Single-Cell Laser Ablations and Dorsoventral Patterning in the Amphipod Parhyale Hawaiensis

In bilaterians, establishing the correct spatial positioning of structures along the two main body axes is one of the fundamental tasks of embryonic development. Dorsoventral (DV) patterning typically involves the localized secretion of Bone Morphogenetic Proteins (BMPs) and their antagonists on opposing sides of the embryo. In the fruit fly Drosophila melanogaster for example, a nuclear activity gradient of the Dorsal protein establishes the expression of the BMP protein decapentaplegic ( dpp) in a dorsal domain and the BMP antagonist short-gastrulation (sog) in a ventral domain. BMP antagonism specifies the neurogenic ectoderm, while cells of the ventral midline play an accessory role in nervous system patterning by secreting the EGF ligand, Spitz. Here, I present a new arthropod model for DV patterning in the amphipod Parhyale hawaiensis and show that the ventral midline is a central feature of DV patterning in this system. Using laser microsurgery, I demonstrate that the Parhyale ventral midline acts as an inductive signaling center and establishes ventral cell fates in surrounding tissue. In the absence of ventral midline cells, embryos are dorsalized; they fail to generate a central nervous system and the normally bilateral limb buds are fused ventrally. I also show that the midline is self-propagating; through induction, it is able to generate new midline de novo as the embryo matures. This allows the embryo to generate DV pattern in the naive ectoderm of the extending germ band in the apparent absence of the Dorsal gradient characteristic of Drosophila. Despite the differing developmental roles of the ventral midline in Parhyale and Drosophila, I show that they are homologous structures with a similar basic molecular profile. The Parhyale single-minded gene (Ph-sim) is required for the specification and function of midline cells, similar to its role in Drosophila. Ph-sim RNAi causes severe dorsalization and embryos fail to specify nervous system or limbs posterior to the second Antennal segment. EGF signaling also appears to be a property of the Parhyale midline, but may not be responsible for its inductive role in DV patterning. Instead, I propose that the patterning properties of the Parhyale midline are mediated by BMP inhibition, possibly by Parhyale sog. It is also likely that sog was expressed in the ventral midline in the last common ancestor of insects and crustaceans (Pancrustacea). This hypothesis provides a new basis for understanding the evolutionary transition that has occurred in the insect lineage, where the Dorsal activity gradient has become a central feature of DV patterning.