Expression of meis and hoxa11 in dipnoan and teleost fins provides new insights into the evolution of vertebrate appendages

Abstract

Background: The concerted activity of Meis and Hoxa11 transcription factors is essential for the subdivision of tetrapod limbs into proximo-distal (PD) domains; however, little is know about the evolution of this patterning mechanism. Here, we aim to study the expression of meis and hoxa11 orthologues in the median and paired rayed fins of zebrafish and in the lobed fins of the Australian lungfish.

Results: First, a late phase of expression of meis1.1 and hoxa11b in zebrafish dorsal and anal fins relates with segmentation of endochondral elements in proximal and distal radials. Second, our zebrafish in situ hybridization results reveal spatial and temporal changes between pectoral and pelvic fins. Third, in situ analysis of meis1, meis3 and hoxa11 genes in Neoceratodus pectoral fins identifies decoupled domains of expression along the PD axis.

Conclusions: Our data raise the possibility that the origin of stylopod and zeugopod lies much deeper in gnathostome evolution and that variation in meis and hoxa11 expression has played a substantial role in the transformation of appendage anatomy. Moreover, these observations provide evidence that the Meis/Hoxa11 profile considered a hallmark of stylopod/zeugopod patterning is present in Neoceratodus.

Keywords: Fin-to-limb transition; Gene expression; Hoxa11; Meis; Neoceratodus; Zebrafish.

Fig. 1

meis1.1 and hoxa11b expression in zebrafish fins. a–f Dorsal, g, h anal and i–l pelvic fins. a–d, f, h, i–l WISH and e, g Alcian Blue staining followed by WISH. a–c, e, g, h Arrowheads indicate fin mesenchyme cells expressing meis1.1 and hoxa11b. c The white arrow indicates unsegmented radial; c the black arrows indicate WISH staining in dorsal somite cells. a–d, j, l The dashed lines indicate distal limit of endochondrogenic mesoderm below the finfold in a–d dorsal and j, l pelvic fins. e–h The solid lines indicate the boundary between proximal and distal radials in e, f dorsal and g, h anal fins. a–f, i–l Anterior to left, distal to top; g, h anterior to left, distal to bottom. Fish length is a–c 3.6–4.4 mm, d 4.0–5.2 mm, e–h 5.6–6.1 mm, i, k 4.1–5.3 mm, j, l 5.0–6.4 mm. Abbreviations: dr, distal radial; pr, proximal radial. Scale bars are 100 μm (a, b, f, g), 50 μm (c–e, h–l), and 70 μm (k–n)

Fig. 2

meis1 and meis3 expression in Neoceratodus lobed fins. Whole-mount in situ hybridization in a, c, e–l pectoral and b, d pelvic lobed fins. a, b White arrowheads indicate meis1 transcript under the AER in a pectoral and b pelvic fin buds. c–g, i–k meis3 expression in the whole bud of c pectoral and d pelvic fins. b, d The white dashed lines highlight the margin of paired pelvic fin buds. a–d Square brackets indicate the AER. The black dashed lines indicate the stylopod-zeugopod boundary at chondrogenesis stages. e–g, i–k From st. 46 to st. 50, e, i meis1 and meis3 transcripts are first localized in the proximal cells of the pectoral lobed fin, f, j after they extend gradually in a PD striped pattern around cartilage condensations, g, k and then they show intersegmental expression (asterisks). h, l Double Alcian Blue/WISH labelings show proximal expression across the humerus region and the first transversal stripe near radius/ulna cartilages. Abbreviations: AER, apical ectodermal ridge; c, cartilage; h, humerus; m, mesenchyme; r, radius; u, ulna. Anterior to left; distal to top. Scale bars are 100 µm (a–d), 80 µm (e, f, i) and 50 µm (g, h, j–l)

Fig. 3

hoxa11 expression in Neoceratodus lobed fins. WISH in pectoral lobed fins from early budding to early chondrogenesis. a Distribution of hoxa11 transcript in the posterior bud. b–d The arrowheads indicate expression in anterior cells at the distal margin of the lungfish pectoral fin. b–d The arrows indicate a PD discontinuity of low, if any, expression in the early phase of hoxa11 activity. e, f Comparison of single WISH and Alcian Blue staining indicates that transcription is depleted in those cells that will ultimately build the humerus (below dashed line). Abbreviations: h, humerus; r, radius; u, ulna. Distal to top, anterior to left. Scale bars are 100 µm (a–d) and 50 µm (e, f)

Fig. 4

Late-phase Meis and Hoxa11 expression in vertebrate appendages. The tree shows phylogenetic relationships of chondrichthyans, sarcopterygians and actinopterygians. Meis (yellow) and Hoxa11 (purple) expression domains at late stage of fin/limb development superimposed on the appendicular skeleton for each taxon