The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1.

Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We describe cloning of a candidate dt gene, dystonin, that is predominantly expressed in the dorsal root ganglia and other sites of neurodegeneration in dt mice. Dystonin encodes an N-terminal actin binding domain and a C-terminal portion comprised of the hemidesmosomal protein, bullous pemphigoid antigen 1 (bpag1). dt and bpag1 are part of the same transcription unit which is partially deleted in a transgenic strain of mice, Tg4, that harbours an insertional mutation at the dt locus, and in mice that carry a spontaneous dt mutation, dtAlb. We also demonstrate abnormal dystonin transcripts in a second dt mutant, dt24J. We conclude that mutations in the dystonin gene are the primary genetic lesion in dt mice.

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development.

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.

Genomic imprinting of Mash2, a mouse gene required for trophoblast development.

The mouse gene Mash2 encodes a transcription factor required for development of trophoblast progenitors. Mash2-homozygous mutant embryos die at 10 days postcoitum from placental failure. Here we show that Mash2 is genomically imprinted. First, Mash2+/- embryos inheriting a wild-type allele from their father die at the same stage as -/- embryos, with a similar placental phenotype. Second, the Mash2 paternal allele is initially expressed by groups of trophoblast cells at 6.5 and 7.5 days post-coitum, but appears almost completely repressed by 8.5 days post-coitum. Finally, we have genetically and physically mapped Mash2 to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor-2 and H19.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

The missense mutation W290R in Fgfr2 causes developmental defects from aberrant IIIb and IIIc signaling.

Missense mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) have been identified in human craniosynostotic syndromes such as Crouzon (CS) and Pfeiffer (PS). FGFR2 has two major isoforms, IIIb and IIIc, generated through alternative splicing with their own temporal, spatial, and ligand-binding specificities. In this study, we report the identification and characterization of a missense mutation in codon 290 of murine Fgfr2 (W290R). The defects in W290R mutants are suggestive of disruption of signalling in both IIIb and IIIc isoforms of the Fgfr2 gene. Heterozygous mutants presented with features resembling those found in patients with CS. Fgfr2(W290R) homozygotes displayed constitutive FGFR2 activation with increased, but correct tissue-specific, expression of the IIIb and IIIc isoforms in many of the defective organs. Our Fgfr2(W290R) mouse model thus represents an excellent mouse model of CS to probe the many questions around the pathogenesis of craniosynostotic birth defects consequent to defects in FGF signaling.

FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak.

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. Here, we demonstrate that FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, we show that FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, we provide evidence that the attenuation of Wnt3a signaling observed in Fgfr1 -/- embryos can be rescued by lowering E-cadherin levels. We propose that modulation of cytoplasmic beta-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak.

Interspecific chimeras in mammals: successful production of live chimeras between Mus musculus and Mus caroli.

Live chimeras between two species of mouse, Mus musculus and Mus caroli, were produced by blastocyst injection. These chimeras were entirely similar to M. musculus in equilibrium with M. musculus chimeras in their somatic tissue organization. This is the first report of completely normal development of interspecific chimeras in mammals.

Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.

A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.

Liver organogenesis promoted by endothelial cells prior to vascular function.

The embryonic role of endothelial cells and nascent vessels in promoting organogenesis, prior to vascular function, is unclear. We find that early endothelial cells in mouse embryos surround newly specified hepatic endoderm and delimit the mesenchymal domain into which the liver bud grows. In flk-1 mutant embryos, which lack endothelial cells, hepatic specification occurs, but liver morphogenesis fails prior to mesenchyme invasion. We developed an embryo tissue explant system that permits liver bud vasculogenesis and show that in the absence of endothelial cells, or when the latter are inhibited, there is a selective defect in hepatic outgrowth. We conclude that vasculogenic endothelial cells and nascent vessels are critical for the earliest stages of organogenesis, prior to blood vessel function.

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene.

Transgenic mice carrying the gamma 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of beta-galactosidase activity. These results suggest that gamma 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse gamma 2-crystallin gene. In a broader context, this study also demonstrates the utility of beta-galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.

Promotion of trophoblast stem cell proliferation by FGF4.

The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.

Regulation of a transcription factor network required for differentiation and metabolism.

Hepatocyte nuclear factors (HNFs) are a heterogeneous class of evolutionarily conserved transcription factors that are required for cellular differentiation and metabolism. Mutations in HNF-1alphaand HNF-4alpha genes impair insulin secretion and cause type 2 diabetes. Regulation of HNF-4/HNF-1 expression by HNF-3alpha and HNF-3beta was studied in embryoid bodies in which one or both HNF-3alpha or HNF-3beta alleles were inactivated. HNF-3beta positively regulated the expression of HNF-4alpha/HNF-1alpha and their downstream targets, implicating a role in diabetes. HNF-3beta was also necessary for expression of HNF-3alpha. In contrast, HNF-3alpha acts as a negative regulator of HNF-4alpha/HNF-1alpha demonstrating that HNF-3alpha and HNF-3beta have antagonistic transcriptional regulatory functions in vivo. HNF-3alpha does not appear to act as a classic biochemical repressor but rather exerts its negative effect by competing for HNF-3 binding sites with the more efficient activator HNF-3beta. In addition, the HNF-3alpha/HNF-3beta ratio is modulated by the presence of insulin, providing evidence that the HNF network may have important roles in mediating the action of insulin.

Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox.

The two mouse genes, En-1 and En-2, that are homologs of the Drosophila segmentation gene engrailed, show overlapping spatially restricted patterns of expression in the neural tube during embryogenesis, suggestive of a role in regional specification. Mice homozygous for a targeted mutation that deletes the homeobox were viable and showed no obvious defects in embryonic development. This may be due to functional redundancy of En-2 and the related En-1 gene product during embryogenesis. Consistent with this hypothesis, the mutant mice showed abnormal foliation in the adult cerebellum, where En-2, and not En-1, is normally expressed.

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.

Cardiac malformation in neonatal mice lacking connexin43.

Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism.

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

Gene disruption in mammals.

In the past year, the first phenotypes have been reported for mutations targeted to developmentally relevant genes by homologous recombination in embryonic stem cells. The results indicate that the genetic circuitry of mammalian development is complex and will require more sophisticated analysis than simple gene disruption. Improvements in the technology of targeted mutagenesis may assist in such analysis.

Fibroblast growth factors in mammalian development.

Polypeptide growth factors are secreted signalling molecules that function as intercellular communicators. Detailed analyses of the expression and function of members of the fibroblast growth factor (FGF) family and their recepotors have demonstrated that the FGF signalling pathways play essential roles in regulating cellular proliferation, differentiation and tissue patterning during vertebrate embryogenesis. Recent studies on the molecular basis of human dysmorphic syndromes have revealed that aberrant FGF signalling during limb and skeletal development can lead to pathogenesis.

Forward genetic screen of mouse reveals dominant missense mutation in the P/Q-type voltage-dependent calcium channel, CACNA1A.

Dominant mutations of the P/Q-type Ca(2+) channel (CACNA1A) underlie several human neurological disorders, including episodic ataxia type 2, familial hemiplegic migraine 1 (FHM1) and spinocerebellar ataxia 6, but have not been found previously in the mouse. Here we report the first dominant ataxic mouse model of Cacna1a mutation. This Wobbly mutant allele of Cacna1a was identified in an ethylnitrosourea (ENU) mutagenesis dominant behavioral screen. Heterozygotes exhibit ataxia from 3 weeks of age and have a normal life span. Homozygotes have a righting reflex defect from postnatal day 8 and later develop severe ataxia and die prematurely. Both heterozygotes and homozygotes exhibit cerebellar atrophy with focal reduction of the molecular layer. No obvious loss of Purkinje cells or decrease in size of the granule cell layer was observed. Real-time polymerase chain reaction revealed altered expression levels of Cacna1g, Calb2 and Th in Wobbly cerebella, but Cacna1a messenger RNA and protein levels were unchanged. Positional cloning revealed that Wobbly mice have a missense mutation leading to an arginine to leucine (R1255L) substitution, resulting in neutralization of a positively charged amino acid in repeat III of voltage sensor segment S4. The dominance of the Wobbly mutation more closely resembles patterns of CACNA1A mutation in humans than previously described mouse recessive mutants (tottering, leaner, rolling Nagoya and rocker). Positive-charge neutralization in S4 has also been shown to underlie several cases of human dominant FHM1 with ataxia. The Wobbly mutant thus highlights the importance of the voltage sensor and provides a starting point to unravel the neuropathological mechanisms of this disease.

Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles.

The N-myc proto-oncogene is expressed in many organs of the mouse embryo, suggesting that it has multiple functions. A null mutation leads to mid-gestation lethality [1-4], obscuring the later roles of the gene in organogenesis. We have generated a multi-purpose gene alteration by combining the potential for homologous and site-specific recombination in a single targeting vector, and using the selectable marker for neomycin-resistance, neo, to downregulate gene activity. This allowed us to create a series of alleles that led to different levels of N-myc expression. The phenotypes revealed a spectrum of developmental problems. The hypomorphic allele produced can be repaired in situ by Cre-recombinase-mediated DNA excision. We show here for the first time the use of a single targeting vector to generate an allelic series. This, and the possibility of subsequent lineage-specific or conditional allele repair in situ, represent new genome modification strategies that can be used to investigate multiple functions of a single gene.