Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation.

The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1.

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

The transcription factor Sox10 is a key regulator of peripheral glial development.

The molecular mechanisms that determine glial cell fate in the vertebrate nervous system have not been elucidated. Peripheral glial cells differentiate from pluripotent neural crest cells. We show here that the transcription factor Sox10 is a key regulator in differentiation of peripheral glial cells. In mice that carry a spontaneous or a targeted mutation of Sox10, neuronal cells form in dorsal root ganglia, but Schwann cells or satellite cells are not generated. At later developmental stages, this lack of peripheral glial cells results in a severe degeneration of sensory and motor neurons. Moreover, we show that Sox10 controls expression of ErbB3 in neural crest cells. ErbB3 encodes a Neuregulin receptor, and down-regulation of ErbB3 accounts for many changes in development of neural crest cells observed in Sox10 mutant mice. Sox10 also has functions not mediated by ErbB3, for instance in the melanocyte lineage. Phenotypes observed in heterozygous mice that carry a targeted Sox10 null allele reproduce those observed in heterozygous Sox10(Dom) mice. Haploinsufficiency of Sox10 can thus cause pigmentation and megacolon defects, which are also observed in Sox10(Dom)/+ mice and in patients with Waardenburg-Hirschsprung disease caused by heterozygous SOX10 mutations.

Neuregulin, a factor with many functions in the life of a schwann cell.

The signalling system comprising the ligand Neuregulin-1, and its receptors, ErbB2 and ErbB3, plays multiple and important roles in glial development. These include functions in early development of neural crest cells, in expansion of the Schwann cell precursor pool and in myelination. Neuregulin is one of the crucial axon-derived signals that influence development of Schwann cells. These are specialized cells that ensheath peripheral axons and provide electrical insulation. Schwann cells have also long been implicated in providing more than a simple ensheathing function. Compelling evidence for this has emerged from the analysis of mice lacking these cells, resulting from a non-functional or compromised Neuregulin signalling system. They serve as a model to study glia-nerve interactions in vivo and indicate that Schwann cells provide important neurotrophic signals, and also cues that regulate perineurium development and nerve fasciculation.

Genes that control the development of migrating muscle precursor cells.

Skeletal muscles in vertebrates, despite their functional and biochemical similarities, are generated via diverse developmental mechanisms. A major subclass of hypaxial muscle groups is derived from long-range migrating progenitor cells that delaminate from the dermomyotome. The development of this lineage is controlled by Pax3, the c-Met tyrosine kinase receptor, its ligand SF/HGF (scatter factor/hepatocyte growth factor) and the homeobox factor Lbx1. These molecules are essential for establishment of the precursor pool, delamination, migration and target finding. Progress has been made in understanding patterning of the muscles, which requires a precise control of proliferation and differentiation of myogenic precursor cells.

E-cadherin and adenomatous polyposis coli mutations are synergistic in intestinal tumor initiation in mice.

BACKGROUND & AIMS: Inactivation of the adenomatous polyposis coli (APC) gene is observed at early stages of intestinal tumor formation, whereas loss of E-cadherin is usually associated with tumor progression. Because both proteins compete for the binding to beta-catenin, an essential component of the Wnt signaling pathway, reduction of E-cadherin levels in an Apc mouse model could influence both tumor initiation and progression. In addition, loss or haploinsufficiency of E-cadherin may affect tumorigenesis by altering its cell-adhesive and associated functions. METHODS: Apc1638N mice were bred with animals carrying a targeted E-cadherin knockout mutation. RESULTS: Double heterozygous animals showed a significant 9-fold and 5-fold increase of intestinal and gastric tumor numbers, respectively, compared with Apc1638N animals. The intestinal tumors of both groups showed no significant differences in grading and staging. Loss of heterozygosity analysis at the Apc and E-cadherin loci in both intestinal and gastric Apc(+/1638N)/E-cad(+/-) tumors revealed loss of the wild-type Apc allele in most cases, whereas the wild-type E-cadherin allele was always retained. This was supported by a positive, although reduced, staining for E-cadherin of intestinal tumor sections. CONCLUSIONS: Introduction of the E-cadherin mutation in Apc1638N animals enhances Apc-driven tumor initiation without clearly affecting tumor progression.

Essential role of Gab1 for signaling by the c-Met receptor in vivo.

The docking protein Gab1 binds phosphorylated c-Met receptor tyrosine kinase directly and mediates signals of c-Met in cell culture. Gab1 is phosphorylated by c-Met and by other receptor and nonreceptor tyrosine kinases. Here, we report the functional analysis of Gab1 by targeted mutagenesis in the mouse, and compare the phenotypes of the Gab1 and c-Met mutations. Gab1 is essential for several steps in development: migration of myogenic precursor cells into the limb anlage is impaired in Gab1-/- embryos. As a consequence, extensor muscle groups of the forelimbs are virtually absent, and the flexor muscles reach less far. Fewer hindlimb muscles exist, which are smaller and disorganized. Muscles in the diaphragm, which also originate from migratory precursors, are missing. Moreover, Gab1-/- embryos die in a broad time window between E13.5 and E18.5, and display reduced liver size and placental defects. The labyrinth layer, but not the spongiotrophoblast layer, of the placenta is severely reduced, resulting in impaired communication between maternal and fetal circulation. Thus, extensive similarities between the phenotypes of c-Met and HGF/SF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1-/-:c-Met+/- embryos. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin receptors, which require IRS1 and IRS2 as specific docking proteins.

The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons.

During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies.

A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool.

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.

Requirement for beta-catenin in anterior-posterior axis formation in mice.

The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Tauhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. Beta-catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated beta-catenin-deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for beta-catenin. Our data demonstrate that beta-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required in the embryonic ectoderm.

The role of Lbx1 in migration of muscle precursor cells.

The homeobox gene Lbx1 is expressed in migrating hypaxial muscle precursor cells during development. These precursors delaminate from the lateral edge of the dermomyotome and form distinct streams that migrate over large distances, using characteristic paths. The targets of migration are limbs, septum transversum and the floor of the first branchial arch where the cells form skeletal muscle of limbs and shoulders, diaphragm and hypoglossal cord, respectively. We used gene targeting to analyse the function of Lbx1 in the mouse. Myogenic precursor cells delaminate from the dermomyotome in Lbx1 mutants, but migrate in an aberrant manner. Most critically affected are migrating cells that move to the limbs. Precursor cells that reach the dorsal limb field are absent. In the ventral limb, precursors are present but distributed in an abnormal manner. As a consequence, at birth some muscles in the forelimbs are completely lacking (extensor muscles) or reduced in size (flexor muscles). Hindlimb muscles are affected strongly, and distal limb muscles are more affected than proximal ones. Other migrating precursor cells heading towards the floor of the first branchial arch move along the appropriate path in Lbx1 mutants. However, these cells migrate less efficiently and reduced numbers of precursors reach their distal target. At birth, the internal lingual muscle is therefore reduced in size. We suggest that Lbx1 controls the expression of genes that are essential for the recognition or interpretation of cues that guide migrating muscle precursors and maintain their migratory potential.

A role of the cryptic gene in the correct establishment of the left-right axis.

During vertebrate embryogenesis, a left-right axis is established. The heart, associated vessels and inner organs adopt asymmetric spatial arrangements and morphologies. Secreted growth factors of the TGF-beta family, including nodal, lefty-1 and lefty-2, play crucial roles in establishing left-right asymmetries [1] [2] [3]. In zebrafish, nodal signalling requires the presence of one-eyed pinhead (oep), a member of the EGF-CFC family of membrane-associated proteins [4]. We have generated a mutant allele of cryptic, a mouse EGF-CFC gene [5]. Homozygous cryptic mutants developed to birth, but the majority died during the first week of life because of complex cardiac malformations such as malpositioning of the great arteries, and atrial-ventricular septal defects. Moreover, laterality defects, including right isomerism of the lungs, right or left positioning of the stomach and splenic hypoplasia were observed. Nodal gene expression in the node was initiated in cryptic mutant mice, but neither nodal, lefty-2 nor Pitx2 were expressed in the left lateral plate mesoderm. The laterality defects observed in cryptic(-/-) mice resemble those of mice lacking the type IIB activin receptor or the homeobox-containing factor Pitx2 [6] [7] [8] [9], and are reminiscent of the human asplenic syndrome [10]. Our results provide genetic evidence for a role of cryptic in the signalling cascade that determines left-right asymmetry.

MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis.

MAPKAP kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAP kinase. By introducing a targeted mutation into the mouse MK2 gene, we have determined the physiological function of MK2 in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is due to a reduction of approximately 90% in the production of tumor necrosis factor-alpha (TNF-alpha) and not to a change in signalling from the TNF receptor. The level and stability of TNF-alpha mRNA is not reduced and TNF-alpha secretion is not affected. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNF-alpha at a post-transcriptional level.

Peripheral nervous system defects in erbB2 mutants following genetic rescue of heart development.

The ErbB2 tyrosine kinase functions as coreceptor for the neuregulin receptors ErbB3 and ErbB4 and can participate in signaling of EGF receptor (ErbB1), interleukin receptor gp130, and G-protein coupled receptors. ErbB2(-/-) mice die at midgestation because of heart malformation. Here, we report a genetic rescue of their heart development by myocardial expression of erbB2 cDNA that allows survival of the mutants to birth. In rescued erbB2 mutants, Schwann cells are lacking. Motoneurons form and can project to muscle, but nerves are poorly fasciculated and disorganized. Neuromuscular junctions form, as reflected in clustering of AChR and postsynaptic expression of the genes encoding the alpha-AChR, AChE, epsilon-AChR, and the RI subunit of the cAMP protein kinase. However, a severe loss of motoneurons on cervical and lumbar, but not on thoracic levels occurs. Our results define the roles of Schwann cells during motoneuron and synapse development, and reveal different survival requirements for distinct motoneuron populations.

The role of SF/HGF and c-Met in the development of skeletal muscle.

Hypaxial skeletal muscles develop from migratory and non-migratory precursor cells that are generated by the lateral lip of the dermomyotome. Previous work shows that the formation of migratory precursors requires the c-Met and SF/HGF genes. We show here that in mice lacking c-Met or SF/HGF, the initial development of the dermomyotome proceeds appropriately and growth and survival of cells in the dermomyotome are not affected. Migratory precursors are also correctly specified, as monitored by the expression of Lbx1. However, these cells remain aggregated and fail to take up long range migration. We conclude that parallel but independent cues converge on the migratory hypaxial precursors in the dermomyotomal lip after they are laid down: a signal given by SF/HGF that controls the emigration of the precursors, and an as yet unidentified signal that controls Lbx1. SF/HGF and c-Met act in a paracrine manner to control emigration, and migratory cells only dissociate from somites located close to SF/HGF-expressing cells. During long range migration, prolonged receptor-ligand-interaction appears to be required, as SF/HGF is expressed both along the routes and at the target sites of migratory myogenic progenitors. Mice that lack c-Met die during the second part of gestation due to a placental defect. Rescue of the placental defect by aggregation of tetraploid (wild type) and diploid (c-Met-/-) morulae allows development of c-Met mutant animals to term. They lack muscle groups that derive from migratory precursor cells, but display otherwise normal skeletal musculature.

Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase.

A number of developmental processes that involve cell migration, growth or morphogenesis depend on extracellular signals. A molecule that provides such signals, known as hepatocyte growth factor/scatter factor (HGF/SF), has attracted considerable interest in recent years because of its distinct structure, mechanism of activation and important roles throughout embryogenesis. This review discusses the main features of HGF/SF and its receptor, the product of the c-met protooncogene, and their role in embryogenesis.

Sustained long term potentiation and anxiety in mice lacking the Mas protooncogene.

The Mas protooncogene is a maternally imprinted gene encoding an orphan G protein-coupled receptor expressed mainly in forebrain and testis. Here, we provide evidence for a function of Mas in the central nervous system. Targeted disruption of the Mas protooncogene leads to an increased durability of long term potentiation in the dentate gyrus, without affecting hippocampal morphology, basal synaptic transmission, and presynaptic function. In addition, Mas-/- mice show alterations in the onset of depotentiation. The permissive influence of Mas ablation on hippocampal synaptic plasticity is paralleled by behavioral changes. While spatial learning in the Morris water maze is not significantly influenced, Mas-deficient animals display an increased anxiety as assessed in the elevated-plus maze. Thus, Mas is an important modulating factor in the electrophysiology of the hippocampus and is involved in behavioral pathways in the adult brain.

The ErbB2 and ErbB3 receptors and their ligand, neuregulin-1, are essential for development of the sympathetic nervous system.

Neuregulins (NDF, heregulin, GGF ARIA, or SMDF) are EGF-like growth and differentiation factors that signal through tyrosine kinase receptors of the ErbB family. Here, we report a novel phenotype in mice with targeted mutations in the erbB2, erbB3, or neuregulin-1 genes. These three mutations cause a severe hypoplasia of the primary sympathetic ganglion chain. We provide evidence that migration of neural crest cells to the mesenchyme lateral of the dorsal aorta, in which they differentiate into sympathetic neurons, depends on neuregulin-1 and its receptors. Neuregulin-1 is expressed at the origin of neural crest cells. Moreover, a tight link between neuregulin-1 expression, the migratory path, and the target site of sympathogenic neural crest cells is observed. Sympathetic ganglia synthesize catecholamines in the embryo and the adult. Accordingly, catecholamine levels in mutant embryos are severely decreased, and we suggest that the lack of catecholamines contributes to the embryonal lethality of the erbB3 mutant mice. Thus, neuregulin-1, erbB2, and erbB3 are required for the formation of the sympathetic nervous system; the block in development observed in mutant mice is caused by a lack of neural crest precursor cells in the anlage of the primary sympathetic ganglion chain. Together with previous observations, these findings establish the neuregulin signaling system as a key regulator in the development of neural crest cells.