Various in vitro studies have suggested that ErbB4 (HER4) is a receptor for the neuregulins, a family of closely related proteins implicated as regulators of neural and muscle development, and of the differentiation and oncogenic transformation of mammary epithelia. Here we demonstrate that ErbB4 is an essential in vivo regulator of both cardiac muscle differentiation and axon guidance in the central nervous system (CNS). Mice lacking ErbB4 die during mid-embryogenesis from the aborted development of myocardial trabeculae in the heart ventricle. They also display striking alterations in innervation of the hindbrain in the CNS that are consistent with the restricted expression of the ErbB4 gene in rhombomeres 3 and 5. Similarities in the cardiac phenotype of ErbB4 and neuregulin gene mutants suggest that ErbB4 functions as a neuregulin receptor in the heart; however, differences in the hindbrain phenotypes of these mutants are consistent with the action of a new ErbB4 ligand in the CNS.

We have generated and analysed null mutations in the mouse genes encoding three structurally related receptors with tyrosine kinase activity: Tyro 3, Axl, and Mer. Mice lacking any single receptor, or any combination of two receptors, are viable and fertile, but male animals that lack all three receptors produce no mature sperm, owing to the progressive death of differentiating germ cells. This degenerative phenotype appears to result from a failure of the tropic support that is normally provided by Sertoli cells of the seminiferous tubules, whose function depends on testosterone and additional factors produced by Leydig cells. Tyro 3, Axl and Mer are all normally expressed by Sertoli cells during postnatal development, whereas their ligands, Gas6 and protein S, are produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter. Here we show that the concerted activation of Tyro 3, Axl and Mer in Sertoli cells is critical to the role that these cells play as nurturers of developing germ cells. Additional observations indicate that these receptors may also be essential for the tropic maintenance of diverse cell types in the mature nervous, immune and reproductive systems.

Hepatocyte growth factor (HGF) and its receptor, the Met tyrosine kinase, are determinants of placenta, liver, and muscle development. Here, we show that Met function in vivo requires signaling via two carboxy-terminal tyrosines. Mutation of both residues in the mouse genome caused embryonal death, with placenta, liver, and limb muscle defects, mimicking the phenotype of met null mutants. In contrast, disrupting the consensus for Grb2 binding allowed development to proceed to term without affecting placenta and liver but caused a striking reduction in limb muscle coupled to a generalized deficit of secondary fibers. These data show that the requirements for Met signaling vary depending on the tissue and reveal a novel role for HGF/ Met in late myogenesis.

Neurotrophins are a family of soluble ligands that promote the survival and differentiation of peripheral and central neurons and regulate synaptic function. The two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), bind and activate a single high-affinity receptor, TrkB. Experiments in cell culture have revealed that an intact Shc adaptor binding site on TrkB and subsequent activation of the Ras/MAPK pathway are important for neuronal survival and neurite outgrowth. To elucidate the intracellular signaling pathways that mediate the diverse effects of BDNF and NT4 in vivo, we have mutated in the mouse germline the Shc binding site in the trkB gene. This trkB(shc) mutation revealed distinctive responses to BDNF and NT4. While nearly all NT4-dependent sensory neurons were lost in trkB(shc/shc) mutant mice, BDNF-dependent neurons were only modestly affected. Activation of MAP kinases and in vitro survival of cultured trkB(shc/shc) neurons were reduced in response to both neurotrophins, with NT4 being less potent than BDNF, suggesting differential activation of TrkB by the two ligands. Moreover, while the Ras/MAPK pathway is required for in vitro differentiation of neuronal cells, trkB(shc/shc) mutant mice do not show any defects in BDNF-dependent differentiation of CNS neurons or in the function of sensory neurons that mediate innocuous touch.

Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lynup/up mice). Lynup/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lynup/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cγ2 in resting Lynup/up B cells. Similarly, Lynup/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lynup/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.