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D H Rowitch

Publications and source records attributed to D H Rowitch.

At least 19 recordsLinked to original sources
PubMed2001

Olig bHLH proteins interact with homeodomain proteins to regulate cell fate acquisition in progenitors of the ventral neural tube.

BACKGROUND: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons. RESULTS: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region. CONCLUSIONS: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.

Animals↗
PubMed2001

Oligodendrocyte lineage genes (OLIG) as molecular markers for human glial brain tumors.

The most common primary tumors of the human brain are thought to be of glial cell origin. However, glial cell neoplasms cannot be fully classified by cellular morphology or with conventional markers for astrocytes, oligodendrocytes, or their progenitors. Recent insights into central nervous system tumorigenesis suggest that novel molecular markers might be found among factors that have roles in glial development. Oligodendrocyte lineage genes (Olig1/2) encode basic helix-loop-helix transcription factors. In the rodent central nervous system, they are expressed exclusively in oligodendrocytes and oligodendrocyte progenitors, and Olig1 can promote formation of an chondroitin sulfate proteoglycon-positive glial progenitor. Here we show that human OLIG genes are expressed strongly in oligodendroglioma, contrasting absent or low expression in astrocytoma. Our data provide evidence that neoplastic cells of oligodendroglioma resemble oligodendrocytes or their progenitor cells and may derive from cells of this lineage. They further suggest the diagnostic potential of OLIG markers to augment identification of oligodendroglial tumors.

Astrocytoma↗
PubMed2001

Characterization of Pax-2 regulatory sequences that direct transgene expression in the Wolffian duct and its derivatives.

The Pax family of transcription factors plays important roles in vertebrate organogenesis. Pax-2 is a critical factor in the development of the mammalian urogenital system. Pax-2 is expressed in the epithelia of the ureter, the Müllerian duct, and the Wolffian duct and in the nephrogenic mesenchyme. Gene targeting in the mouse as well as natural mutations in mouse and man have demonstrated the requirement of Pax-2 in the development of these structures. Little is known about the molecular mechanisms regulating Pax-2 expression in the developing urogenital system. As a first step to reveal these mechanisms and to search for the elements and factors controlling Pax-2 expression we have characterized regulatory sequences of the Pax-2 gene in an in vivo reporter assay in the mouse. An 8.5-kb genomic region upstream of the Pax-2 transcription start site directed reporter gene activity in the epithelium of the pronephric duct at 8.25 days postcoitum (dpc) and in the Wolffian duct starting from 9.0 dpc. Expression in the Wolffian duct and its derivatives, the ureter, the collecting duct system, the seminal vesicles, the vas deferens, and the epididymis, was maintained at least until 18.5 dpc. Hence, an element(s) in the 8.5-kb upstream region is sufficient to initiate and maintain Pax-2 expression in the Wolffian duct and its derivatives. In order to more precisely map the Wolffian duct regulatory sequences, a deletion analysis of the 8.5-kb upstream region was performed in a transient in vivo reporter assay. A 0.4-kb subfragment was required for marker gene expression in the Wolffian duct. Misexpression of fgf8 under the control of the 8.5-kb upstream region resulted in polycystic kidneys, demonstrating the general usefulness of Pax-2 regulatory sequences in misexpression of foreign genes in the ureter and collecting duct system of the kidney in transgenic approaches in mice.

Animals↗
PubMed2001

Six3 promotes the formation of ectopic optic vesicle-like structures in mouse embryos.

A few years ago, three novel murine homeobox genes closely related to the Drosophila sine oculis (so) gene (Six1-3) were isolated and were all included in the Six/so gene family. Because of its early expression in the developing eye field, Six3 was initially thought to be the functional ortholog of the Drosophila so gene. This hypothesis was further supported by the demonstration that ectopic Six3 expression in medaka fish (Oryzias latipes) promotes the formation of ectopic lens and retina tissue. Here, we show that similar to Drosophila, where the eyeless/Pax6 gene regulates the eye-specific expression of so, Six3 expression in the murine lens placodal ectoderm is also controlled by Pax6. We also show that ectopic Six3 expression promotes the formation of ectopic optic vesicle-like structures in the hindbrain-midbrain region of developing mouse embryos.

Amino Acid Sequence↗
PubMed2001

Sonic hedgehog is required during an early phase of oligodendrocyte development in mammalian brain.

Oligodendrocyte precursor development in the embryonic spinal cord is thought to be regulated by the secreted signal, Sonic hedgehog (Shh). Such precursors can be identified by the expression of Olig genes, encoding basic helix-loop-helix factors, in the spinal cord and brain. However, the signaling pathways that govern oligodendrocyte precursor (OLP) development in the rostral central nervous system are poorly understood. Here, we show that Shh is required for oligodendrocyte development in the mouse forebrain and spinal cord, and that Shh proteins are both necessary and sufficient for OLP production in cortical neuroepithelial cultures. Moreover, adenovirus-mediated Olig1 ectopic expression can promote OLP formation independent of Shh activity. Our results demonstrate essential functions for Shh during early phases of oligodendrocyte development in the mammalian central nervous system. They further suggest that a key role of Shh signaling is activation of Olig genes.

Animals↗
PubMed2001

Oligodendrocyte development in the spinal cord and telencephalon: common themes and new perspectives.

There are clear parallels between oligodendrocyte development in the spinal cord and forebrain. However, there is new evidence that in both of these regions oligodendrocyte lineage development may be more complex than we earlier thought. This stems from the recent identification of three new transcription factor genes, Olig1, Olig2 and Sox10, that are expressed from the early stages of oligodendrocyte lineage development. In this article, we highlight the common themes underlying specification and early development of oligodendrocytes in the spinal cord and telencephalon. Then, we discuss recent studies of Sox10 and the Olig genes and their implications for oligodendrocyte specification. We conclude that although the mechanisms of oligodendrogenesis appear to be fundamentally similar at different rostro-caudal levels of the neuraxis, there are still many unanswered questions about the details of oligodendrocyte specification.

Animals↗
PubMed2001

Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon.

In the caudal neural tube, oligodendrocyte progenitors (OLPs) originate in the ventral neuroepithelium under the influence of Sonic hedgehog (SHH), then migrate throughout the spinal cord and brainstem before differentiating into myelin-forming cells. We present evidence that oligodendrogenesis in the anterior neural tube follows a similar pattern. We show that OLPs in the embryonic mouse forebrain express platelet-derived growth factor alpha-receptors (PDGFRA), as they do in more caudal regions. They first appear within a region of anterior hypothalamic neuroepithelium that co-expresses mRNA encoding SHH, its receptor PTC1 (PTCH) and the transcription factors OLIG1, OLIG2 and SOX10. Pdgfra-positive progenitors later spread through the forebrain into areas where Shh is not expressed, including the cerebral cortex. Cyclopamine inhibited OLP development in cultures of mouse basal forebrain, suggesting that hedgehog (HH) signalling is obligatory for oligodendrogenesis in the ventral telencephalon. Moreover, Pdgfra-positive progenitors did not appear on schedule in the ventral forebrains of Nkx2.1 null mice, which lack the telencephalic domain of Shh expression. However, OLPs did develop in cultures of Nkx2.1(-/-) basal forebrain and this was blocked by cyclopamine. OLPs also developed in neocortical cultures, even though Shh transcripts could not be detected in the embryonic cortex. Here, too, the appearance of OLPs was suppressed by cyclopamine. In keeping with these findings, we detected mRNA encoding SHH and Indian hedgehog (IHH) in both Nkx2.1(-/-) basal forebrain cultures and neocortical cultures. Overall, the data are consistent with the idea that OLPs in the telencephalon, possibly even some of those in the cortex, develop under the influence of SHH in the ventral forebrain.

Animals↗
PubMed2001

Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development.

beta-Catenin is a central component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. We have investigated the role of beta-catenin during brain morphogenesis, by specifically inactivating the beta-catenin gene in the region of Wnt1 expression. To achieve this, mice with a conditional ('floxed') allele of beta-catenin with required exons flanked by loxP recombination sequences were intercrossed with transgenic mice that expressed Cre recombinase under control of Wnt1 regulatory sequences. beta-Catenin gene deletion resulted in dramatic brain malformation and failure of craniofacial development. Absence of part of the midbrain and all of the cerebellum is reminiscent of the conventional Wnt1 knockout (Wnt1(-/-)), suggesting that Wnt1 acts through beta-catenin in controlling midbrain-hindbrain development. The craniofacial phenotype, not observed in embryos that lack Wnt1, indicates a role for beta-catenin in the fate of neural crest cells. Analysis of neural tube explants shows that (beta-catenin is efficiently deleted in migrating neural crest cell precursors. This, together with an increased apoptosis in cells migrating to the cranial ganglia and in areas of prechondrogenic condensations, suggests that removal of beta-catenin affects neural crest cell survival and/or differentiation. Our results demonstrate the pivotal role of beta-catenin in morphogenetic processes during brain and craniofacial development.

Animals↗
PubMed2000

Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system.

During development, basic helix-loop-helix (bHLH) proteins regulate formation of neurons from multipotent progenitor cells. However, bHLH factors linked to gliogenesis have not been described. We have isolated a pair of oligodendrocyte lineage genes (Olg-1 and Olg-2) that encode bHLH proteins and are tightly associated with development of oligodendrocytes in the vertebrate central nervous system (CNS). Ectopic expression of Olg-1 in rat cortical progenitor cell cultures promotes formation of oligodendrocyte precursors. In developing mouse embryos, Olg gene expression overlaps but precedes the earliest known markers of the oligodendrocyte lineage. Olg genes are expressed at the telencephalon-diencephalon border and adjacent to the floor plate, a source of the secreted signaling molecule Sonic hedgehog (Shh). Gain- and loss-of-function analyses in transgenic mice demonstrate that Shh is both necessary and sufficient for Olg gene expression in vivo.

Age Factors↗
PubMed2000

Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors.

Sonic hedgehog (Shh) signal transduction via the G-protein-coupled receptor, Smoothened, is required for proliferation of cerebellar granule neuron precursors (CGNPs) during development. Activating mutations in the Hedgehog pathway are also implicated in basal cell carcinoma and medulloblastoma, a tumor of the cerebellum in humans. However, Shh signaling interactions with cell cycle regulatory components in neural precursors are poorly understood, in part because appropriate immortalized cell lines are not available. We have utilized primary cultures from neonatal mouse cerebella in order to determine (i) whether Shh initiates or maintains cell cycle progression in CGNPs, (ii) if G(1) regulation by Shh resembles that of classical mitogens, and (iii) whether individual D-type cyclins are essential components of Shh proliferative signaling in CGNPs. Our results indicate that Shh can drive continued cycling in immature, proliferating CGNPs. Shh treatment resulted in sustained activity of the G(1) cyclin-Rb axis by regulating levels of cyclinD1, cyclinD2, and cyclinE mRNA transcripts and proteins. Analysis of CGNPs from cyclinD1(-/-) or cyclinD2(-/-) mice demonstrates that the Shh proliferative pathway does not require unique functions of cyclinD1 or cyclinD2 and that D-type cyclins overlap functionally in this regard. In contrast to many known mitogenic pathways, we show that Shh proliferative signaling is mitogen-activated protein kinase independent. Furthermore, protein synthesis is required for early effects on cyclin gene expression. Together, our results suggest that Shh proliferative signaling promotes synthesis of regulatory factor intermediates that upregulate or maintain cyclin gene expression and activity of the G(1) cyclin-Rb axis in proliferating granule neuron precursors.

Animals↗
PubMed2000

Fate of the mammalian cardiac neural crest.

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

Animals↗
PubMed2000

Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis.

Neural crest cells are multipotential stem cells that contribute extensively to vertebrate development and give rise to various cell and tissue types. Determination of the fate of mammalian neural crest has been inhibited by the lack of appropriate markers. Here, we make use of a two-component genetic system for indelibly marking the progeny of the cranial neural crest during tooth and mandible development. In the first mouse line, Cre recombinase is expressed under the control of the Wnt1 promoter as a transgene. Significantly, Wnt1 transgene expression is limited to the migrating neural crest cells that are derived from the dorsal CNS. The second mouse line, the ROSA26 conditional reporter (R26R), serves as a substrate for the Cre-mediated recombination. Using this two-component genetic system, we have systematically followed the migration and differentiation of the cranial neural crest (CNC) cells from E9.5 to 6 weeks after birth. Our results demonstrate, for the first time, that CNC cells contribute to the formation of condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel's cartilage, mandible, the articulating disc of temporomandibular joint and branchial arch nerve ganglia. More importantly, there is a dynamic distribution of CNC- and non-CNC-derived cells during tooth and mandibular morphogenesis. These results are a first step towards a comprehensive understanding of neural crest cell migration and differentiation during mammalian craniofacial development. Furthermore, this transgenic model also provides a new tool for cell lineage analysis and genetic manipulation of neural-crest-derived components in normal and abnormal embryogenesis.

Animals↗
PubMed1999

Pax-2 regulatory sequences that direct transgene expression in the developing neural plate and external granule cell layer of the cerebellum.

Expression of Pax-2 in the mouse gastrula is the first marker of the midbrain-hindbrain region. To address roles played by transcription factors in the process of neural plate pattern formation and to facilitate gain-of-function approaches in the study of midbrain-hindbrain and cerebellar development, we characterized regulatory sequences at the Pax-2 locus using an in vivo transgenic mouse reporter assay. An 8.5 kb fragment of genomic DNA located upstream of Pax-2 directed lacZ expression prior to neurulation (7.5 days post-coitum, dpc) in a region fated to become midbrain and hindbrain, and subsequently in developing neuroepithelium. While similar to the pattern of Pax-2 expression, reporter gene activity extended beyond the boundaries of Pax-2 expression, most probably reflecting purdurance of beta-galactosidase activity and an absence of DNA sequences that restrict Pax-2 expression to rhombomere 1 by 9. 5 dpc. In the fetal and neonatal brain, Pax-2-lacZ activity was confined largely to Purkinje cells and the external granule cell layer (EGL) of the cerebellum. A 4 kb regulatory element, in contrast, initiated neural expression at 8.25 dpc in the anterior hindbrain, but recapitulated all later aspects of Pax-2-lacZ activity observed with the larger transgene. These results indicate the presence of regulatory sequences upstream of the Pax-2 locus capable of directing gene expression in the developing midbrain, first rhombomere of the hindbrain, and its principal derivative, the cerebellum. Successful misexpression of Sonic hedgehog demonstrates that Pax-2 regulatory sequences should prove generally useful for transgenic gain-of-function approaches in mice.

Animals↗
PubMed1999

Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells.

Activation of the Sonic hedgehog (Shh) signal transduction pathway is essential for normal pattern formation and cellular differentiation in the developing CNS. However, it is also thought to be etiological in primitive neuroectodermal tumors. We adapted GAL4/UAS methodology to ectopically express full-length Shh in the dorsal neural tube of transgenic mouse embryos commencing at 10 d postcoitum (dpc), beyond the period of primary dorsal-ventral pattern formation and floorplate induction. Expression of Shh was maintained until birth, permitting us to investigate effects of ongoing exposure to Shh on CNS precursors in vivo. Proliferative rates of spinal cord precursors were twice that of wild-type littermates at 12.5 dpc. In contrast, at late fetal stages (18.5 dpc), cells that were Shh-responsive but postmitotic were present in persistent structures reminiscent of the ventricular zone germinal matrix. This tissue remained blocked in an undifferentiated state. These results indicate that cellular competence restricts the proliferative response to Shh in vivo and provide evidence that proliferation and differentiation can be regulated separately in precursor cells of the spinal cord. Thus, Hedgehog signaling may contribute to CNS tumorigenesis by directly enhancing proliferation and preventing neural differentiation in selected precursor cells.

Animals↗
PubMed1999

Cystic malformation of the posterior cerebellar vermis in transgenic mice that ectopically express Engrailed-1, a homeodomain transcription factor.

In WEXPZ-En-1 transgenic mice, Engrailed-1, a homeodomain-containing transcription factor, is ectopically expressed in the developing brain under control of the Wnt-1 enhancer. En-1 is a developmental regulatory control gene which has an essential role in the formation of the midbrain and cerebellum. Approximately 28% of WEXPZ-En-1 + mice develop cystic malformations of the posterior lobe of the cerebellar vermis, fourth ventricular dilatation, and postnatal hydrocephalus. These anatomic features are also found among the spectrum of posterior fossa malformations in humans. Expression characteristics of the WEXP transgene suggest that the neuropathology observed in WEXPZ-En-1+ mice stems from overexpression of En-1 during fetal and neonatal phases of cerebellar development. These observations raise the possibility that abnormal regulation of Engrailed genes, or targets of Engrailed, may be involved in the pathogenesis of cystic central nervous system malformations of the posterior fossa in humans.

Animals↗
PubMed1998

Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.

Animals↗
PubMed1998

Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate.

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(-/-) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

Animals↗