Complete Loss of Netrin-1 Results in Embryonic Lethality and Severe Axon Guidance Defects without Increased Neural Cell Death.

Jenea M Bin,Emilie Dumontier,Artur Kania,Jean-Francois Cloutier,Timothy E Kennedy,Karen Lai Wing Sun,Dong Han,Louis-Philippe Croteau

doi:10.1016/j.celrep.2015.07.028

Abstract

Netrin-1 regulates cell migration and adhesion during the development of the nervous system, vasculature, lung, pancreas, muscle, and mammary gland. It is also proposed to function as a dependence ligand that inhibits apoptosis; however, studies disagree regarding whether netrin-1 loss-of-function mice exhibit increased cell death. Furthermore, previously studied netrin-1 loss-of-function gene-trap mice express a netrin-1-β-galactosidase protein chimera with potential for toxic gain-of-function effects, as well as a small amount of wild-type netrin-1 protein. To unambiguously assess loss of function, we generated netrin-1 floxed and netrin-1 null mouse lines. Netrin-1(-/-) mice die earlier and exhibit more severe axon guidance defects than netrin-1 gene-trap mice, revealing that complete loss of function is more severe than previously reported. Netrin-1(-/-) embryos also exhibit increased expression of the netrin receptors DCC and neogenin that are proposed dependence receptors; however, increased apoptosis was not detected, inconsistent with netrin-1 being an essential dependence receptor ligand in the embryonic spinal cord.

Highlights

Netrin-1 is a secreted protein that regulates cell migration, cellcell interactions, and cell-extracellular matrix adhesion during the embryonic development of multiple tissues, including the nervous system, vasculature, lung, pancreas, muscle, and mammary gland (Lai Wing Sun et al, 2011)
The protein chimera presents the netrin-1 sequence on the extracellular face of the plasma membrane, raising the possibility that residual netrin-1 function may persist, even in mice homozygous for the insertion
Generation of the Netrin-1 Null Mouse Line To assess the consequences of complete loss of netrin-1 function, we generated a conditional netrin-1 allele by introducing two unidirectional loxP sites flanking the first coding exon of the netrin-1 gene

Summary

Introduction

Netrin-1 is a secreted protein that regulates cell migration, cellcell interactions, and cell-extracellular matrix adhesion during the embryonic development of multiple tissues, including the nervous system, vasculature, lung, pancreas, muscle, and mammary gland (Lai Wing Sun et al, 2011). The gene-trap line (netrin-1bgeo) that has been used to study netrin-1 loss of function contains a b-galactosidase transgene inserted in the second intron of the netrin-1 gene (Skarnes et al, 1995). This results in the expression of a truncated netrin1-b-galactosidase chimeric protein that includes the amino-terminal half of netrin-1. The protein chimera presents the netrin-1 sequence on the extracellular face of the plasma membrane, raising the possibility that residual netrin-1 function may persist, even in mice homozygous for the insertion. The netrin-1bgeo mice are hypomorphs that likely maintain some level of residual netrin-1 function

Results

Discussion

Conclusion