Fgf4 maintains Hes7 levels critical for normal somite segmentation clock function
- Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, United States
- Optical Microscopy and Analysis Laboratory, Frederick National Laboratory for Cancer Research, United States
- Institute for Frontier Life and Medical Sciences, Kyoto University, Japan
Figures
Figure 1 with 3 supplements
Fgf4 mutants have defects in vertebral and somite patterning.
(A, A’) Max intensity projection (MIP) of HCR staining of Fgf4 and Fgf8 mRNA expression (A) or Fgf4 only (A’) in the PSM of a five somite stage wildtype embryo (dorsal view). PS: primitive streak, …
Figure 1—figure supplement 1
Mispatterning of rostral somites correct as development proceeds.
Graph depicting the percentage of abnormal somites scored with a mispatterned Uncx mRNA WISH pattern at the somite position listed on the x-axis. Somites 1–6 were scored in somite stage 7 Fgf4 …
Figure 1—figure supplement 2
Mesp2 pattern is normal at 24–26 somite stage in Fgf4 mutants.
(A–B’) HCR staining showing no difference in the patterns of Mesp2 and Tbx6 expression between control and Fgf4 mutant 24–26 somite stage embryos. A and B: MIP, lateral view with anterior to the …
Figure 1—figure supplement 3
Fgf4 expression in mutant and wildtype embryos.
HCR staining of the indicated genes in control and Fgf4 mutant embryos at 6ss (A, A’, B, B’) and somite stage 24 (C, C’, D, D’). The probe (Fgf4rec) hybridizes only to sequences between the loxP …
Figure 2
Fgf4 mutants maintain a normal wavefront.
(A, B) HCR staining of 5–6 somite stage control (A, n = 8) and Fgf4 mutant embryos (B, n = 11). In mutants, Msgn1 expression was similar between mutants and controls, but Mesp2 expression was …
Figure 3
Fgf8 is sufficient for maintaining canonical FGF-responsive gene expression in Fgf4 mutants.
(A, B) HCR staining of 5–6 somite stage control (n = 6) and Fgf4 mutant (n = 3) embryos showing comparable expression of Fgf8, quantified in (C). (C) Quantification of Fgf8 mRNA expression, …
Figure 4 with 1 supplement
Pattern of Notch signaling is abnormal in Fgf4 mutants.
(A) Quantification of immunostained NICD fluorescent signal specifically in the mesoderm of 5–6 somite stage embryos. Note, there is no significant difference between Fgf4 mutants (n = 5) and …
Figure 4—figure supplement 1
Lfng expression is abnormally patterned in Fgf4 mutants.
(A, B) HCR staining of Lfng and Tbx6 expression in control (A) and Fgf4 mutant embryos (B), showing abnormal expression of Lfng in mutants. (A’, B’) Spot models of Lfng expression visualized using …
Figure 5 with 5 supplements
Hes7 expression is abnormally patterned and reduced in Fgf4 mutants.
(A–F) HCR staining for Hes7 expression in 5–6 somite stage control and Fgf4 mutant embryos sorted by phase of oscillation, showing abnormal expression of Hes7 in mutants. (G) Distribution of phases …
Figure 5—figure supplement 1
Hes7 expression is abnormal in Fgf4 mutants.
(A–D) Chromogenic whole mount in situ hybridization of Hes7 mRNA in 5–6 somite stage control and Fgf4 mutant embryos. Development of chromogenic signal was either not saturated (developed for ~1 hr …
Figure 5—figure supplement 2
Using Hes7 null homozygotes to adjust Imaris baseline cutoff.
(A, B) HCR staining of 5–6 somite stage control (n = 3) and Hes7 mutant (n = 3) embryos using probes complimentary to the deleted region of Hes7. Note that the probe does not detect any product …
Figure 5—figure supplement 3
Imaris-modeling HCR Expression.
(A) HCR staining of Hes7 and Tbx6 in a 5–6 somite stage wildtype embryo; dorsal view, anterior left. (B) Imaris-generated surface model of Tbx6 expression domain from A. (C) Imaris-generated spot …
Figure 5—figure supplement 4
Hes7 expression in Fgf4 mutants is reduced, irrespective of oscillation phase.
Quantification of Hes7 expression within the PSM of control and Fgf4 mutants at 5–6 somite stage. Data and embryos are identical to Figure 5H, except that fluorescence intensity quantification …
Figure 5—figure supplement 5
Hes7 expression is normal at 24–26 somite stage in Fgf4 mutants.
(A–F) HCR staining of representative 24–26 somite stage control and Fgf4 mutant embryos showing normal patterns of Hes7 expression for each phase of oscillation. MIP, lateral view with anterior to …
Figure 6
Reduced Hes7 expression with less distinct peaks and troughs in the Fgf4 mutant PSM.
(A, B) Spot models based on HCR analysis of Hes7 expression, in 5–6 somite stage control and Fgf4 mutant embryos within the PSM, as defined by the Tbx6 expression domain. Localized Hes7 expression …
Figure 7 with 1 supplement
Removal of one Hes7 allele exacerbates vertebral defects in Fgf4 mutants.
(A–C) Histograms representing the vertebral column and associated ribs (C = cervical, T = thoracic, L = lumbar), showing variety and location of vertebral defects at E18.5 in the genotypes indicated …
Figure 7—figure supplement 1
Removal of one Hes7 allele has no effect on vertebral defects in Fgf8 mutants.
Histograms representing the vertebral column and associated ribs (C = cervical, L = thoracic, L = lumbar), showing variety and location of vertebral defects at E18.5 in the genotypes indicated (A, n …
Tables
Table 1
Experimental Crosses.
| Experimental cross | Experimental genotype (Freq) | Control genotype (Freq) |
|---|---|---|
| Fgf4flox/flox x TCreTG/TG; Fgf4Δ/wt | TCreTG/0; Fgf4flox/Δ (1/2) | TCreTG/0; Fgf4flox/wt (1/2) |
| Fgf8flox/flox x TCreTG/TG; Fgf8Δ/wt | TCreTG/0; Fgf8flox/Δ (1/2) | TCreTG/0; Fgf8flox/wt (1/2) |
| Hes7Δ/wt x Hes7Δ/wt | Hes7Δ/Δ (1/4) | Hes7wt/wt (1/4) |
| Fgf4flox/flox; Hes7Δ/wt x TCreTG/TG; Fgf4Δ/wt | TCreTG/0; Fgf4flox/Δ; Hes7Δ/wt (1/4) | TCreTG/0; Fgf4flox/wt (1/4) TCreTG/0; Fgf4flox/wt; Hes7Δ/wt (1/4) TCreTG/0; Fgf4flox/Δ (1/4) |
| Fgf8flox/flox; Hes7Δ/wt x TCreTG/TG; Fgf8Δ/wt | TCreTG/0; Fgf8flox/Δ; Hes7Δ/wt (1/4) | TCreTG/0; Fgf8flox/wt (1/4) TCreTG/0; Fgf8flox/wt; Hes7Δ/wt (1/4) TCreTG/0; Fgf8flox/Δ (1/4) |
-
Experimental cross is always written ‘female x male’.
Δ = ‘deletion’ or null.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background Mus musculus | Fgf4Flox/delta | PMID:10802662 | MGI:95518 | |
| Strain, strain background Mus musculus | Fgf8flox/delta | PMID:9462741 | MGI:99604 | |
| Strain, strain background Mus musculus | Hes7delta | PMID:11641270 | MGI:2135679 | |
| Strain, strain background Mus musculus | TCre | PMID:16049111 | MGI:3605847 | |
| Chemical compound, drug | Alcian Blue 8GX | Sigma | A5268-25G | |
| Chemical compound, drug | Alizarin Red | Sigma | A-5533 | |
| Chemical compound, drug | BM Purple | Roche | 11 442 074 001 | |
| Chemical compound, drug | Dapi | Life Technologies | D21490 | |
| Chemical compound, drug | Iohexol (Nycodenz) | Accurate Chemical and Scientific Corp | AN1002424 | |
| Chemical compound, drug | Ultra-low gelling temperature agarose | Sigma | A5030 | |
| Chemical compound, drug | Hydrogen peroxide | Sigma | 516813–500 ML | |
| Chemical compound, drug | Ammonium Chloride | Mallinckrodt | 3384 | |
| Chemical compound, drug | N-Methylacetamide | Sigma | AN1002424 | |
| Chemical compound, drug | Triton-X-100 | Sigma | T8787 | |
| Chemical compound, drug | fluorescein | Sigma | 46960 | |
| Chemical compound, drug | acid blue 9 | TCI | CI42090 | |
| Chemical compound, drug | rose bengal | Sigma | 198250 | |
| Commercial assay or kit | V3.0 HCR Kit | Molecular Instruments | Wholemount Mouse Embryo | |
| Software, algorithm | Imaris Software | Bitplane Inc | Imaris V9.2.1 | |
| Antibody | Anti-NICD (rabbit polyclonal) | CST | #4147 | (1:100) |
| Antibody | Anti-rabbit alexa-647 (goat polyclonal) | Thermo Fisher Scientific | A32733 | (1:100) |
| Antibody | Anti-Dig (sheep FAB fragments) | Roche | 11 093 274 910 | (1:4000) |
| Other | Fetal calf serum | Thermo Fisher Scientific | 16140071 |
