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H Westphal

Publications and source records attributed to H Westphal.

At least 55 records · Page 3Linked to original sources
PubMed1998

Genomic structure and chromosomal localization of the mouse LIM domain-binding protein 1 gene, Ldb1.

The LIM domain is a structural motif that is well conserved throughout evolution in a variety of factors known to play important roles in development and cell regulation. Ldb genes encode LIM domain-binding (Ldb) factors. Here we report on the structural organization and chromosomal localization of the mouse Ldb1 gene. It contains at least 10 exons and spans approximately 4 kb of genomic DNA. The transcription initiation site is located 462 bp upstream of the translation initiation codon ATG as determined by 5'-RACE. Sequencing analysis of the 5'-flanking region shows TATA and CCAAT motifs as well as potential binding sites for GATA, CF-1, PEA3, HRE, APRRE, RARE, Myc, and c-Jun. Ldb1 maps to the distal region of mouse chromosome 19 that is syntenic with human chromosome 10q.

Animals↗
PubMed1998

Expression and regulation of GFRalpha3, a glial cell line-derived neurotrophic factor family receptor.

We report the identification of an additional member of the glial cell line-derived neurotrophic factor (GDNF) family receptor, termed GFRalpha3, that is homologous to the previously identified GDNF and neurturin ligand binding receptors GFRalpha1 and GFRalpha2. GFRalpha3 is 32% and 37% identical to GFRalpha1 and GFRalpha2, respectively. RNase protection assays show that whereas gfralpha1 and gfralpha2 are abundant in both developing and adult brain, gfralpha3 is exclusively expressed during development. All receptors are widely present in both the developing and adult peripheral nervous system and in peripheral organs. For instance, in situ hybridization shows that the developing liver, stomach, intestine, kidney, and sympathetic chain, which all contain ret-expressing cells, transcribe unique complementary and overlapping patterns of most or all of the GDNF family receptors and ligands. In sensory neurons of the trigeminal ganglion gfralpha2 and gfralpha3 are expressed in different subpopulations of neurons, whereas gfralpha1 is coexpressed in some gfralpha2 and gfralpha3-positive neurons. We find that the gfralpha1 population of trigeminal neurons is absent in GDNF null mutant mice, suggesting that GDNF signals in vivo by interacting with GFRalpha1. Thus, our results show that there are at least three members in the GDNF family of ligand binding receptors and that these receptors may be crucial in conferring ligand specificity in vivo. The unique complementary and overlapping expression of gfralpha3 implies distinct functions in the developing and adult mouse from that of GFRalpha1 and GFRalpha2.

Amino Acid Sequence↗
PubMed1998

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.

Animals↗
PubMed1998

Altered brain serotonin homeostasis and locomotor insensitivity to 3, 4-methylenedioxymethamphetamine ("Ecstasy") in serotonin transporter-deficient mice.

The sodium-dependent, high affinity serotonin [5-hydroxytryptamine (5-HT)] transporter (5-HTT) provides the primary mechanism for inactivation of 5-HT after its release into the synaptic cleft. To further evaluate the function of the 5-HTT, the murine gene was disrupted by homologous recombination. Despite evidence that excess extracellular 5-HT during embryonic development, including that produced by drugs that inhibit the 5-HTT, may lead to severe craniofacial and cardiac malformations, no obvious developmental phenotype was observed in the 5-HTT-/- mice. High affinity [3H]5-HT uptake was completely absent in 5-HTT-/- mice, confirming a physiologically effective knockout of the 5-HTT gene. 5-HTT binding sites labeled with [125I] 3 beta-(4'-iodophenyl)tropan-2 beta-carboxylic acid methyl ester were reduced in a gene dose-dependent manner, with no demonstrable binding in 5-HTT-/- mutants. In adult 5-HTT-/- mice, marked reductions (60-80%) in 5-HT concentrations were measured in several brain regions. While (+)-amphetamine-induced hyperactivity did not differ across genotypes, the locomotor enhancing effects of (+)-3, 4-methylenedioxymethamphetamine, a substituted amphetamine that releases 5-HT via a transporter-dependent mechanism, was completely absent in 5-HTT-/- mutants. Together, these data suggest that the presence of a functional 5-HTT is essential for brain 5-HT homeostasis and for 3,4-methylenedioxymethamphetamine-induced hyperactivity.

Animals↗
PubMed1998

Insulin-like growth factor-I affects perinatal lethality and postnatal development in a gene dosage-dependent manner: manipulation using the Cre/loxP system in transgenic mice.

Insulin-like growth factor-I (IGF-I) is essential for cell growth, differentiation and postnatal development. A null mutation in igf-1 causes intrauterine growth retardation and perinatal lethality. The present study was designed to test the lower limit of igf-1 gene dosage that ensures survival and postnatal growth by using the Cre/loxP system. Mice with variable reductions in IGF-I levels were generated by crossing EIIa-cre transgenic mice and mice with loxP-flanked igf-1 locus (igf-1/flox). EIIa-cre mice express bacteriophage P1 Cre (causes recombination) recombinase under the adenovirus promoter EIIa, during early embryonic development before implantation, and cause genomic recombination of the igf-1/flox locus. Mice with the most extensive recombination die immediately after birth, while the survivors have significant growth retardation in proportion to the reduction in their igf-1 gene. Interestingly, this gene dosage effect on body weight was not very significant before weaning. However, when the young animals were weaned at 3 weeks, the igf-1 gene dosage was the only independent predictor of the weight gain between 3 and 6 weeks among the parameters tested. Although growth retarded, mice with Cre-induced partial igf-1 deficiency were fertile and gave birth to null mice. Thus Cre-induced genomic recombination using the EIIa promoter occurs during development and creates distinct phenotypes compared with the conventional null mutation. This variability allows for postnatal survival and will enable one to begin to explore the role of the endocrine vs. paracrine effects of IGF-I.

Animals↗
PubMed1998

Formation of Rathke's pouch requires dual induction from the diencephalon.

Targeted disruption of the homeobox gene T/ebp (Nkx2.1, Ttf1, Titf1) in mice results in ablation of the pituitary. Paradoxically, while T/ebp is expressed in the ventral diencephalon during forebrain formation, it is not expressed in Rathke's pouch or in the pituitary gland at any time of embryogenesis. Examination of pituitary development in the T/ebp homozygous null mutant embryos revealed that a pouch rudiment is initially formed but is eliminated by programmed cell death before formation of a definitive pouch. In the diencephalon of the mutant, Bmp4 expression is maintained, whereas Fgf8 expression is not detectable. These data and additional genetic and molecular observations suggest that Rathke's pouch develops in a two-step process that requires at least two sequential inductive signals from the diencephalon. First, BMP4 is required for induction and formation of the pouch rudiment, a role confirmed by analysis of Bmp4 homozygous null mutant embryos. Second, FGF8 is necessary for activation of the key regulatory gene Lhx3 and subsequent development of the pouch rudiment into a definitive pouch. This study provides firm molecular genetic evidence that morphogenesis of the pituitary primordium is induced in vivo by signals from the adjacent diencephalon.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Nonmuscle myosin II-B is required for normal development of the mouse heart.

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B-/- embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B+/- mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 +/- 1.8 micron (right ventricle) and 7.8 +/- 1.3 micron (left ventricle) in NMHC-B+/+ and B+/- mice to 14.7 +/- 1.1 micron and 13.8 +/- 2.3 micron, respectively, in NMHC-B-/- mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 +/- 0.2 micron for NMHC-B+/+ and B+/- vs. 7. 2 +/- 0.6 micron for NMHC-B-/- mice (P < 0.01)). Six of seven NMHC-B-/- newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B-/- mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.

Animals↗
PubMed1997

Neurturin and glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta), novel proteins related to GDNF and GDNFR-alpha with specific cellular patterns of expression suggesting roles in the developing and adult nervous system and in peripheral organs.

Cloning strategies were used to identify a gene termed glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta) related to GDNFR-alpha. In situ hybridization was then used to map cellular expression of the GDNF-related trophic factor neurturin (NTN) and GDNFR-beta mRNA in developing and adult mice, and comparisons with GDNFR-alpha and RET were made. Neurturin is expressed in postnatal cerebral cortex, striatum, several brainstem areas, and the pineal gland. GDNFR-beta mRNA was more widely expressed in the developing and adult CNS, including cerebral cortex, cerebellum, thalamus, zona incerta, hypothalamus, brainstem, and spinal cord, and in subpopulations of sensory neurons and developing peripheral nerves. NTN colocalized with RET and GDNFR-alpha in ureteric buds of the developing kidney. The circular muscle layer of the developing intestines, smooth muscle of the urether, and developing bronchiolae also expressed NTN. GDNFR-beta was found in myenteric but not submucosal intestinal plexuses. In developing salivary glands NTN had an epithelial expression, whereas GDNFR-beta was expressed in surrounding tissue. Neurturin and GDNFR-beta were present in developing sensory organs. In the gonads, NTN appeared to be expressed in Sertoli cells and in the epithelium of the oviduct, whereas GDNFR-beta was expressed by the germ cell line. Our findings suggest multiple roles for NTN and GDNFR-beta in the developing and adult organism. Although NTN and GDNFR-beta expression patterns are sometimes complementary, this is not always the case, suggesting multiple modi operandi of GDNF and NTN in relation to RET and the two binding proteins, GDNFR-alpha and GDNFR-beta.

Animals↗
PubMed1997

Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice.

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel-Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/-) mice appeared phenotypically normal, VHL -/- mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL -/- embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL -/- mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL -/- embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.

Animals↗
PubMed1997

Lhx4, a LIM homeobox gene.

LIM homeobox genes are well conserved in evolution and play important roles as transcriptional regulators of embryonic development. Here we report on the structure of LIM domains of the mouse Lhx4 (Gsh4) gene. The cDNA was generated by modified reverse transcription-PCR from midgestation embryo templates, using a degenerate consensus primer. The deduced amino acid sequence of the first LIM domain reveals 77% identity and that of the second domain reveals 86% identity with the corresponding sequences of the closely related Lhx3 gene. In addition, there is 38-56% similarity to other members of the Lhx gene family. The LIM consensus sequence is well conserved in Lhx4.

Amino Acid Sequence↗
PubMed1997

Mouse model of GM2 activator deficiency manifests cerebellar pathology and motor impairment.

The GM2 activator deficiency (also known as the AB variant), Tay-Sachs disease, and Sandhoff disease are the major forms of the GM2 gangliosidoses, disorders caused by defective degradation of GM2 ganglioside. Tay-Sachs and Sandhoff diseases are caused by mutations in the genes (HEXA and HEXB) encoding the subunits of beta-hexosaminidase A. The GM2 activator deficiency is caused by mutations in the GM2A gene encoding the GM2 activator protein. For degradation of GM2 ganglioside by beta-hexosamindase A, the GM2 activator protein must participate by forming a soluble complex with the ganglioside. In each of the disorders, GM2 ganglioside and related lipids accumulate to pathologic levels in neuronal lysosomes, resulting in clinically similar disorders with an onset in the first year of life, progressive neurodegeneration, and death by early childhood. We previously have described mouse models of Tay-Sachs (Hexa -/-) and Sandhoff (Hexb -/-) diseases with vastly different clinical phenotypes. The Hexa -/- mice were asymptomatic whereas the Hexb -/- mice were severely affected. Through gene disruption in embryonic stem cells we now have established a mouse model of the GM2 activator deficiency that manifests an intermediate phenotype. The Gm2a -/- mice demonstrated neuronal storage but only in restricted regions of the brain (piriform, entorhinal cortex, amygdala, and hypothalamic nuclei) reminiscent of the asymptomatic Tay-Sachs model mice. However, unlike the Tay-Sachs mice, the Gm2a -/- mice displayed significant storage in the cerebellum and defects in balance and coordination. The abnormal ganglioside storage in the Gm2a -/- mice consisted of GM2 with a low amount of GA2. The results demonstrate that the activator protein is required for GM2 degradation and also may indicate a role for the GM2 activator in GA2 degradation.

Animals↗
PubMed1997

A human papillomavirus type 18 E6/E7 transgene sensitizes mouse lens cells to human wild-type p53-mediated apoptosis.

We have studied the concerted action of factors that influence the balance between cell proliferation and cell death in the developing lens of transgenic mice. We show that a human papillomavirus type 18 (HPV18) E6/E7 transgene that predominantly expresses the viral E7 gene product triggers apoptosis in a dose dependent manner, and causes retardation of lens growth or microphakia. E7 is known to inactivate pRB, the product of the retinoblastoma gene, and to enhance the action of p53. Our earlier work had demonstrated that over-expression of p53 itself can cause apoptosis of lens cells, and that a mutant p53 allele can interfere with this process. In the present study, we examined lenses that simultaneously express different constellations of the HPV18 E6/E7, wild-type and mutant human p53, and wild-type human pRB transgenes. We observed that lens cells expressing the HPV18 transgene are more sensitive to wild-type human p53 action than normal lens cells. As a result, there is severe microphakia in lenses that express both the HPV18 and the wild-type p53 transgenes. By contrast, apoptosis was reduced in lenses that co-expressed the HPV18 and either the pRB or the mutant p53 transgene. We conclude that levels of wild-type p53 are critical, and that any excess of p53 or suppression of pRB can cause cell death. Our results encourage attempts to counteract the deleterious action of human papillomaviruses in cervical cancer by a combination of measures that decrease cell proliferation and enhance apoptosis.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins.

The LIM-only protein Lmo2, activated by chromosomal translocations in T-cell leukaemias, is normally expressed in haematopoiesis. It interacts with TAL1 and GATA-1 proteins, but the function of the interaction is unexplained. We now show that in erythroid cells Lmo2 forms a novel DNA-binding complex, with GATA-1, TAL1 and E2A, and the recently identified LIM-binding protein Ldb1/NLI. This oligomeric complex binds to a unique, bipartite DNA motif comprising an E-box, CAGGTG, followed approximately 9 bp downstream by a GATA site. In vivo assembly of the DNA-binding complex requires interaction of all five proteins and establishes a transcriptional transactivating complex. These data demonstrate one function for the LIM-binding protein Ldb1 and establish a function for the LIM-only protein Lmo2 as an obligatory component of an oligomeric, DNA-binding complex which may play a role in haematopoiesis.

Adaptor Proteins, Signal Transducing↗
PubMed1997

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.

Animals↗
PubMed1997

Morphological alterations in the peripheral and central nervous systems of mice lacking glial cell line-derived neurotrophic factor (GDNF): immunohistochemical studies.

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of growth factors with neurotrophic activity on midbrain dopaminergic neurons and on developing and mature motoneurons of the brainstem and spinal cord. To investigate the extent of GDNF dependency of central and peripheral nervous structures during development, we have performed an immunohistochemical analysis of sections from the whole head including brain, peripheral ganglia, developing teeth and tongue, as well as intestines, in mutant mice lacking a part of the third exon that encodes the GDNF protein. As described previously, these null-mutated mice lack most of the enteric nerve plexus and are subject to agenesis or severe dysgenesis of the kidneys. In the present communication, we examined the development of vibrissae and incisor and molar teeth, as well as the innervation of these structures, and found no differences between null-mutated and control mice. A decrease in the immunohistochemical labeling intensity with tyrosine hydroxylase was observed in the superior cervical ganglion (SCG), as well as in the pontine nucleus locus coeruleus, and the sympathetic innervation of blood vessels and glands in the head was significantly decreased. None of the brain nuclei studied exhibited any significant decreases in the total number of neurons, but the packing density of neurons in the nucleus locus coeruleus was decreased. These data indicate that GDNF might be one neurotrophic factor that contributes to the development of central and peripheral noradrenergic neurons.

Animals↗