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. 2016 Jul 28:6:30580.
doi: 10.1038/srep30580.

Molecular developmental mechanism in polypterid fish provides insight into the origin of vertebrate lungs

Norifumi Tatsumi  1 , Ritsuko Kobayashi  1 , Tohru Yano  1 , Masatsugu Noda  1 , Koji Fujimura  1   2 , Norihiro Okada  3   4 , Masataka Okabe  1
Affiliations

Molecular developmental mechanism in polypterid fish provides insight into the origin of vertebrate lungs

Norifumi Tatsumi et al. Sci Rep. .

Abstract

The lung is an important organ for air breathing in tetrapods and originated well before the terrestrialization of vertebrates. Therefore, to better understand lung evolution, we investigated lung development in the extant basal actinopterygian fish Senegal bichir (Polypterus senegalus). First, we histologically confirmed that lung development in this species is very similar to that of tetrapods. We also found that the mesenchymal expression patterns of three genes that are known to play important roles in early lung development in tetrapods (Fgf10, Tbx4, and Tbx5) were quite similar to those of tetrapods. Moreover, we found a Tbx4 core lung mesenchyme-specific enhancer (C-LME) in the genomes of bichir and coelacanth (Latimeria chalumnae) and experimentally confirmed that these were functional in tetrapods. These findings provide the first molecular evidence that the developmental program for lung was already established in the common ancestor of actinopterygians and sarcopterygians.

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Figures

Figure 1
Figure 1. Lung development in Polypterus senegalus.
Specimens at (a) 8 days post fertilization (dpf), (c) 9 dpf, and (e) 13 dpf; and sections at (b) 8 dpf, (d) 9 dpf, and (f–i) 13 dpf stained with hematoxylin and eosin. Red arrows indicate the right lung bud. Scale bars, 100 μm. fg, foregut; lb, lung bud; m, mesenchyme.
Figure 2
Figure 2. Gene expression patterns of Polypterus senegalus embryos.
Gene expression patterns of schematic images in tetrapods and of P. senegalus at (a–e) 8.5 days post fertilization (dpf) and (f–j) 12 dpf for the genes Fgf10 (b,g), Nkx2.1 (c,h), Tbx4 (d,i), and Tbx5 (e,j). Dotted lines in (b–e) indicate the foregut endoderm and lung bud. Scale bars, 100 μm. fg, foregut; lb, lung bud; m, mesenchyme.
Figure 3
Figure 3. The mouse lung mesenchyme-specific enhancer (LME) of the Tbx4 gene.
(a) VISTA plots comparing the mouse LME sequences of the Tbx4 gene in human (Homo sapiens), chicken (Gallus gallus), frog (Xenopus tropicalis), coelacanth (Latimeria chalumnae), bichir (Polypterus senegalus), zebrafish (Danio rerio), and medaka (Oryzias latipes) with that in mouse (Mus musculus). The green dashed box indicates the conserved region in the LME. (b) Multiple sequence alignment of the conserved region among human, mouse, chicken, frog, coelacanth and bichir. A core lung mesenchyme-specific enhancer (C-LME) of 150 bp was found in bichir as well as the sarcopterygians, as shown in orange. The predicted binding sites of the transcription factors are indicated by blue boxes.
Figure 4
Figure 4. Functional assay for the core lung mesenchyme-specific enhancer of the Tbx4 gene.
Various vectors were introduced into the right-hand side of the chicken lung mesenchyme: (a–d) the ptk-EGFP vector alone as a control, (e–h) psTbx4C-LME4×, and (i–l) lcTbx4C-LME4×. pCAGGS-Kusabira-orange was co-introduced to monitor the plasmid-positive area (b,f,j). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (a,e,i). No expression was observed in the control (c), whereas enhanced green fluorescent protein (EGFP) expression from psTbx4C-LME4× and lcTbx4C-LME4× was detected in the lung mesenchyme (g,k). The signals from Kusabira-orange were merged with the EGFP expression (d,h,l). Asterisks indicate the right lung bud. Scale bars, 200 μm.
Figure 5
Figure 5. Summary of the Tbx4 C-LME in osteichthyan evolution.
Possession of the Tbx4 core lung mesenchyme-specific enhancer (C-LME) is summarized on a simplified phylogenetic tree of osteichthyes. The blue line shows the possession of Tbx4 C-LME in sarcopterygians and actinopterygians, corresponding to the possession of lungs. Lungfish are assumed to possess the Tbx4 C-LME due to the existence in coelacanths. The blue squares indicate presumptive positions for functional loss of the Tbx4 C-LME during the evolution of actinopterygians. We indicate the point at which this loss may have occurred. The red arrow indicates the timing of 3R-WGD (teleost specific whole-genome duplication).
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