Abstract:
The segmentation of the axial skeleton and peripheral nervous system involves a complex integration of multiple patterning molecules. For the latter, axon-repelling molecules in the posterior half-sclerotome are particularly important. This study built on a previously performed mouse microarray screen for novel candidate genes in the posterior half-sclerotome. Multiple candidates were selected for whole-mount in situ hybridization in chick. Two were expressed in the posterior half-sclerotome: thrombin receptor (F2R) and fibronectin leucine rich transmembrane protein-2 (Flrt2). Flrt2 was selected for siRNA-mediated knockdown and a new in ovo transfection technique for somites successfully developed. Scrambled siRNA-transfection did not affect morphogenesis, somite patterning or axon guidance. However, Flrt2 siRNA-transfection resulted in defects in notochord, dermomyotome and neural tube morphogenesis, and in the de-fasciculation and mis-targeting of spinal axons into the posterior half-sclerotome and dermomyotome. Hence, Flrt2 may be a chemorepellent for spinal axons.
An unidentified peanut agglutinin (PNA)-binding glycoprotein in the posterior half-sclerotome was previously shown to repel spinal axons. In this project, the expression of a family of mucin-type O-glycosylation enzymes (which could glycosylate the PNA-binding protein) was investigated by whole-mount in situ hybridization in chick, but none was differentially expressed in the posterior half-sclerotome. One candidate for the PNA-binding glycoprotein, Presenilin1, was investigated because of previously published loss of spinal nerve segmentation in Presenilin1 mutants. However, analysis of Presenilin1-hypomorphic mutant mouse embryos showed this was not the PNA-binding molecule. Live-immunostaining for a second candidate, prolyl 4-hydroxylase, beta polypeptide (P4HB), showed its expression coincided with PNA-binding at the surface of posterior half-sclerotome cells. P4HB siRNA-transfection into somites reduced PNA binding and disrupted spinal axon segmentation and expression of a posterior sclerotome marker, Uncx4.1. Overall, these results suggest that P4HB is a strong candidate to be the key PNA-binding glycoprotein in the posterior half-sclerotome that repels spinal axons.
