Biomedical subjects

Heiner Westphal

Publications and source records attributed to Heiner Westphal.

At least 19 recordsLinked to original sources

PubMed2007

Multidrug resistance protein 4 protects bone marrow, thymus, spleen, and intestine from nucleotide analogue-induced damage.

Nucleoside-based analogues are mainstays in the treatment of cancer, viral infections, and inflammatory diseases. Recent studies showing that the ATP-binding cassette transporter, multidrug resistance protein 4, is able to efflux nucleoside and nucleotide analogues from transfected cells suggests that the pump may affect the efficacy of this class of agents. However, the in vivo pharmacologic functions of the pump are largely unexplored. Here, using Mrp4(-/-) mice as a model system, and the nucleotide analogue, 9'-(2'-phosphonylmethoxyethyl)-adenine (PMEA) as a probe, we investigate the ability of Mrp4 to function in vivo as an endogenous resistance factor. In the absence of alterations in plasma PMEA levels, Mrp4-null mice treated with PMEA exhibit increased lethality associated with marked toxicity in several tissues. Affected tissues include the bone marrow, spleen, thymus, and gastrointestinal tract. In addition, PMEA penetration into the brain is increased in Mrp4(-/-) mice. These findings indicate that Mrp4 is an endogenous resistance factor, and that the pump may be a component of the blood-brain barrier for nucleoside-based analogues. This is the first demonstration that an ATP-binding cassette transporter can affect in vivo tissue sensitivity towards this class of agents.

Adenine↗

PubMed2006

Lhx1 and Lhx5 maintain the inhibitory-neurotransmitter status of interneurons in the dorsal spinal cord.

Lhx1 and Lhx5 are co-expressed in multiple interneuron cell types in the developing spinal cord. These include early-born dI4 and dI6 inhibitory interneurons, as well as late-born inhibitory dILA neurons (dILA), all of which express the paired-domain transcription factor Pax2. Although it appears that Lhx1 and Lhx5 do not control the initial specification of the neuronal cell types in which they are expressed, we have found a cell-autonomous requirement for either Lhx1 or Lhx5 to maintain the expression of Pax2, Pax5 and Pax8 in dorsal inhibitory neurons at later developmental stages. Lhx1; Lhx5 double-knockout mice exhibit a downregulation of Gad1 and Viaat (Slc32a1) from E13.5 onwards that is closely associated with a decrease in Pax2 expression. Pax2 is a key factor for dorsal GABAergic identity, with the expression of Pax5 and Pax8 being differentially dependent on Pax2 in the dorsal horn. In summary, our findings support a model in which the differentiation of GABAergic interneurons in the dorsal cord depends on Pax2, with Lhx1 and Lhx5 helping to activate and maintain Pax2 expression in these cells. Lhx1 and Lhx5 therefore function together with Pax2, Pax5 and Pax8 to establish a GABAergic inhibitory-neurotransmitter program in dorsal horn interneurons.

Animals↗

PubMed2006

Genetic interaction of Gsc and Dkk1 in head morphogenesis of the mouse.

Mouse embryos lacking Gsc and Dkk1 function display severe deficiencies in craniofacial structures which are not found in either Dkk1 homozygous null or Gsc homozygous null mutant embryos. Loss of Gsc has a dosage-related effect on the severity of head truncation phenotype in Dkk1 heterozygous embryos. The synergistic effect of these mutations in enhancing head truncation provides direct evidence of a genetic interaction between Gsc and Dkk1, which display overlapping expression in the prechordal mesoderm. In the absence of Gsc activity, the expression of Dkk1, WNT genes and a transgenic reporter for WNT signalling are altered. Our results show that Gsc and Dkk1 functions are non-redundant in the anterior mesendoderm for normal anterior development and Gsc may influence Wnt signalling as a negative regulator.

Journal Article↗

PubMed2006

Reduced expression of the LIM-homeobox gene Lhx3 impairs growth and differentiation of Rathke's pouch and increases cell apoptosis during mouse pituitary development.

The formation of the anterior and intermediate lobes of the pituitary gland is a multi-step process regulated by cell-cell interactions involving a number of signaling pathways and by cascades of cell-intrinsic transcription factors. The LIM-homeodoamin protein Lhx3 has previously been shown to play an essential role in the growth of Rathke's pouch, a primordium of the anterior and intermediate lobes of the pituitary. However, the mechanisms underlying the function and regulation of Lhx3 remain to be elucidated. Here we report that a targeted insertion of a DNA fragment in the 3'-untranslated region of the Lhx3 gene reduces the expression of both Lhx3 mRNA and protein in Rathke's pouch. Mutant mice homozygous for this Lhx3 allele show severe hypoplasia of the pouch, a defect identical to that observed in Lhx3-null mutants. To gain insights into the mechanism of Lhx3 function in pituitary development, we further analyzed the Lhx3 deficient mutants by examination of early pituitary marker expression, cell proliferation, and cell apoptosis. Our results revealed an increase in cell apoptosis and a loss of Islet1 and Calbindin marker expression in Rathke's pouch of these mutants. Recently, increased cell apoptosis in Rathke's pouch has been described in mutant mice impaired in the function of the bicoid-like homeodomain proteins Pitx1 and Pitx2. In those mutants, the expression of Lhx3 is absent. Our results thus underscore the view that Lhx3 functions downstream of the Pitx factors in the same transcriptional cascade that controls growth and early cell differentiation of the developing pituitary gland.

Animals↗

PubMed2006

Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8.

Mammalian oogenesis requires oocyte-specific transcriptional regulators. The full complement of oocyte-specific transcription factors is unknown. Here, we describe the finding that Sohlh1, a spermatogenesis and oogenesis basic helix-loop-helix transcription factor in females, is preferentially expressed in oocytes and required for oogenesis. Sohlh1 disruption perturbs follicular formation in part by causing down-regulation of two genes that are known to disrupt folliculogenesis: newborn ovary homeobox gene (Nobox) and factor in the germ-line alpha (Figla). In addition, we show that Lhx8 is downstream of Sohlh1 and critical in fertility. Thus, Sohlh1 and Lhx8 are two germ cell-specific, critical regulators of oogenesis.

Animals↗

PubMed2006

Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium.

The Dkk family of secreted cysteine-rich proteins regulates Wnt/beta-catenin signaling by interacting with the Wnt co-receptor Lrp5/6. Here, we show that Dkk2-mediated repression of the Wnt/beta-catenin pathway is essential to promote differentiation of the corneal epithelial progenitor cells into a non-keratinizing stratified epithelium. Complete transformation of the corneal epithelium into a stratified epithelium that expresses epidermal-specific differentiation markers and develops appendages such as hair follicles is achieved in the absence of the Dkk2 gene function. We show that Dkk2 is a key regulator of the corneal versus epidermal fate of the ocular surface epithelium.

Animals↗

PubMed2006

Rho family Guanine nucleotide exchange factor Brx couples extracellular signals to the glucocorticoid signaling system.

Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lysophosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein alpha(13)-, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcriptosome activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes.

A Kinase Anchor Proteins↗

PubMed2006

Cloning and characterization of the 5'-flanking region of the Ehox gene.

The paired-like homeobox-containing gene Ehox plays a role in embryonic stem cell differentiation and is highly expressed in the developing placenta and thymus. To understand the mechanisms of regulation of Ehox gene expression, the 5'-flanking region of the Ehox gene was isolated from a mouse BAC library. 5'-RACE analysis revealed a single transcriptional start site 130 nucleotides upstream of the translation initiation codon. Transient transfection with a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the nt -84 to -68 region contained a positive cis-acting element for efficient expression of the Ehox gene. Mutational analysis of this region and oligonucleotide competition in the electrophoretic mobility shift assay revealed the presence of a CCAAT box, which is a target for transcription nuclear factor Y (NFY). NFY is essential for positive gene regulation. No tissue-specific enhancer was identified in the 1.9-kb 5'-flanking region of the Ehox gene. Ehox is expressed during the early stages of embryo development, specifically in the brain at 9.5 dpc, as well as during the late stages of embryo development. These results suggest that NFY is an essential regulatory factor for Ehox transcriptional activity, which is important for the post-implantation stage of the developing embryo.

5' Flanking Region↗

PubMed2006

Characterization of a mouse strain expressing Cre recombinase from the 3' untranslated region of the dopamine transporter locus.

Dopamine (DA) neurotransmission has been implicated in several neurological and psychiatric disorders. The dopamine transporter (DAT) is highly expressed in dopaminergic neurons of the ventral mesencephalon and regulates neurotransmission by transporting DA back into the presynaptic terminals. To mediate restricted DNA recombination events into DA neurons using the Cre/loxP technology, we have generated a knockin mouse expressing Cre recombinase under the transcriptional control of the endogenous DAT promoter. To minimize interference with DAT function by preservation of both DAT alleles, Cre recombinase expression was driven from the 3' untranslated region (3'UTR) of the endogenous DAT gene by means of an internal ribosomal entry sequence. Crossing this murine line with a LacZ reporter showed colocalization of DAT immunocytochemistry and beta-galactosidase staining in all regions analyzed. This knockin mouse can be used for generating tissue specific knockouts in mice carrying genes flanked by loxP sites, and will facilitate the analysis of gene function in dopaminergic neurons.

3' Untranslated Regions↗

PubMed2006

Sfrp5 is not essential for axis formation in the mouse.

Secreted frizzled related protein (Sfrp) genes encode extracellular factors that can modulate Wnt signaling. During early post-implantation mouse development Sfrp5 is expressed in the anterior visceral endoderm (AVE) and the ventral foregut endoderm. The AVE is important in anterior-posterior axis formation and the ventral foregut endoderm contributes to multiple gut tissues. Here to determine the essential role of Sfrp5 in early mouse development we generated Sfrp5-deficient mice by gene targeting. We report that Sfrp5-deficient mice are viable and fertile. To determine whether the absence of an axis phenotype might be due to genetic redundancy with Dkk1 in the AVE we generated Sfrp5;Dkk1 double mutant mice. AVE development and primitive streak formation appeared normal in Sfrp5(-/-);Dkk1(-/-) embryos. These results indicate that Sfrp5 is not essential for axis formation or foregut morphogenesis in the mouse and also imply that Sfrp5 and Dkk1 together are not essential for AVE development.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Generation and characterization of dickkopf3 mutant mice.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues.

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

Alleles↗

PubMed2005

Ssdp1 regulates head morphogenesis of mouse embryos by activating the Lim1-Ldb1 complex.

The transcriptional activity of LIM-homeodomain (LIM-HD) proteins is regulated by their interactions with various factors that bind to the LIM domain. We show that reduced expression of single-stranded DNA-binding protein 1 (Ssdp1), which encodes a co-factor of LIM domain interacting protein 1 (Ldb1), in the mouse mutant headshrinker (hsk) disrupts anterior head development by partially mimicking Lim1 mutants. Although the anterior visceral endoderm and the anterior definitive endoderm, which together comprise the head organizer, were able to form normally in Ssdp1(hsk/hsk) mutants, development of the prechordal plate was compromised. Head development is partially initiated in Ssdp1(hsk/hsk) mutants, but neuroectoderm tissue anterior to the midbrain-hindbrain boundary is lost, without a concomitant increase in apoptosis. Cell proliferation is globally reduced in Ssdp1(hsk/hsk) mutants, and approximately half also exhibit smaller body size, similar to the phenotype observed in Lim1 and Ldb1 mutants. We also show that Ssdp1 contains an activation domain and is able to enhance transcriptional activation through a Lim1-Ldb1 complex in transfected cells, and that Ssdp1 interacts genetically with Lim1 and Ldb1 in both head development and body growth. These results suggest that Ssdp1 regulates the development of late head organizer tissues and body growth by functioning as an essential activator component of a Lim1 complex through interaction with Ldb1.

Animals↗

PubMed2005

Analysis of the in vivo functions of Mrp3.

Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3(-/-) mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide ( approximately 2.0- to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3(-/-) mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3(-/-) and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.

Animals↗

PubMed2005

Restoring stemness.

This essay is focused on a specific line of research toward regenerative therapies that is based on the use of embryonic stem cells but tries to avoid cloning techniques that are the heart of current ethical debates.

Animals↗

PubMed2004

Simultaneous absence of dopamine D1 and D2 receptor-mediated signaling is lethal in mice.

Dopamine (DA) controls a wide variety of physiological functions in the central nervous system as well as in the neuroendocrine and gastrointestinal systems. DA signaling is mediated by five cloned receptors named D1-D5. Knockout mouse models for the five receptors have been generated, and, albeit impaired for some important DA-mediated functions, they are viable and can reproduce. D1 and D2 receptors are the most abundant and widely expressed DA receptors. Cooperative/synergistic effects mediated by these receptors have been suggested, in particular, in the control of motor behaviors. To analyze the extent of such interrelationship, we have generated double D1/D2 receptor mutants. Interestingly, in contrast to single knockouts, we found that concurrent ablation of the D1 and D2 receptors is lethal during the second or third week after birth. This dramatic phenotype is likely to be related to altered feeding behavior and dysfunction of the gastrointestinal system, especially because major anatomical changes were not identified in the brain. Similarly, in the absence of functional D1, heterozygous D2 mutants (D1r(-/-);D2r(+/-)) showed severe growth retardation and did not survive their postweaning period. The analysis of motor behavior in D1r/D2r compound mutants showed that loss of D2-mediated functions reduces motor abilities, whereas the effect of D1r ablation on locomotion strongly depends on the experimental paradigms used. These studies highlight the interrelationship between D1 and D2 receptor-mediated control of motor activity, food intake, and gastrointestinal functions, which has been elusive in the single-gene ablation studies.

Animals↗

PubMed2004

Profound impairment in social recognition and reduction in anxiety-like behavior in vasopressin V1a receptor knockout mice.

Considerable evidence suggests that arginine vasopressin (AVP) is critically involved in the regulation of many social and nonsocial behaviors, including emotionality. The existence of two AVP receptors in the brain, namely the V1a and V1b subtypes, and the lack of clear pharmacological data using selective agonists or antagonists, make it difficult to determine which receptor is responsible for the AVP-mediated effects on behavior. Here we report the behavioral effects of a null mutation in the V1a receptor (V1aR) in male mice. Male mice lacking functional V1aR (V1aRKO) exhibit markedly reduced anxiety-like behavior and a profound impairment in social recognition. V1aRKO performed normally on spatial and nonsocial olfactory learning and memory tasks. Acute central administration of AVP robustly stimulated stereotypical scratching and autogrooming in wild-type (WT), but not V1aRKO males. AVP and oxytocin (OT) mRNA and OT receptor-binding levels were similar in WT and V1aRKO mice. Given the current findings, the V1aR may provide a novel potential pharmacological target for social and affective disorders including autism, and anxiety disorders.

Animals↗

PubMed2004

A functional floxed allele of Pkd1 that can be conditionally inactivated in vivo.

Gene targeting has been used to create a variety of lines of mice with Pkd1 mutations that share many common features. Homozygous Pkd1 mutants invariably develop pancreatic and renal cysts if they survive to day 15.5 post coitum and die in either the fetal or the perinatal period. In contrast, mice with heterozygous mutations of Pkd1 are generally normal and have few if any renal cysts. These features have limited the utility of these models as tools to study the pathogenesis of cyst formation and the effect of various therapeutic interventions on disease progression. This report describes a new line of mice with a floxed allele of Pkd1 (Pkd1(cond)) that has an FRT-flanked neomycin cassette inserted into intron 1 and lox P sites inserted into intron 1 and intron 4. The Pkd1(cond) allele is fully functional, and homozygotes are viable and healthy. It is shown that the lox P and FRT sites can be selectively induced to recombine to produce two new alleles, Pkd1(del2-4) and Pkd1(cond-Deltaneo), by crossing to animals that express either the cre or FLPe recombinase, respectively. It is found that Pkd1(del2-4) allele functions as a true null, whereas presence or absence of the neomycin gene has no functional effects. It also is shown that somatic loss of Pkd1 results in renal and hepatic cysts. This new line of mice will be invaluable in the study of Pkd1 biology and serve as a powerful new tool that can be used to study the pathogenesis of autosomal dominant polycystic kidney disease.

Alleles↗