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Gene |
Clone |
Species |
Stages |
Anatomy |
not
|
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laevis
|
NF stage 10.5
to
NF stage 11
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dorso-lateral marginal zone
,
dorsal marginal zone
,
upper blastopore lip
,
presumptive axial mesoderm
|
not
|
|
laevis
|
NF stage 14
|
pre-chordal neural plate
,
notochord
,
circumblastoporal collar
,
axial mesoderm
,
neural plate
,
[+]
|
foxd4l1.1
|
|
laevis
|
NF stage 10.5
to
NF stage 11
|
dorsal marginal zone
|
foxd4l1.1
|
|
laevis
|
NF stage 14
|
notochord
,
neural plate
,
floor plate
,
neural tube
|
foxa4
|
|
laevis
|
NF stage 10.5
to
NF stage 11
|
dorsal marginal zone
,
blastopore lip
|
foxa4
|
|
laevis
|
NF stage 14
|
notochord
,
neural plate
,
floor plate
,
neural tube
|
foxa1
|
|
laevis
|
NF stage 10.5
to
NF stage 11
|
dorsal marginal zone
,
presumptive axial mesoderm
|
foxa1
|
|
laevis
|
NF stage 14
|
notochord
,
axial mesoderm
,
neural groove
,
neural plate
,
floor plate
,
[+]
|
shh
|
|
laevis
|
NF stage 10.5
to
NF stage 11
|
mesoderm
,
dorsal marginal zone
,
upper blastopore lip
|
shh
|
|
laevis
|
NF stage 16
|
notochord
,
hypochord
,
neural groove
,
floor plate
,
neural tube
|
enc1.2
|
|
laevis
|
NF stage 10.5
to
NF stage 11
|
dorsal marginal zone
,
upper blastopore lip
,
presumptive axial mesoderm
|
enc1.2
|
|
laevis
|
NF stage 14
|
pre-chordal neural plate
,
notochord
,
axial mesoderm
,
floor plate
,
neural tube
|
|
|
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. |
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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. |
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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. |
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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. |
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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. |