Genetic evidence: zebrafish hoxba and hoxbb clusters are essential for the anterior-posterior positioning of pectoral fins

Morimichi Kikuchi
Renka Fujii
Daiki Kobayashi
Yuki Kawabe
Haruna Kanno
Sohju Toyama
Farah Tawakkal
Kazuya Yamada
Akinori Kawamura author has email address

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama, Japan

https://doi.org/10.7554/eLife.105889.1

Open access
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Figures and data

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-M) 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.

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.

Transformation of pectoral fin progenitor cells into cardiac cells in hoxba;hoxbb homozygous embryos.
(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.

Zebrafish hoxba;hoxbb mutants lack a response to 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.

The 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.

Screening for hox genes responsible for the specification of zebrafish pectoral fins.
(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 Table S1. (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.

hoxb4a-b5a^del/del;hoxb5b^del/del larvae partially recapitulate the absence of the pectoral fins.
(A-C) Dorsal views of zebrafish hoxba^-/-;hoxb5b^del/del larvae at 3 dpf. (D, E) Expression patterns of tbx5a in sibling wild-type and hoxba^-/-;hoxb5b^del/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 hoxb5a ^del/del;hoxb5b^del/dellarvae. (H) Expression patterns of zebrafish hoxb4a at the 10-somite stage. (I-K) Dorsal view of frameshift-induced hoxb4a^-/-;hoxb5a^-/-;hoxb5b^-/- and hoxb4a-b5a ^del/del;hoxb5b^del/del larvae. All images are captured from the dorsal side. Arrowheads indicate the presumptive positions of pectoral fin buds. Scale bars represent 100 μm.

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