Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTPsigma

The LAR-family protein tyrosine phosphatase sigma (PTPsigma, encoded by the gene Ptprs) consists of a cell adhesion-like extracellular domain composed of immunoglobulin and fibronectin type-III repeats, a single transmembrane domain and two intracellular catalytic domains. It was previously shown to be expressed in neuronal and lung epithelial tissues in a developmentally regulated manner. To study the role of PTPsigma in mouse development, we inactivated Ptprs by gene targeting. All Ptprs+/- mice developed normally, whereas 60% of Ptprs-/- mice died within 48 hours after birth. The surviving Ptprs-/- mice demonstrated stunted growth, developmental delays and severe neurological defects including spastic movements, tremor, ataxic gait, abnormal limb flexion and defective proprioception. Histopathology of brain sections revealed reduction and hypocellularity of the posterior pituitary of Ptprs-/- mice, as well as a reduction of approximately 50-75% in the number of choline acetyl transferase-positive cells in the forebrain. Moreover, peripheral nerve electrophysiological analysis revealed slower conduction velocity in Ptprs-/- mice relative to wild-type or heterozygous animals, associated with an increased proportion of slowly conducting, small-diameter myelinated fibres and relative hypomyelination. By approximately three weeks of age, most remaining Ptprs-/- mice died from a wasting syndrome with atrophic intestinal villi. These results suggest that PTPsigma has a role in neuronal and epithelial development in mice.     

Disruption of CREB function in brain leads to neurodegeneration

Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To define its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem(-/-) background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.     

Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal interface of mouse placenta

Eutherian placenta, an organ that emerged in the course of mammalian evolution, provides essential architecture, the so-called feto-maternal interface, for fetal development by exchanging nutrition, gas and waste between fetal and maternal blood. Functional defects of the placenta cause several developmental disorders, such as intrauterine growth retardation in humans and mice. A series of new inventions and/or adaptations must have been necessary to form and maintain eutherian chorioallantoic placenta, which consists of capillary endothelial cells and a surrounding trophoblast cell layer(s). Although many placental genes have been identified, it remains unknown how the feto-maternal interface is formed and maintained during development, and how this novel design evolved. Here we demonstrate that retrotransposon-derived Rtl1 (retrotransposon-like 1), also known as Peg11 (paternally expressed 11), is essential for maintenance of the fetal capillaries, and that both its loss and its overproduction cause late-fetal and/or neonatal lethality in mice.     

Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.     

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.     

Meiotic arrest and aneuploidy in MLH3-deficient mice

MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis. The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2 6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process. Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis. Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice. To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3(-/-) mice. Here we show that Mlh3(-/-) mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I. Mlh3(-/-) spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization. Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.     

A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.     

Regulation of left-right patterning in mice by growth/differentiation factor-1

The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1) is a TGF-beta family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous systemin late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdfl is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1-/- mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1-/- embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2), nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.     

beta-cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter beta-cell mass

Regulation of glucose homeostasis by insulin depends on the maintenance of normal beta-cell mass and function. Insulin-like growth factor 1 (Igf1) has been implicated in islet development and differentiated function, but the factors controlling this process are poorly understood. Pancreatic islets produce Igf1 and Igf2, which bind to specific receptors on beta-cells. Igf1 has been shown to influence beta-cell apoptosis, and both Igf1 and Igf2 increase islet growth; Igf2 does so in a manner additive with fibroblast growth factor 2 (ref. 10). When mice deficient for the Igf1 receptor (Igf1r(+/-)) are bred with mice lacking insulin receptor substrate 2 (Irs2(-/-)), the resulting compound knockout mice show a reduction in mass of beta-cells similar to that observed in pancreas of Igf1r(-/-) mice (ref. 11), suggesting a role for Igf1r in growth of beta-cells. It is possible, however, that the effects in these mice occur secondary to changes in vascular endothelium or in the pancreatic ductal cells, or because of a decrease in the effects of other hormones implicated in islet growth. To directly define the role of Igf1, we have created a mouse with a beta-cell-specific knockout of Igf1r (betaIgf1r(-/-)). These mice show normal growth and development of beta-cells, but have reduced expression of Slc2a2 (also known as Glut2) and Gck (encoding glucokinase) in beta-cells, which results in defective glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, Igf1r is not crucial for islet beta-cell development, but participates in control of differentiated function.     

Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis

Inhibition of telomerase is proposed to limit the growth of cancer cells by triggering telomere shortening and cell death. Telomere maintenance by telomerase is sufficient, in some cell types, to allow immortal growth. Telomerase has been shown to cooperate with oncogenes in transforming cultured primary human cells into neoplastic cells, suggesting that telomerase activation contributes to malignant transformation. Moreover, telomerase inhibition in human tumour cell lines using dominant-negative versions of TERT leads to telomere shortening and cell death. These findings have led to the proposition that telomerase inhibition may result in cessation of tumour growth. The absence of telomerase from most normal cells supports the potential efficacy of anti-telomerase drugs for tumour therapy, as its inhibition is unlikely to have toxic effects. Mice deficient for Terc RNA (encoding telomerase) lack telomerase activity, and constitute a model for evaluating the role of telomerase and telomeres in tumourigenesis. Late-generation Terc-/- mice show defects in proliferative tissues and a moderate increase in the incidence of spontaneous tumours in highly proliferative cell types (lymphomas, teratocarcinomas). The appearance of these tumours is thought to be a consequence of chromosomal instability in these mice. These observations have challenged the expected effectiveness of anti-telomerase-based cancer therapies. Different cell types may nonetheless vary in their sensitivity to the chromosomal instability produced by telomere loss or to the activation of telomere-rescue mechanisms. Here we show that late-generation Terc-/- mice, which have short telomeres and are telomerase-deficient, are resistant to tumour development in multi-stage skin carcinogenesis. Our results predict that an anti-telomerase-based tumour therapy may be effective in epithelial tumours.     

Retinoic acid rescues inner ear defects in Hoxa1 deficient mice

Little is known about the genetic pathways involved in the early steps of inner ear morphogenesis. Hoxa1 is transiently expressed in the developing hindbrain; its targeted inactivation in mice results in severe abnormalities of the otic capsule and membranous labyrinth. Here we show that a single maternal administration of a low dose of the vitamin A metabolite retinoic acid is sufficient to compensate the requirement for Hoxa1 function. It rescues cochlear and vestibular defects in mutant fetuses without affecting the development of the wildtype fetuses. These results identify a temporal window of susceptibility to retinoids that is critical for mammalian inner ear specification, and provide the first evidence that a subteratogenic dose of vitamin A derivative can be effective in rescuing a congenital defect in the mammalian embryo.     

A mutation in stratifin is responsible for the repeated epilation (Er) phenotype in mice

Stratifin (Sfn, also called 14-3-3sigma) is highly expressed in differentiating epidermis and mediates cell cycle arrest. Sfn is repressed in cancer, but its function during development is uncharacterized. We identified an insertion mutation in the gene Sfn in repeated epilation (Er) mutant mice by positional cloning. Er/+ mice expressed a truncated Sfn protein, which probably contributes to the defects in Er/Er and Er/+ epidermis and to cancer development in Er/+ mice.     

Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system

Mouse embryos deficient in Gata3 die by 11 days post coitum (d.p.c.) from pathology of undetermined origin. We recently showed that Gata3-directed lacZ expression of a 625-kb Gata3 YAC transgene in mice mimics endogenous Gata3 expression, except in thymus and the sympathoadrenal system. As this transgene failed to overcome embryonic lethality (unpublished data and ref. 3) in Gata3-/- mice, we hypothesized that a neuroendocrine deficiency in the sympathetic nervous system (SNS) might cause embryonic lethality in these mutants. We find here that null mutation of Gata3 leads to reduced accumulation of Th (encoding tyrosine hydroxylase, Th) and Dbh (dopamine beta-hydroxylase, Dbh) mRNA, whereas several other SNS genes are unaffected. We show that Th and Dbh deficiencies lead to reduced noradrenaline in the SNS, and that noradrenaline deficiency is a proximal cause of death in mutants by feeding catechol intermediates to pregnant dams, thereby partially averting Gata3 mutation-induced lethality. These older, pharmacologically rescued mutants revealed abnormalities that previously could not be detected in untreated mutants. These late embryonic defects include renal hypoplasia and developmental defects in structures derived from cephalic neural crest cells. Thus we have shown that Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis.     

Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9.

The initial stages of pancreatic development occur early during mammalian embryogenesis, but the genes governing this process remain largely unknown. The homeodomain protein Pdx1 is expressed in the developing pancreatic anlagen from the approximately 10-somite stage, and mutations in the gene Pdx1 prevent the development of the pancreas. The initial stages of pancreatic development, however, still occur in Pdx1-deficient mice. Hlxb9 (encoding Hb9; ref. 6) is a homeobox gene that in humans has been linked to dominant inherited sacral agenesis and we show here that Hb9 is expressed at early stages of mouse pancreatic development and later in differentiated beta-cells. Hlxb9 has an essential function in the initial stages of pancreatic development. In absence of Hlxb9 expression, the dorsal region of the gut epithelium fails to initiate a pancreatic differentiation program. In contrast, the ventral pancreatic endoderm develops but exhibits a later and more subtle perturbation in beta-cell differentiation and in islet cell organization. Thus, dorsally Hlxb9 is required for specifying the gut epithelium to a pancreatic fate and ventrally for ensuring proper endocrine cell differentiation.     

Vitamin A controls epithelial/mesenchymal interactions through Ret expression

Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.     

Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome

Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in fibrillin-1 (encoded by FBN1 in humans and Fbn1 in mice), a matrix component of extracellular microfibrils. A distinct subgroup of individuals with Marfan syndrome have distal airspace enlargement, historically described as emphysema, which frequently results in spontaneous lung rupture (pneumothorax; refs. 1-3). To investigate the pathogenesis of genetically imposed emphysema, we analyzed the lung phenotype of mice deficient in fibrillin-1, an accepted model of Marfan syndrome. Lung abnormalities are evident in the immediate postnatal period and manifest as a developmental impairment of distal alveolar septation. Aged mice deficient in fibrillin-1 develop destructive emphysema consistent with the view that early developmental perturbations can predispose to late-onset, seemingly acquired phenotypes. We show that mice deficient in fibrillin-1 have marked dysregulation of transforming growth factor-beta (TGF-beta) activation and signaling, resulting in apoptosis in the developing lung. Perinatal antagonism of TGF-beta attenuates apoptosis and rescues alveolar septation in vivo. These data indicate that matrix sequestration of cytokines is crucial to their regulated activation and signaling and that perturbation of this function can contribute to the pathogenesis of disease.     

A mutation in Sec15l1 causes anemia in hemoglobin deficit (hbd) mice

Hemoglobin deficit (hbd) mice carry a spontaneous mutation that impairs erythroid iron assimilation but does not cause other defects. Normal delivery of iron to developing erythroid precursors is highly dependent on the transferrin cycle. Through genetic mapping and complementation experiments, we show that the hbd mutation is an in-frame deletion of a conserved exon of the mouse gene Sec15l1, encoding one of two Sec15 proteins implicated in the mammalian exocyst complex. Sec15l1 is linked to the transferrin cycle through its interaction with Rab11, a GTPase involved in vesicular trafficking. We propose that inactivation of Sec15l1 alters recycling of transferrin cycle endosomes and increases the release of transferrin receptor exocytic vesicles. This in turn decreases erythroid iron uptake. Determining the molecular basis of the hbd phenotype provides new insight into the intricate mechanisms necessary for normal erythroid iron uptake and the function of a mammalian exocyst protein.