Functional Morphology of Hatching: Ontogeny and Distribution of Hatching Gland Cells in Red‐Eyed Treefrogs and a New Marker for Anuran Hatching Enzyme

Environmentally cued hatching (ECH) is widespread in animals and requires regulation of hatching mechanisms. Enzymatic digestion of the egg membrane is a common hatching mechanism in vertebrates and invertebrates. In amphibians and fishes, hatching enzymes (HE) are synthesized and released by hatching gland cells (HGC), whose functional ontogeny determines when hatching can occur. Ontogenetic studies of HGC development or HE expression are limited, based largely on external cell morphology; few markers for HGC or HE are available, and those appear specific for Xenopus. Moreover, mechanisms regulating HE release are unknown in anurans. To investigate variation in the hatching process, we need tools to identify and analyze its components. Agalychnis callidryas (Hylidae) is a well-established model of ECH, showing plastically timed, acute HE release, unlike the gradual release described for some aquatic anurans. We developed a new antibody marker for A. callidryas HE that also labels HGC/HE in glassfrogs (Centrolenidae). As glassfrogs and treefrogs diverged 62 mya, the antibody may be broadly useful in anurans. We used the AcHE antibody to examine the development and distribution of HGC and accumulation of HE, two key elements of hatching mechanisms, in A. callidryas. We found a much larger number (ca. 4200) and broader distribution of HGC than has been documented in any amphibian, with HGC densely but non-contiguously distributed over the front of the head and eyes and scattered along the dorsal midline. HE expression begins before hatching competence and is strong throughout the plastic hatching period, unlike HE gene expression which diminishes after competence. The distribution and expression ontogeny of A. callidryas' HE/HGC appear related to their hatching performance, plasticity, and embryo morphology. The AcHE antibody will enable comparative research to elucidate co-variation in the functional morphology, performance, and ecological context of hatching.