|
Display additional annotations [+]
|
|
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. |
|
Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
not
|
|
laevis
|
NF stage 10.5
to
NF stage 11
|
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
,
[+]
|
ntn1
|
|
laevis
|
NF stage 14
to
NF stage 16
|
pre-chordal neural plate
,
neural plate
,
floor plate
,
neural fold
|
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. |
|
|
|
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. |
|
Display additional annotations [+]
|
|
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. |
|
Display additional annotations [+]
|
|
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. |