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. 2020 Sep;22(5):384-402.
doi: 10.1111/ede.12354.

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes

Emily C Funk  1   2 , Catriona Breen  1 , Bhargav D Sanketi  3 , Natasza Kurpios  3 , Amy McCune  1
Affiliations

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes

Emily C Funk et al. Evol Dev. 2020 Sep.

Abstract

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe-finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray-finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.

Keywords: Bmp16; development; gas bladder; lungs; ray‐finned fishes.

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Figures

FIGURE 1
FIGURE 1
Basic morphology and phylogenetic distribution of lungs and the gas bladder. (a) Diagram of a typical teleost fish showing the dorsal location of the gas bladder situated above the gut and below the spine (Pough, Heiser, & McFarland, 1996). (b) A highly pruned phylogeny of bony vertebrates showing taxa of interest. The two major lineages of the bony vertebrate clade are the lobe-finned fishes (about 30,500 species), which includes tetrapods (e.g., mouse), and the ray-finned fishes (over 30,000 species; Nelson et al., 2016). Within the ray-finned fishes, bichirs are the only ray-finned fish that possess lungs (blue), while bowfin and teleosts possess a gas bladder (orange). Based on phylogenetic distribution of air-filled organs, the lungs are the ancestral state for the bony vertebrates and the gas bladder originated in the lineage containing bowfin and teleosts (e.g., zebrafish). (c) Dorsal versus ventral outgrowth of lungs and gas bladders in key taxa. Taxa are illustrated in the left column. To the right of each organism, in the middle column, is a line drawing showing a lateral view of the dissected foregut and lungs/gas bladder (after Dean, 1895). In the right-most column, a transverse-sectional schematic of the foregut and lungs or gas bladder is shown (after Dean, 1895). As shown, zebrafish and bowfin have a dorsally budding gas bladder. Bichir, Australian lungfish, and mouse have ventrally budding lungs. df, dorsal fin; fg, foregut; GB, gas bladder; in, intenstine; L: lungs; pd, pneumatic duct; pe, pectoral fin; sc, spinal cord; t, trachea
FIGURE 2
FIGURE 2
Diagram of known gene interactions regulating lung development. (a) At embryonic day 9 (E9) in mouse, the lung field is established. (b) At E9.5, the nascent lungs bud ventrally from the anterior foregut. During these early stages of lung development, E9 and E9.5 (a and b), Nkx2.1 (yellow), the first marker of lung develoment, is expressed ventrally in the foregut endoderm and indicates the site of lung budding. Sox2 (blue) is expressed dorsally in the foregut endoderm and is mutually inhibitory with Nkx2.1. Bmp4 in the mesoderm interacts with its receptors, Bmpr1a and Bmpr1b, in the endoderm to inhibit the expression of Sox2 ventrally allowing for the expression of Nkx2.1. (c) At E12.5, during lung branching, Bmp4 (dark green) becomes expressed at the distal tips of the branching lung buds, and Fgf10 (light green) is expressed in the mesoderm surrounding the distal tips. Bmp4 and Fgf10 interact to promote bud outgrowth and branching. Endo, endoderm; Meso, mesoderm
FIGURE 3
FIGURE 3
Sox2 and Nkx2.1 expression in bichir lungs. For each pair of panels, the top panel shows gene expression in bright red overlain by bright blue DAPI staining of cell nuclei. Bottom panels show gene expression alone in bright red. Compasses indicate orientation of sections. (a) Sagittal section of Sox2 expression in the foregut and lungs at stage 35 during outgrowth. The position of the transverse section is marked on the sagittal section (dotted line) and on the inset diagram of a larval bichir. (b) Sox2 expression in the lungs at Stage 35 is shown in transverse section. These sections show that Sox2 is expressed throughout the foregut (fg) but absent from the lung buds (L). (c) Nkx2.1 expression at Stage 34, right after lung budding. At Stage 34, Nkx2.1 is expressed throughout the foregut (fg) and lung bud (L) but strongest expression is in the ventral foregut and lung bud. (d) Nkx2.1 expression at Stage 35, during lung outgrowth. At Stage 35, Nkx2.1 continues to be expressed throughout the foregut and lungs with strongest expression in the ventral foregut and weaker expression in the dorsal foregut and lungs. A, anterior; D, dorsal; L, left; n, notochord; P, posterior; R, right; V, ventral
FIGURE 4
FIGURE 4
Sox2 and Nkx2.1 expression in bowfin gas bladder. For each pair of panels, the top or left panel shows gene expression in bright red overlain by bright blue DAPI staining of cell nuclei. Bottom or right panels show gene expression alone in bright red. Compasses indicate orientation of sections. (a) Sox2 expression in sagittal view in bowfin at Stage 26, right after gas bladder budding. The position of the transverse section is marked on the sagittal section (dotted line) and on the inset diagram of a larval bowfin. (b) Sox2 expression at Stage 26 displayed in transverse section. As shown, Sox2 is expressed throughout the foregut (fg) and the gas bladder bud (GB), and expression extends beyond the pneumatic duct (pd) connection to the foregut. (c) Nkx2.1 expression at Stage 26, right after gas bladder budding shows clearly the ventrally restricted expression of Nkx2.1 in the foregut and absence of Nkx2.1 expression in the gas bladder bud. (d) Nkx2.1 expression at Stage 27, during gas bladder outgrowth showing strong Nkx2.1 expression ventrally in the foregut as well as very weak expression in the gas bladder. A, anterior; D, dorsal; L, left; n, notochord; P, posterior; R, right; V, ventral
FIGURE 5
FIGURE 5
Sox2 and Nkx2.1 expression in zebrafish gas bladder. For each pair of panels, the top or left panel shows gene expression in bright red overlain by bright blue DAPI staining of cell nuclei. Bottom or right panels show gene expression alone in bright red. Compasses indicate orientation of sections. (a) Sagittal view of Sox2 expression in zebrafish at 3 dpf, right after gas bladder budding. The position of the transverse section is marked on the sagittal section (dotted line) and on the inset diagram of a larval zebrafish. (b) Sox2 expression at 3 dpf shown in transverse section. As shown, Sox2 is expressed throughout the foregut (fg) and the gas bladder bud (GB). However, Sox2 expression is absent from the gut beyond the pneumatic duct (pd). (c) Nkx2.1b expression in zebrafish at 2 dpf, initial gas bladder budding. (d) Nkx2.1 expression at 3 dpf, right after budding. Nkx2.1b expression is expanded to the entire foregut and the gas bladder bud. A, anterior; D, dorsal; L, left; n, notochord; P, posterior; R, right; V, ventral
FIGURE 6
FIGURE 6
Bmp4 expression and Smad phosphorylation in bichir lungs. For each pair of panels, top panels shown protein expression in bright red overlain with DAPI in blue. Bottom panels show protein expression alone in bright red. All panels are shown in transverse section, with the compass indicating orientation shown in upper right panel. Inset in panel a shows a diagram of a larval bichir with position of anterior and posterior sections indicated. (a) Bmp4 expression in the anterior region of bichir lungs at Stage 36 during lung outgrowth. (b) Bmp4 expression in the posterior region of the lungs at Stage 36. Bmp4 is expressed in the foregut (fg) and lung endoderm with strong expression in the lungs. (c) Smad phosphorylation, shown in bright red, is detected in anterior region of bichir lungs at Stage 36 during lung outgrowth. (d) Smad phosphorylation is detected in posterior region of the lungs at Stage 36. Smad phosphorylation, indicating Bmp4 activity, is detected strongly in the ventral mesenchyme surrounding the lung buds. D, dorsal; L, left; lL, left lung; R, right; rL, right lung; V, ventral
FIGURE 7
FIGURE 7
Bmp16 gene tree. Reconstructed gene tree of relationships of Bmp16, Bmp4, and Bmp2 among bony vertebrates. The Bmp2 cluster is shown in blue. The Bmp4 cluster is shown in orange. The Bmp16 cluster is shown in purple. The sea lamprey Bmp2/4 genes (black) are used as the outgroup. Note that the putative bowfin Bmp16 is nested within the Bmp16 clade
FIGURE 8
FIGURE 8
Bmp16 expression and Smad phosphorylation in bowfin gas bladder. For each column, top panels show gene expression in bright red overlaid with DAPI in blue. Middle panels show gene expression alone in bright red. The bottom panels depict graphically the gene expression patterns shown as photomicrographs above for Bmp16 (dark green) and pSmad (orange). All panels are transverse sections at Stage 27 during outgrowth. The anterior-posterior location of the transverse sections is indicated on the larval bowfin diagrams shown above the panels. (a) Bmp16 expression in the anterior region of bowfin gas bladder. (b) Smad phosphorylation is detected in the anterior region of the gas bladder. (c) Bmp16 expression in the posterior region of the gas bladder in bowfin. (d) Smad phosphorylation is detected in the posterior region of the gas bladder. Bmp16 is expressed strongly in the anterior gas bladder (GB); however, more posteriorly, where the gas bladder expands laterally, Bmp16 expression is absent. Bmp16 activity, as indicated by Smad phosphorylation, is strong in the dorsal mesenchyme surrounding the gas bladder, both anteriorly and posteriorly. n, notochord; pd, pneumatic duct
FIGURE 9
FIGURE 9
Gene expression changes during the lung-to-gas bladder transition in ray-finned fishes. Using a pruned bony-vertebrate phylogeny as an evolutionary framework, expression patterns of Sox2 (red), Nkx2.1 (purple), Bmp4 (light green), and Bmp16 (dark green) are shown for transverse sections of the foregut and either lungs or gas bladder of each taxon. Expression of Nkx2.1 and Sox2 are conserved for mouse and bichir. Bmp4 is expressed in the lung endoderm during outgrowth of both lungs and gas bladder, but Bmp4 is restricted to the distal tips of lung branch in mouse and not bichir. Expression of Sox2 is expanded in taxa with gas bladders (bowfin, zebrafish) relative to taxa with lungs (mouse, bichir). Expression of Nkx2.1 is expanded in taxa with gas bladders (bowfin, zebrafish) relative to taxa with lungs (mouse, bichir). Bmp16, rather than Bmp4 is expressed in taxa with gas bladders. Hypothesized expression patterns in the common ancestor of bony vertebrates, inferred from shared patterns between bichir and tetrapods, are indicated at the base of the phylogeny. The zebrafish gas bladder is connected to the foregut, but the pneumatic duct is not shown in the Bmp16 image because the section is posterior to the pneumatic duct. fg, foregut; GB, gas bladder; L,
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