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Gene Expr Patterns
2022 Jan 01;45:119262. doi: 10.1016/j.gep.2022.119262.
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inka1b expression in the headmesoderm is dispensable for facial cartilage development.
Jeon H
,
Jin S
,
Choe CP
.
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Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the headmesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and headmesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the headmesoderm might not be essential for head development in zebrafish.
Fig. 1. Phylogenetic analysis of vertebrate Inka family proteins. This phylogenetic tree is constructed using maximum likelihood (ML) based on multiple sequence alignment for the CDS amino acid sequences. Bootstrap values are shown at branches. In this ML tree, zebrafish Inka1b is grouped with the bony fish Inka1 with a 100% bootstrap value, and zebrafish Inka2 is included in the vertebrate Inka2 group with a 100% of bootstrap value. The fish Inka1 group is marked with red lines, and green lines indicate the tetrapod Inka1 group. Vertebrate Inka2 group is presented with blue lines. Note that Callorhinchus milii (cartilaginous fish) Inka1 is not grouped with the bony fish Inka1. Also, no Inka1a equivalent has yet been described in Oryzias melastigma, but the Inka1 ortholog has been designated Inka1b. Cm, Callorhinchus milii (elephant shark); Om, Oryzias melastigma (Indian medaka); Dr, Danio rerio (zebrafish); Xl, Xenopus laevis (African clawed frog); Mm, Mus musculus (house mouse); Hs, Homo sapiens (human).
Fig. 2. Expression of inka1b at the stage of arch segmentation. (A–D) Fluorescence in situ hybridization of inka1b (green) in conjunction with immunohistochemistry of GFP (red) in wild-type Tg(her5:GFP) reporter lines marking the pharyngeal endoderm and pouches. (A) Arrowheads indicate inka1b expression observed in the adjacent regions to the first (p1) and second (p2) pouches at 18 hpf. Arrows show inka1b expression in small patches located to the posterior of the second pouch. (B) inka1b expression in the adjacent regions to the third pouch (p3) and posterior cell mass (CM) at 24 hpf, is marked with arrowhead and arrow, respectively. (C) Arrowheads indicate inka1b expression adjacent to the third (p3) and fourth (p4) pouches at 28 hpf, with an arrow marking new expression of inka1b appearing in the adjacent to the cell mass. (D) inka1b expression in the neighboring regions of pouches 3–5 at 32 hpf, is marked with arrowheads. (B–D) inka1b expression in the potential cranial nerveganglia is indicated with asterisks. (A′-D′) Green channel only. Scale bar: 40 μm. Anterior is to the left and dorsal is to the top.
Fig. 3. Expression of inka1b in the headmesoderm. (A–D) Fluorescence in situ hybridization of inka1b (green) in wild-type Tg(sox10:GFP) (red in A and B) and Tg(nkx2.5:GFP) (red in C and D) transgenic animals. (A, B) Arrowheads mark inka1b expression adjacent to the sox10-positive pharyngeal arches (a2-a5) at 24 and 28 hpf. (C, D) Arrowheads indicate inka1b expression overlapped with the nkx2.5-positive mesoderm (m3 and m4), with arrows showing inka1b expression preceding that of the nkx2.5:GFP expression in the headmesoderm at 24 and 32 hpf. (A–D) inka1b expression in the potential cranial nerveganglia is indicated with asterisks. (A′-D′) Green channel only. (A″-D″) Red channel only. Scale bars: 40 μm (A and C). Anterior is to the left.
Fig. 4. Generation of a loss-of-function allele of inka1b gene. (A) Structure of inka1b gene. inka1b gene consists of three exons, with the third exon bearing sequences for the most protein-coding region (black box). 5′- and 3′-UTR are displayed as open boxes. The gRNA target site is marked in yellow. (B) A loss-of-function allele of inka1b gene. Sequence alignment shows the deletion mutation of the inka1b mutant allele. In the wild-type inka1b sequence, the gRNA target and PAM sites are marked in red and blue, respectively. The electrophoretogram shows the lesion in the inka1b mutant allele. (C) Schematic of the Inka1b protein encoded by the wild-type and mutant alleles. The Inka-box domain shown by black box in wild-type Inka1b protein is partially missing (grey box) in the mutant Inka1b protein. The 14-3-3 binding site is marked in green.
Fig. 5. Normal development of pouches and facial cartilages in inka1b mutants. (A–H) In situ hybridization of inka1b (green) in wild-type and inka1b homozygous mutant siblings during arch segmentation. Arrowheads and asterisks indicate inka1b expression in the headmesoderm and cranial nerveganglia, respectively. While inka1b expression in the head is evident in wild-type animals, its expression was greatly reduced in inka1b homozygous mutants. Number of embryos examined: A (29), B (37), C (42), D (35), E (26), F (34), G (35), H (31). Scale bar: 40 μm. (I) Quantification of inka1b mRNA transcripts. Expression in wild types was scaled to 1. Relative expression of inka1b mRNA in inka1b mutants at each time point was greatly reduced compared to wild-type siblings. Data represent mean ± s.e.m. Asterisks indicate statistical differences at P < 0.05 as determined by one-way ANOVA test. (J, K) Alcama immunohistochemistry (green) shows five pouches (p1-p5), bearing a bi-layered structure in wild-type and inka1b homozygous mutant animals at 32 hpf. All 118 wild-type siblings and 41 inka1b homozygous mutants show normal pouches. Cranial nerveganglia are indicated with asterisks. Scale bar: 40 μm. (L, M) Ventral view of dissected facial cartilages. Each element of facial cartilages is marked in wild-type and inka1b homozygous mutant animals. The facial cartilages are normal in all 82 wild-type siblings and 24 inka1b homozygous mutants. m, Meckel's cartilage; pq, palatoquadratecartilage; hs, hyosymplectic cartilage; ch, ceratohyalcartilage; cb, ceratobranchial cartilage. Scale bar: 100 μm. (A-H, J-M) Anterior is to the left.
Supplementary Figure S1. Sequence alignments for Inka proteins. Multiple sequence alignment for the coding sequence (CDS) of
vertebrate Inka family proteins. The Inka box domain and 14-3-3 binding site are in blue and red, respectively. The conserved amino
acids in all vertebrate Inka family proteins are highlighted in yellow. Dr, Danio rerio (zebrafish); Om, Oryzias melastigma (Indian
medaka); Cm, Callorhinchus milii (elephant shark); Xl, Xenopus laevis (African clawed frog); Mm, Mus musculus (house mouse); Hs,
Homo sapiens (human).
Supplementary Figure S2. Normal development of cranial nerveganglia and blood vessels in inka1b mutants. (A, B) Alcama
immunohistochemistry (green) shows normal cranial nerveganglia (arrowheads) adjacent to the ear in wild-type (n=54) and inka1b
homozygous mutant (n=21) animals at 30 hpf. (C, D) Tg(flk:GFP) transgenic reporters visualize blood vessels in the head of wild-type
and inka1b homozygous mutant animals at 30 hpf. Blood vessels (arrowheads) adjacent to the pouches are normal in all wild-type
(n=43) and inka1b homozygous mutant (n=17) animals. Scale bars: 40 μm. Anterior is to the left.
