Sonic hedgehog is essential to foregut development.

Congenital malformation of the foregut is common in humans, with an estimated incidence of 1 in 3000 live births, although its aetiology remains largely unknown. Mice with a targeted deletion of Sonic hedgehog (Shh) have foregut defects that are apparent as early as embryonic day 9.5, when the tracheal diverticulum begins to outgrow. Homozygous Shh-null mutant mice show oesophageal atresia/stenosis, tracheo-oesophageal fistula and tracheal and lung anomalies, features similar to those observed in humans with foregut defects. The lung mesenchyme shows enhanced cell death, decreased cell proliferation and downregulation of Shh target genes. These results indicate that Shh is required for the growth and differentiation of the oesophagus, trachea and lung, and suggest that mutations in SHH and its signalling components may be involved in foregut defects in humans.

Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene.

The family of basic helix-loop-helix (bHLH) genes comprises transcription factors involved in many aspects of growth and development. We have previously described two bHLH transcription factors, Nhlh1 and Nhlh2 (originally named NSCL1 and NSCL2). The nucleotide and predicted protein sequences of Nhlh1 and Nhlh2 are homologous within their bHLH domain where there are only three conservative amino acid differences. During murine embryogenesis, Nhlh1 and Nhlh2 share an overlapping but distinct pattern of expression in the developing nervous system. To improve our understanding of the role of these genes during neurogenesis, we have generated mice containing targeted deletions of both genes and here describe our results for Nhlh2. Loss of Nhlh2 results in a disruption of the hypothalamic-pituitary axis in mice. Male Nhlh2-/- mice are microphallic, hypogonadal and infertile with alterations in circulating gonadotropins, a defect in spermatogenesis and a loss of instinctual male sexual behaviour. Female Nhlh2-/- mice reared alone are hypogonadal, but when reared in the presence of males, their ovaries and uteri develop normally and they are fertile. Both male and female homozygotes exhibit progressive adult-onset obesity. Nhlh2 is expressed in the ventral-medial and lateral hypothalamus, Rathke's pouch and in the anterior lobe of the adult pituitary. Our results support a role for Nhlh2 in the onset of puberty and the regulation of body weight metabolism.

Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism.

Tay-Sachs and Sandhoff diseases are clinically similar neurodegenerative disorders. These two sphingolipidoses are characterized by a heritable absence of beta-hexosaminidase A resulting in defective GM2 ganglioside degradation. Through disruption of the Hexa and Hexb genes in embryonic stem cells, we have established mouse models corresponding to each disease. Unlike the two human disorders, the two mouse models show very different neurologic phenotypes. Although exhibiting biochemical and pathologic features of the disease, the Tay-Sachs model showed no neurological abnormalities. In contrast, the Sandhoff model was severely affected. The phenotypic difference between the two mouse models is the result of differences in the ganglioside degradation pathway between mice and humans.

Differential effects of zeta and eta transgenes on early alpha/beta T cell development.

The zeta-family dimers (zeta, eta, and gamma) are a group of structurally and functionally related proteins that are expressed in developing thymocytes and function as signal transducing subunits of the T cell antigen receptor (TCR) and certain Ig Fc receptors. Zeta, eta, and gamma each contain one or more copies of a conserved tyrosine-based activation motif (TAM) that is known to be required for signal transduction. To examine the developmental importance of multiple or individual TAM elements we generated transgenic mice that express: (a) full-length (FL) zeta-chain (3 TAMs); (b) eta-chain, a naturally occurring variant of zeta that is derived from alternative splicing (2 TAMs); or (c) truncated zeta-chain (CT108; 1 TAM), under the control of the human CD2 promoter and regulatory elements. Unexpectedly, we found that overexpression of the FL zeta chain caused premature termination of RAG-1 and RAG-2 expression, prevented productive rearrangement of the TCR-alpha and TCR-beta genes and blocked entry of thymocytes into the CD4/CD8 developmental pathway. In contrast, we found that overexpression of eta or CT108 had no effect on normal thymocyte maturation. These results suggest that an early signaling pathway exists in precursor TCR- thymocytes that can regulate RAG-1 and RAG-2 expression and is differentially responsive to individual members of the zeta-family dimers.

Impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking CC chemokine receptor 1.

CC chemokine receptor 1 (CCR1) is expressed in neutrophils, monocytes, lymphocytes, and eosinophils, and binds the leukocyte chemoattractant and hematopoiesis regulator macrophage inflammatory protein (MIP)-1alpha, as well as several related CC chemokines. Four other CCR subtypes are known; their leukocyte and chemokine specificities overlap with, but are not identical to, CCR1, suggesting that CCR1 has both redundant and specific biologic roles. To test this, we have developed CCR1-deficient mice (-/-) by targeted gene disruption. Although the distribution of mature leukocytes was normal, steady state and induced trafficking and proliferation of myeloid progenitor cells were disordered in -/- mice. Moreover, mature neutrophils from -/- mice failed to chemotax in vitro and failed to mobilize into peripheral blood in vivo in response to MIP-1alpha. Consistent with this, -/- mice had accelerated mortality when challenged with Aspergillus fumigatus, a fungus controlled principally by neutrophils. To test the role of CCR1 in granuloma formation, we injected Schistosoma mansoni eggs intravenously, and observed a 40% reduction in the size of lung granulomas in -/- mice compared to +/+ littermates. This was associated with increased interferon-gamma and decreased interleukin-4 production in -/- versus +/+ lung lymph node cells stimulated with egg-specific antigen, suggesting that CCR1 influences the inflammatory response not only through direct effects on leukocyte chemotaxis, but also through effects on the type 1-type 2 cytokine balance. Thus CCR1 has nonredundant functions in hematopoiesis, host defense, and inflammation.

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.

Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences.

Transgenic mice were generated with pRSV-CAT, a chimeric gene construct containing the long terminal repeat of Rous sarcoma virus (RSV) linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT expression, detected in adult animals of five independent strains, was preferentially directed to organs rich in tendon, bone, and muscle. This pattern reflects the disease specificity of the intact virus and suggests that the tissue tropism of RSV is determined at least in part by the presence of endogenous tissue-specific factors that can promote expression of genetic information linked to the long terminal repeat. In two of the mouse strains, insertion of the pRSV-CAT DNA resulted in developmental abnormalities. One of these strains was characterized by a dominant trait of embryonic lethality, the other by a recessive trait of fused toes in all four feet.

Oncogenesis of the lens in transgenic mice.

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.

Multistep control of pituitary organogenesis.

Lhx3 and Lhx4 (Gsh4), two closely related LIM homeobox genes, determine formation of the pituitary gland in mice. Rathke's pouch is formed in two steps-first as a rudiment and later as a definitive pouch. Lhx3 and Lhx4 have redundant control over formation of the definitive pouch. Lhx3 controls a subsequent step of pituitary fate commitment. Thereafter, Lhx3 and Lhx4 together regulate proliferation and differentiation of pituitary-specific cell lineages. Thus, Lhx3 and Lhx4 dictate pituitary organ identity by controlling developmental decisions at multiple stages of organogenesis.

Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5.

The mammalian hippocampus contains the neural circuitry that is crucial for cognitive functions such as learning and memory. The development of such circuitry is dependent on the generation and correct placement of the appropriate number and types of neurons. Mice lacking function of the LIM homeobox gene Lhx5 showed a defect in hippocampus development. Hippocampal neural precursor cells were specified and proliferated, but many of them failed to either exit the cell cycle or to differentiate and migrate properly. Lhx5 is therefore essential for the regulation of precursor cell proliferation and the control of neuronal differentiation and migration during hippocampal development.

Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin.

Despite myriads of biological activities ascribed to uteroglobin (UG), a steroid-inducible secreted protein, its physiological functions are unknown. Mice in which the uteroglobin gene was disrupted had severe renal disease that was associated with massive glomerular deposition of predominantly multimeric fibronectin (Fn). The molecular mechanism that normally prevents Fn deposition appears to involve high-affinity binding of UG with Fn to form Fn-UG heteromers that counteract Fn self-aggregation, which is required for abnormal tissue deposition. Thus, UG is essential for maintaining normal renal function in mice, which raises the possibility that an analogous pathogenic mechanism may underlie genetic Fn-deposit human glomerular disease.

T cell development in mice that lack the zeta chain of the T cell antigen receptor complex.

The zeta subunit of the T cell antigen receptor complex is required for targeting nascent receptor complexes to the cell surface and for receptor-mediated signal transduction. To examine the significance of the zeta subunit in T cell development, mice deficient for zeta expression were generated by gene targeting. These zeta-/- mice had few CD4+CD8+ thymocytes, and the generation of CD4+ and CD8+ single positive T cells was impaired but not completely abrogated. Peripheral T cells were present but were unusual in that they expressed small amounts of CD5 and few T cell receptors. Thus, zeta chain expression influences thymocyte differentiation but is not absolutely required for the generation of single positive T cells.

Specification of pituitary cell lineages by the LIM homeobox gene Lhx3.

During pituitary organogenesis, the progressive differentiation of distinct pituitary-specific cell lineages from a common primordium involves a series of developmental decisions and inductive interactions. Targeted gene disruption in mice showed that Lhx3, a LIM homeobox gene expressed in the pituitary throughout development, is essential for differentiation and proliferation of pituitary cell lineages. In mice homozygous for the Lhx3 mutation, Rathke's pouch formed but failed to grow and differentiate; such mice lacked both the anterior and intermediate lobes of the pituitary. The determination of all pituitary cell lineages, except the corticotrophs, was affected, suggesting that a distinct, Lhx3-independent ontogenetic pathway exists for the initial specification of this lineage.

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

Role for GDNF in biochemical and behavioral adaptations to drugs of abuse.

The present study examined a role for GDNF in adaptations to drugs of abuse. Infusion of GDNF into the ventral tegmental area (VTA), a dopaminergic brain region important for addiction, blocks certain biochemical adaptations to chronic cocaine or morphine as well as the rewarding effects of cocaine. Conversely, responses to cocaine are enhanced in rats by intra-VTA infusion of an anti-GDNF antibody and in mice heterozygous for a null mutation in the GDNF gene. Chronic morphine or cocaine exposure decreases levels of phosphoRet, the protein kinase that mediates GDNF signaling, in the VTA. Together, these results suggest a feedback loop, whereby drugs of abuse decrease signaling through endogenous GDNF pathways in the VTA, which then increases the behavioral sensitivity to subsequent drug exposure.

Functions of LIM-homeobox genes.

Homeobox genes play fundamental roles in development. They can be subdivided into several subfamilies, one of which is the LIM-homeobox subfamily. The primary structure of LIM-homeobox genes has been remarkably conserved through evolution. Have their functions similarly been conserved? A host of new data has been derived from mutational analysis in diverse organisms, such as nematodes, flies and vertebrates. These studies have revealed a prominent involvement of LIM-homeodomain proteins in tissue patterning and differentiation, and their function in neural patterning is evident in all organisms studied to date. Here, we summarize the recent findings on LIM-homeobox gene function, compare the function of these genes from different organisms and describe specific co-factor requirements.

Early steps in pituitary organogenesis.

Significant advances have been made in defining the transcription cascade that is responsible for the early steps of pituitary formation and the environmental signals that induce, pattern and specify the pituitary gland and its cell types. It is now possible to outline the molecular mechanisms underlying the formation of the pituitary gland, as well as the initial development of organ-specific cell types.

Functional overlap between murine Inpp5b and Ocrl1 may explain why deficiency of the murine ortholog for OCRL1 does not cause Lowe syndrome in mice.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked human genetic disorder characterized by mental retardation, congenital cataracts, and renal tubular dysfunction. The Lowe syndrome gene, OCRL1, encodes a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in the Golgi complex. The pathogenesis of Lowe syndrome due to deficiency of a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in the Golgi complex is unknown. We have used targeted disruption in embryonic stem cells to make mice deficient in Ocrl1, the mouse homologue for OCRL1, as an animal model for the disease. Surprisingly, mice deficient in Ocrl1 do not develop the congenital cataracts, renal Fanconi syndrome, or neurological abnormalities seen in the human disorder. We hypothesized that Ocrl1 deficiency is complemented in mice by inositol polyphosphate 5-phosphatase (Inpp5b), an autosomal gene that encodes a phosphatidylinositol bisphosphate 5-phosphatase highly homologous to Ocrl1. We created mice deficient in Inpp5b; the mice were viable and fertile without phenotype except for testicular degeneration in males beginning after sexual maturation. We crossed mice deficient in Ocrl1 to mice deficient in Inpp5b. No liveborn mice or embryos lacking both enzymes were found, demonstrating that Ocrl1 and Inpp5b have overlapping functions in mice and suggesting that the lack of phenotype in Ocrl1-deficient mice may be due to compensating Inpp5b function.