Sarcopterygian fin ontogeny elucidates the origin of hands with digits

Abstract

How the hand and digits originated from fish fins during the Devonian fin-to-limb transition remains unsolved. Controversy in this conundrum stems from the scarcity of ontogenetic data from extant lobe-finned fishes. We report the patterning of an autopod-like domain by hoxa13 during fin development of the Australian lungfish, the most closely related extant fish relative of tetrapods. Differences from tetrapod limbs include the absence of digit-specific expansion of hoxd13 and hand2 and distal limitation of alx4 and pax9, which potentially evolved through an enhanced response to shh signaling in limbs. These developmental patterns indicate that the digit program originated in postaxial fin radials and later expanded anteriorly inside of a preexisting autopod-like domain during the evolution of limbs. Our findings provide a genetic framework for the transition of fins into limbs that supports the significance of classical models proposing a bending of the tetrapod metapterygial axis.

Fig. 1. Homology between fins and tetrapods limbs.

Sarcopterygian fins (Australian lungfish fin shown) resemble tetrapod limbs, and proximally clear homologs of the humerus, radius, and ulna can be identified (pink field) (5, 8), an organization that is absent from ray finned fish (actinopterygians). In the distal region, they, however, lack the tetrapod-specific cross-articular anatomy (bend arrow) by which the long bones of the hand articulate with the radius and ulna via the nodular bones of the wrist (black). Hence, the evolutionary origin of the hand and digits at the fin-to-limb transition remains unresolved (yellow field).

Fig. 2. “Hand” and “digit” domains in embryonic pectoral fins of the Australian lungfish.

Gene expression was detected using whole-mount in situ hybridization during stages 42 to 48/49. Names for colorimetrically detected genes are indicated on the left. Fluorescent detection is shown for the oldest stage in the rightmost column with detection for col2a in red, the anti-muscle sarcomere antibody MF20 in azure, actinodin1/2 in green (performed for hoxa13 sample only), and fin contours are shown using Hoechst staining (dark blue). The proximal boundary of hoxa13 expression is indicated using a white dotted line. The radius (“r”) and ulna (“u”) are indicated in the rightmost column. Stages 45 to 49 fins were dissected and flat mounted, whereas stages 42 to 44 fins were imaged in position on the embryo. Additional expression data are provided in figs. S1 and S2. Anterior is to the left. Scale bars, 200 μm.

Fig. 3. Expression of shh, gli3, and gremlin1 during lungfish development.

Shh expression is observed in a posterior ZPA during stage 44 (black arrowhead) but subsides at stage 45. Gli3 is detected in a weak domain with anterior bias during fin budding (stage 42), leaving a clear posterior domain (black arrowhead) in which it is not expressed, and its expression is hence complementary with the early expression of hand2 (Fig. 2) similar to what is observed in tetrapod limbs (32). Expression of the BMP antagonist gremlin1 becomes expressed in a central fin domain along both anterior and posterior aspects of the forming metapterygial axis (dotted line in stage 45). Fins were dissected and imaged on an agarose dish. Additional data are provided for stages 42 to 45 in fig. S1. Anterior is to the left. Scale bars, 200 μm.

Fig. 4. A compound origin for hands with digits.

During development, hoxa13 demarcates a similar endochondral autopodial domain in lungfish and tetrapods (top, orange), whereas this gene is primarily expressed in the dermal skeleton in ray-finned fish. Lungfish, however, lack the progressive posteriorization that patterns tetrapod digits (bottom, blue). Instead, genes such as hand2 and hoxd13 remain restricted to the posterior side of the fin, while an anterior domain patterned by alx4, which in tetrapods becomes excluded from the distal limb, extends along the proximal-distal fin axis. This terminal phase of posteriorization is therefore a distinguishing feature of tetrapods. Together, this suggests separated evolutionary trajectories for hand and digit domains and supports an inferred bending of the tetrapod metapterygial axis and homology between digits and postaxial fin radials (3, 4, 48). A generalized expression signature for ray-finned fish was reconstructed using (12, 22, 23) for hoxa13, (12, 42) for hoxd13, (18, 43) and fig. S3 for hand2, and (43) and fig. S3 for alx4. A teleost fin skeleton is shown as a generalized representation for ray-finned fish (also see main text).