Max Hämmerle
scientific collaborator and MSc alumni (Advisor: Georg Brenneis)
at the Department of Evolutionary Biology, Unit for Integrative Zoology 

Integrative study of muscle formation and appendage patterning genes sheds light on sea spider leg development

Abstract
One of the most striking features of the marine sea spiders (Pycnogonida) are their four pairs of long walking legs, which can dwarf the remaining body and resulted in their alternative name Pantopoda (=all-legs). During the anamorphic development of pycnogonids, leg development occurs consecutively at the posterior body terminus, whereby an almost complete leg abruptly emerges from a tiny cuticularized limb bud after a single molt. This externally explosive mode of leg formation has significantly hindered the study of proximo-distal (P-D) appendage patterning, as the leg tissues are drastically compressed in the limb bud cuticle prior to release. To overcome these challenges, we combined two modern approaches to interrogate leg development in the sea spider Pycnogonum litorale. Taking advantage of the strictly segmental array of intrinsic leg musculature in adults, we employed fluorescent F-actin staining and myosin heavy chain immunolabeling to follow muscle differentiation as a proxy for podomere location. Complementing these morphological landmarks, we applied HCR-FISH to visualize the expression of key genes that underline the P-D patterning of arthropod appendages.

Our study resolves the 3D-folded P-D axis of the compressed leg beneath the limb bud cuticle and shows that muscle differentiation in proximal and distal podomeres predates that in medial leg regions. Moreover, we document a previously unknown muscle group stretching over two podomere borders. Embedding these muscle data in the gene expression domains permits to resolve the P-D regionalization at (near) podomere resolution, providing important novel data for comparative studies on leg development in arthropods.