Fgf8 signaling for development of the midbrain and hindbrain

The Wnt-1 (int-1) proto-oncogene, which encodes a putative signaling molecule, is expressed exclusively in the developing central nervous system and adult testes. To examine the role of Wnt-1, we generated six independent embryonic stem cell lines in which insertion of a neoR gene by homologous recombination inactivated a Wnt-1 allele. Germline chimeras were generated from two lines, and progeny from matings between heterozygous parents were examined. In all day 9.5 fetuses homozygous for mutated Wnt-1 alleles, most of the midbrain and some rostral metencephalon were absent. The remainder of the neural tube and all other tissues were normal. In late-gestation homozygotes, there was virtually no midbrain and no cerebellum, while the rest of the fetus was normal. Homozygotes are born, but die within 24 hr. Thus the normal role of Wnt-1 is in determination or subsequent development of a specific region of the central nervous system.

Receptor tyrosine kinases (RTKs) control a wide variety of processes in multicellular organisms, including proliferation, differentiation, migration and survival. Their activity is tightly controlled through the coordinated action of both positive and negative regulators that function at multiple levels of the signal transduction cascade, and at different time points within the growth-factor-induced response. When this process goes awry, the outcome can be developmental defects and malignancy. Sprouty (Spry) proteins represent a major class of ligand-inducible inhibitors of RTK-dependent signaling pathways. New biochemical and genetic evidence indicates specific roles of the Spry genes in development and multiple modes of action of the Spry proteins in regulation of the RTK-induced response.

The int-1 proto-oncogene was first identified as a gene activated in virally induced mouse mammary tumours. Expression studies, however, suggest that the normal function of this gene may be in spermatogenesis and in the development of the central nervous system. Genes sharing sequence similarity with int-1 have been found throughout the animal kingdom. For example, int-1 has 54% amino-acid identity to the Drosophila segment polarity gene wingless (wg). Both the int-1 and wg gene products seem to be secreted proteins, presumably involved in cell-cell signalling. We have now explored the function of int-1 in the mouse by disrupting one of the two int-1 alleles in mouse embryo-derived stem cells using positive-negative selection. This cell line was used to generate a chimaeric mouse that transmitted the mutant allele to its progeny. Mice heterozygous for the int-1 null mutation are normal and fertile, whereas mice homozygous for the mutation may exhibit a range of phenotypes from death before birth to survival with severe ataxia. The latter pathology in mice and humans is often associated with defects in the cerebellum. Examination of int-1-/int-1- mice at several stages of embryogenesis revealed severe abnormalities in the development of the mesencephalon and metencephalon indicating a prominent role for the int-1 protein is in the induction of the mesencephalon and cerebellum.