (D–F) and stained for notochord (Tor70, brown, A–C). Loss of Hh signaling results in reduced (E) or broken (B) staining of floor plate markers in the brain in 48% of cyclopamine-treated embryos (N = 149). Shh mRNA does not cause dorsal expansion of floor plate except in the brain (arrows, C and F). Pink in C, F is staining for co-injected lineage tracer.
	Fig. S5. Notch and Shh signaling operate in parallel to specify floor plate, but Shh signaling is not required for Notch repression of notochord. Quantification of results shown in Fig. 6. Embryos were injected and/or treated as indicated. At stage 20–22, embryos were fixed, bisected, stained for lacZ lineage tracer, and assayed for FP by Netrin ISH (A, C) and for notochord by Tor70 staining (B). We scored FP development by the intensity of Netrin staining and notochord development by the size of the Tor70+ structure. Total percentages of embryos of each phenotype (see Fig. 6) are represented by stacked column graphs. N is the number of bisected embryos scored in at least 2 independent experiments. For determination of statistical significance, χ2 tests were performed pairwise between 1) controls and single reagents and 2) double treatments and each single component. * significant difference from control, p < 0.05. # significant difference from NICD/SDBM, p < 0.05. + significant difference from other reagent, p < 0.05.
	Fig. S6. Analysis of floor plate markers reveals two separate populations of medial floor plate. In situ hybridization for indicated markers was performed at stage 10.5–11 (A, C, E, G, I, K, M) and neurula stages (st.14—B, D, F, J, L, N, O; st.16—H, P). Prime panels (B', etc.) show transverse bisections of matching neurula stages (notochord outlined in red). Markers expressed in the organizer (marked by asterisk) at gastrula stages are expressed throughout the dorsal midline and in a narrow domain in the floor plate (A–N') at neurula stages. Netrin expression begins at early neurula stages (st.14, O) in a wider domain than early markers (O', P' compared to B'–N'). We have designated the narrow, early-forming FP the 1° MFP and the wider, later-forming FP the 2° FP.
	enc1.2 (ectodermal-neural cortex (with BTB-like domain), gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.
	enc1.2 (ectodermal-neural cortex (with BTB-like domain), gene 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior up.

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