HoxB-derived hoxba and hoxbb clusters are essential for the anterior–posterior positioning of zebrafish pectoral fins
- Division of Life Science, Graduate School of Science and Engineering, Saitama University, Japan
Figures
Figure 1
Lack of pectoral fins in hoxba;hoxbb cluster-deleted mutants.
(A–G) Dorsal views of live zebrafish larvae at 3 dpf, obtained from intercrosses between hoxba;hoxbb cluster-deficient hemizygous fish. Arrowheads indicate the positions of the pectoral fins. (H–L) Expression patterns of tbx5a in the pectoral fin bud (arrowhead) at 30 hpf. Dorsal views are shown, and the genotype of each specimen was determined. For each genotype, reproducibility was confirmed with at least three different specimens. Genotyping revealed that all the embryos lacking pectoral fins (n = 15) were hoxba;hoxbb double homozygotes. Scale bars are 100 µm.
Figure 2
Significantly decreased expression of tbx5a in the pectoral fin buds is specific to hoxba;hoxbb cluster-deleted mutants.
(A–I) Expression patterns of tbx5a in the pectoral fin buds of combinatorial deletion mutants of zebrafish hox clusters. Dorsal views of embryos at 30 hpf are displayed. After capturing images, the genotype of each specimen was determined. For each genotype, reproducibility was confirmed with at least three different specimens. Scale bars are 100 µm.
Figure 3
Loss of fin progenitors with posterior expansion of cardiac marker expression in hoxba;hoxbb mutants.
(A–F) Expression patterns of tbx5a were compared between sibling wild-type and hoxba;hoxbb homozygous embryos during embryogenesis. The range of tbx5a expression in the lateral mesoderm is indicated by a bracket. The progenitor cells of pectoral fins are indicated by an arrowhead. (G, H) Expression patterns of nkx2.5 were compared between sibling and hoxba;hoxbb mutants. Enlarged views are shown in (G’, H’). The different regions of nkx2.5 expression between wild-type and mutants are indicated by brackets. (I, J) Expression patterns of cmlc2 are shown. All images were captured from the dorsal side. For each stage, reproducibility was confirmed with at least three different specimens. Scale bars indicate 100 µm.
Figure 4 with 1 supplement
Zebrafish hoxba;hoxbb mutants lack a response to retinoic acid (RA) in fin buds.
(A–D) Exogenous RA exposure in raldh2 mutants can rescue tbx5a expression in fin buds. Arrowheads indicate the positions of the pectoral fin buds. (E–H) RA treatments in hoxba;hoxbb cluster-deleted mutants do not rescue tbx5a expression in fin buds. (I, J) Expression patterns of raldh2 were compared between sibling and hoxba;hoxbb mutants. For each genotype, reproducibility was confirmed with at least three different specimens. All images were captured from the dorsal side. Scale bars represent 100 µm.
Figure 4—figure supplement 1
Generation of the zebrafish raldh2 mutants by CRISPR–Cas9.
(A) The nucleotide sequence surrounding the mutations in raldh2 is shown on the left. The target sequence of the crRNA is emphasized with an underline, while the inserted nucleotide sequence is indicated in orange. Below the DNA sequence, the predicted amino acid is displayed, with red letters highlighting the abnormal amino acid resulting from the frameshift mutation. The asterisk represents the termination codon. (B) A schematic representation of the predicted protein structure is shown, with the red box indicating the abnormal amino acid sequences resulting from the frameshift mutations. The total number of predicted amino acids is displayed to the right of the schematic. (C) The absence of pectoral fins (arrowhead) in raldh2 homozygous mutants is observed dorsally at 3 dpf. (D) The expression of tbx5a in the pectoral fin buds (arrowhead) is significantly reduced in raldh2 mutants. The phenotype of our raldh2sud118 mutants closely resembles the phenotype observed in previously isolated raldh2 mutants.
Figure 5 with 1 supplement
Expression patterns of hox5a and hoxb5b are regulated by RA.
(A) Schematic representation of the 49 hox genes organized into seven hox clusters in zebrafish. hox genes in hoxba and hoxbb clusters are highlighted with orange. (B) Expression profiles of the 49 hox genes in wild-type and raldh2−/− embryos at the 20-somite stage, analyzed by RNA-seq. The average FPKM of each hox gene was compared between sibling and raldh2 mutants. The absence of specific hox genes is indicated by a slash. (C–H) Expression patterns of hoxb5a and hoxb5b in sibling wild-type, raldh2−/−, and RA-treated raldh2−/− embryos at the 10-somite stage. Arrowheads indicate the presumptive positions of pectoral fin buds. For each genotype, reproducibility was confirmed with at least three different specimens. All images are captured from the dorsal side. Scale bars represent 100 µm.
-
Figure 5—source data 1
This source data file contains the numerical values used to generate Figure 5B.
- https://cdn.elifesciences.org/articles/105889/elife-105889-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Volcano plot of the transcriptome analysis between sibling and raldh2 mutants.
The volcano plot illustrates both upregulated and downregulated differentially expressed genes from the comparison between wild-type and raldh2 mutant embryos. Dots with more than a twofold increase in expression are indicated in orange, while those with more than a twofold decrease are shown in light blue. Among them, dots corresponding to hox genes are specifically highlighted.
-
Figure 5—figure supplement 1—source data 1
This source data file provides the underlying numerical data used to create the volcano plot.
- https://cdn.elifesciences.org/articles/105889/elife-105889-fig5-figsupp1-data1-v1.xlsx
Figure 6 with 1 supplement
Screening for hox genes responsible for the zebrafish pectoral fin formation.
(A) Schematic representation of the genetic screening for hox genes involved in the specification of pectoral fins in zebrafish. (B–G) Dorsal views of live mutant larvae at 3 dpf were obtained during the screening. After capturing images, genotyping, and DNA sequencing in target hoxb genes were conducted. For the mutants illustrated in (C–G), hoxb genes are shown with frameshift mutations introduced. Detailed information is provided in Supplementary file 1A. (H–J) Dorsal views of hoxba−/−;hoxb5b−/− and hoxba−/−;hoxb5b−/−;hoxb6b−/− larvae at 3 dpf. (K–M) Expression patterns of tbx5a in hoxba−/−;hoxb5b−/− and hoxba−/−;hoxb5b−/−;hoxb6b−/− mutants at 30 hpf. Arrowheads indicate the presumptive positions of pectoral fin buds. All images are captured from the dorsal side. Scale bars represent 100 µm.
Figure 6—figure supplement 1
Generation of the frameshift-induced hox mutants using CRISPR–Cas9.
Schematic representations show zebrafish hoxb8b, hoxb5b;hoxb6b, and hoxb5b;hoxb6b;hoxb8b mutants. The other frameshift-induced hox mutants were previously described (Maeno et al., 2024). The nucleotide sequence around the mutations is shown on the left. The target sequence of crRNA is emphasized with an orange underline. The inserted nucleotide sequence is emphasized in red. Below the DNA sequence, the predicted amino acid is shown. Red letters indicate the abnormal amino acid caused by the frameshift mutation. On the right, a schema represents the predicted protein structure. The blue box indicates the homeodomain, and the red box indicates the abnormal amino acid sequences from the frameshift mutations. The total number of predicted amino acids is shown on the right of the schema.
Figure 7 with 1 supplement
hoxb4a-b5adel/del;hoxb5bdel/del larvae partially recapitulate the absence of the pectoral fins.
(A–C) Dorsal views of zebrafish hoxba−/−;hoxb5bdel/del larvae at 3 dpf. (D, E) Expression patterns of tbx5a in sibling wild-type and hoxba−/−;hoxb5bdel/del (n = 4) at 30 hpf. Arrowheads indicate the presumptive positions of pectoral fin buds. (F, G) Dorsal view of frameshift-induced hoxb5a−/−;hoxb5b−/− and hoxb5adel/del;hoxb5bdel/del larvae. (H–L) Dorsal view of frameshift-induced hoxb4a−/−;hoxb5a−/−;hoxb5b−/− and hoxb4a-b5adel/del;hoxb5bdel/del larvae. All images are captured from the dorsal side. Arrowheads indicate the presumptive positions of pectoral fin buds. (M–R) Expression patterns of zebrafish hoxb4a, hoxb6a, hoxb6b, hoxb7a, hoxb8a, and hoxb8b at the 10-somite stage. Dorsal views. Arrowheads indicate the presumptive positions of pectoral fin buds. Scale bars represent 100 µm.
Figure 7—figure supplement 1
Generation of locus-deletion mutants using CRISPR–Cas9.
(A, C, E, G) Schematic representations show the genomic structure of zebrafish hoxb4a, hox5a, and hoxb5b. Boxes represent the exons, with the blue boxes indicating the coding regions. Black arrowheads indicate the target sites of crRNA, while red arrowheads represent the primers used for genotyping. (B, D, F, H) Display genomic locus deletions of hox4a, hox5a, hoxb5b, and hoxb4a-b5a deletion mutants. The flanking sequences of the 5′- and 3′-crRNA targets (underline) are shown in blue and green letters, respectively.
Additional files
-
Supplementary file 1
The frameshift mutations introduced in hoxbb genes in the embryos lacking pectoral fins during the screening.
The DNA sequences of hoxb5b, hoxb6b, and hoxb8b were analyzed in 2 dpf embryos apparently lacking pectoral fins. Among the CRISPR–Cas9-induced mutations, those resulting in frameshifts are highlighted in blue.
- https://cdn.elifesciences.org/articles/105889/elife-105889-supp1-v1.xlsx
-
Supplementary file 2
The target-specific sequences of crRNAs used in this study.
- https://cdn.elifesciences.org/articles/105889/elife-105889-supp2-v1.xlsx
-
Supplementary file 3
The sequences of primers used for the genotyping in this study.
- https://cdn.elifesciences.org/articles/105889/elife-105889-supp3-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/105889/elife-105889-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
HoxB-derived hoxba and hoxbb clusters are essential for the anterior–posterior positioning of zebrafish pectoral fins
eLife 14:RP105889.
https://doi.org/10.7554/eLife.105889.3
