Biomedical subjects

W H Klein

Publications and source records attributed to W H Klein.

At least 37 records · Page 2Linked to original sources

PubMed1998

Expression of a gene trap reporter construct in a subset of cells in embryonic sites of hematopoiesis: evidence for alternative rRNA production in hematopoietic cells.

Three mouse lines were generated from independent gene trap events in embryonic stem cells. These lines express a betageo reporter gene in a subset of cells at sites of embryonic hematopoiesis. The 5' breakpoints of all three lines were found to lie in 45S ribosomal RNA transcription units. Expression was apparently linked to metabolic activity in these cells, since the kinetics of expression during embryogenesis matched that of cycling cells with colony forming unit spleen (CFU-S) potential. Expression was not seen in adult tissues unless the animals were treated with hydroxyurea, inducing synchronous entry of quiescent CFU-S into the cell cycle. Our results suggest that there is a subset of hematopoietic stem cells, which when actively proliferating, express the SAbetageo reporter construct from RNA polymerase I transcription units.

Animals↗

PubMed1998

beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo.

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.

Animals↗

PubMed1998

Expression and functional analysis of mouse EXT1, a homolog of the human multiple exostoses type 1 gene.

Hereditary multiple exostoses (EXT) is a genetically heterogeneous, autosomal dominant skeletal disorder. The gene for EXT1 maps to human chromosome 8q24.1 and encodes an evolutionary conserved protein that is a member of a multigene family. The mouse homolog of human EXT1 protein is 99% similar to its human counterpart. Here, we present the expression profiles of the mouse EXT1 gene. EXT1 mRNA is initially expressed at 6.5 days post-coitum (d.p.c.), which coincides with gastrulation of the mouse embryo. Whole mount in situ hybridization with 10.5 to 12.5 d.p.c. mouse embryos showed a high level of expression of EXT1 mRNA in developing limb buds. Epitope tagging experiments revealed the endoplasmic reticulum localization of EXT1 protein. This localization was consistent with a hydrophobic stretch of amino acids present at the N-terminal end of the EXT1 protein. These results provide novel information on the function of EXT1 and the etiology of hereditary multiple exostoses.

3T3 Cells↗

PubMed1998

Overlapping functions of the myogenic bHLH genes MRF4 and MyoD revealed in double mutant mice.

The myogenic basic helix-loop-helix (bHLH) genes - MyoD, Myf5, myogenin and MRF4 - exhibit distinct, but overlapping expression patterns during development of the skeletal muscle lineage and loss-of-function mutations in these genes result in different effects on muscle development. MyoD and Myf5 have been shown to act early in the myogenic lineage to establish myoblast identity, whereas myogenin acts later to control myoblast differentiation. In mice lacking myogenin, there is a severe deficiency of skeletal muscle, but some residual muscle fibers are present in mutant mice at birth. Mice lacking MRF4 are viable and have skeletal muscle, but they upregulate myogenin expression, which could potentially compensate for the absence of MRF4. Previous studies in which Myf5 and MRF4 null mutations were combined suggested that these genes do not share overlapping myogenic functions in vivo. To determine whether the functions of MRF4 might overlap with those of myogenin or MyoD, we generated double mutant mice lacking MRF4 and either myogenin or MyoD. MRF4/myogenin double mutant mice contained a comparable number of residual muscle fibers to mice lacking myogenin alone and myoblasts from those double mutant mice formed differentiated multinucleated myotubes in vitro as efficiently as wild-type myoblasts, indicating that neither myogenin nor MRF4 is absolutely essential for myoblast differentiation. Whereas mice lacking either MRF4 or MyoD were viable and did not show defects in muscle development, MRF4/MyoD double mutants displayed a severe muscle deficiency similar to that in myogenin mutants. Myogenin was expressed in MRF4/MyoD double mutants, indicating that myogenin is insufficient to support normal myogenesis in vivo. These results reveal unanticipated compensatory roles for MRF4 and MyoD in the muscle differentiation pathway and suggest that a threshold level of myogenic bHLH factors is required to activate muscle structural genes, with this level normally being achieved by combinations of multiple myogenic bHLH factors.

Animals↗

PubMed1997

Two Otx proteins generated from multiple transcripts of a single gene in Strongylocentrotus purpuratus.

Orthodenticle-related (Otx) proteins are a highly conserved class of homeobox-containing transcription factors found in a wide range of organisms. They function in numerous developmental events, most prominently, anterior head patterning in insects and vertebrates. In the sea urchin, Strongylocentrotus purpuratus, an orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specific Spec2a gene and more generally the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from other species, we isolated cDNA and genomic clones containing SpOtx sequences. Here, we report that SpOtx exists in two forms (alpha and beta) that are generated by alternative RNA splicing from a single SpOtx gene. SpOtx(alpha) and SpOtx(beta) had identical C-termini and homeoboxes but were entirely different in their N-terminal domains. SpOtx(alpha) mRNAs were transcribed from a single start site and accumulated in all cells during cleavage, but were gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contrast, three distinct SpOtx(beta) mRNAs resulted from two separate transcriptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later were largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx(beta) appeared later in development than SpOtx(alpha). Overexpression of SpOtx(alpha) and SpOtx(beta) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx(alpha) was able to repress the accumulation of SpOtx(beta) transcripts, whereas SpOtx(beta) had no effect on the accumulation of SpOtx(alpha) transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates.

Alternative Splicing↗

PubMed1997

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Animals↗

PubMed1997

Multiple signaling events specify ectoderm and pattern the oral-aboral axis in the sea urchin embryo.

In the sea urchin embryo, the animal-vegetal axis is established during oogenesis and the oral-aboral axis is specified sometime after fertilization. The mechanisms by which either of these axes are specified and patterned during embryogenesis are poorly understood. Here, we investigated the role of cellular interactions in the specification of the ectoderm territories and polarization of the ectoderm along the oral-aboral axis. Isolated animal halves (mesomeres), which are fated to give rise to oral and aboral ectoderm, developed into polarized embryoids that expressed an oral ectoderm-specific marker uniformly. These embryoids also produced neuron-like cells and serotonergic neurons, suggesting that mesomeres are autonomously specified as oral ectoderm. Mesomere-derived embryoids did not express any aboral ectoderm-specific markers, although we previously showed that aboral ectoderm-specific genes can be induced by 25 mM lithium chloride, which also induced endoderm formation (Wikramanayake, A. H., Brandhorst, B. P. and Klein, W. H.(1995). Development 121, 1497-1505). To ascertain if endoderm formation is a prerequisite for induction of aboral ectoderm by lithium and for normal ectoderm patterning in animal halves, we modulated the lithium treatment to ensure that no endoderm formed. Remarkably, treating animal halves with 10 mM LiCl at approximately 7 hours postfertilization resulted in embryoids that displayed oral-aboral axis patterning in the absence of endoderm. Application of 25 mM LiCl to animal halves at approximately 16 hours postfertilization, which also did not induce endoderm, resulted in polarized expression of the aboral ectoderm-specific LpS1 protein, but global expression of the Ecto V antigen and no induction of the stomodeum or ciliary band. These results suggest that at least two signals, a positive inductive signal to specify the aboral ectoderm and a negative suppressive signal to inactivate oral ectoderm-specific genes in the prospective aboral ectoderm territory, are needed for correct spatial expression of oral and aboral ectoderm-specific genes. Transmission of both these signals may be prerequisite for induction of secondary ectodermal structures such as the ciliary band and stomodeum. Thus, differentiation of ectoderm and polarization of the oral-aboral axis in Lytechinus pictus depends on cellular interactions with vegetal blastomeres as well as interactions along the oral-aboral axis.

Animals↗

PubMed1996

Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.

Animals↗

PubMed1996

Extensive motor neuron survival in the absence of secondary skeletal muscle fiber formation.

Mice with a null mutation in the myogenic basic helixloop-helix regulatory gene myogenin have severe developmental muscle defects resulting in loss of secondary muscle fibers and perinatal death. In this study, we used the myogenin mutant mouse as a model to study the effects of the loss of secondary muscle fibers and the contribution of primary muscle fibers on the survival of motor neurons during programmed cell death. We demonstrate that in the absence of secondary skeletal muscle fibers there is complete survival of facial motor nucleus motor neurons and approximately 60% survival of spinal lumbar motor neurons in the myogenin mutant mouse. The surviving spinal motor neurons maintain axonal projections into the hindlimb and display aspects of synaptic contact into the remaining rudimentary fibers. These findings suggest that primary muscle fibers, representing approximately 10% of normal muscle mass, contribute significantly to the control of motor neuron cell survival in mammals.

Animals↗

PubMed1996

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Animals↗

PubMed1996

Transient appearance of Strongylocentrotus purpuratus Otx in micromere nuclei: cytoplasmic retention of SpOtx possibly mediated through an alpha-actinin interaction.

At the 16-cell stage, the sea urchin embryo is partitioned along the animal-vegetal axis into eight mesomeres, four macromeres, and four micromeres. The micromeres, unlike the other blastomeres, are autonomously specified to produce skeletogenic mesenchymal cells and are also required to induce the vegetal-plate territory. A long-held belief is that micromeres inherit localized maternal determinants that endow them with their cell autonomous behavior and inducing capabilities. Here, we present evidence that an orthodenticle-related protein, SpOtx appears transiently in nuclei of micromeres but not in nuclei of mesomeres and macromeres. At later stages of development, SpOtx was translocated into nuclei of all cells. To address the possibility that SpOtx was retained in the cytoplasm at early developmental stages, we searched for cytoplasmic proteins that interact with SpOtx. A proline-rich region of SpOtx resembling an SH3-binding domain was used to screen an embryo cDNA expression library, and a cDNA clone was isolated and shown to be alpha-actinin. A yeast two-hybrid analysis showed a specific interaction between the proline-rich region of SpOtx and a putative SH3 domain of the sea urchin alpha-actinin. Because micromeres lack an actin-based cytoskeleton, the results suggested that, at the vegetal pole of the 16-cell stage embryo, SpOtx was translocated into micromere nuclei, whereas in other blastomeres SpOtx was actively retained in the cytoplasm by binding to alpha-actinin. The transient appearance of SpOtx in micromere nuclei may be associated with the specification of micromere cell fate.

Actinin↗

PubMed1996

Very early and transient vegetal-plate expression of SpKrox1, a Krüppel/Krox gene from Stronglyocentrotus purpuratus.

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Krüppel/Krox-related genes from a Strongylocentrotus purpuratus embryo cDNA library. One newly identified clone, named SpKrox1, contained four zinc fingers and a leucine zipper domain. SpKrox1 expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastrula and pluteus stages. SpKrox1 mRNA was first seen in macromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate cells invaginated into the blastopore, SpKrox1 expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.

Amino Acid Sequence↗

PubMed1996

A G/C-rich DNA-regulatory element controls positive expression of the sea urchin Lytechinus pictus aboral ectoderm-specific LpS1 gene.

The LpS1 beta gene of Lytechinus pictus is activated at the late cleavage stage about 12 hr after fertilization. LpS1 beta transcripts accumulate exclusively in aboral ectoderm lineages. LpS1 beta is thus a classic example of a gene whose expression is tightly controlled both temporally and spatially during early development. Previous studies on the LpS1 beta promoter identified two G-string DNA elements, one proximal and one distal to the LpS1 beta transcriptional start site, which bind to an ectoderm-enriched nuclear factor. In this report, we show that the ectoderm G-string factor binds to a G/C-rich region larger than the G-string itself and that the binding of the G-string factor requires sequences immediately downstream from the G-string. These downstream sequences are essential for full promoter activity. Two regions of 5'-flanking DNA are required for positive control of LpS1 beta, region I from base pairs -762 to -511, and region II, which includes the G/C-rich element, from base pairs -108 to -61. Region I also contains a mesenchyme cell repressor element. The results indicate that LpS1 beta expression is regulated positively in ectoderm cells and negatively in mesenchyme cells. Similar positive and negative control mechanisms regulate the expression of the related Spec genes of Strongylocentrotus purpuratus, but in this gene family the DNA elements are entirely different. We hypothesize that cis-regulatory elements are evolutionarily dynamic and that many different combinations of DNA elements are capable of given rise to aboral ectoderm-specific expression.

Animals↗

PubMed1996

Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein.

While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified. SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene. Here, we present evidence that SpOtx has the potential to alter cell fates. SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation. Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate. At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations. These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor. Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.

Animals↗

PubMed1995

Activation of the myogenin promoter during mouse embryogenesis in the absence of positive autoregulation.

Myogenin, a member of the MyoD family of helix-loop-helix proteins, can induce myogenesis in a wide range of cell types. In addition to activating muscle structural genes, members of the MyoD family can autoactivate their own and cross-activate one another's expression in transfected cells. This has led to the hypothesis that autoregulatory loops among these factors provide a mechanism for amplifying and maintaining the muscle-specific gene expression program in vivo. Here, we make use of myogenin-null mice to directly test this hypothesis. To investigate whether the myogenin protein autoregulates the myogenin gene during embryogenesis, we introduced a myogenin-lacZ transgene into mice harboring a null mutation at the myogenin locus. Despite a severe deficiency of skeletal muscle in myogenin-null neonates, the myogenin-lacZ transgene was expressed normally in myogenic cells throughout embryogenesis. These results show that myogenin is not required for regulation of the myogenin gene and argue against the existence of a myogenin autoregulatory loop in the embryo.

Animals↗

PubMed1995

Myogenin's functions do not overlap with those of MyoD or Myf-5 during mouse embryogenesis.

The four myogenic basic helix-loop-helix proteins, MyoD, myogenin, Myf-5, and MRF4, can each activate skeletal muscle differentiation when introduced into nonmuscle cells. During embryogenesis, each of these genes is expressed in a unique but overlapping pattern in skeletal muscle precursors and their descendants. Gene knockout experiments have shown that MyoD and Myf-5 play seemingly redundant roles in the generation of myoblasts. However, the role of either of these genes during differentiation in vivo has not been determined. In contrast, a myogenin-null mutation blocks differentiation and results in a dramatic decrease in muscle fiber formation, yet the role of myogenin in the generation or maintenance of myoblast populations is not known. Because myogenin possesses the same myogenic activity as MyoD and Myf-5 in vitro and the expression patterns of these three genes overlap in vivo, we sought to determine if myogenin shares certain functions with either MyoD or Myf-5 in vivo. We therefore generated mice with double homozygous null mutations in the genes encoding MyoD and myogenin or Myf-5 and myogenin. These mice showed embryonic and perinatal phenotypes characteristic of the combined defects observed in mice mutant for each gene alone. As shown by histological analysis and expression of muscle-specific genes, the numbers of undifferentiated myoblasts and residual myofibers were comparable between myogenin-mutant homozygotes and the double-mutant homozygotes. Myoblasts isolated from neonates of the combined mutant genotypes underwent myogenesis in tissue culture, indicating that no more than two of the four myogenic factors are required to support muscle differentiation. These results demonstrate that the functions of myogenin do not overlap with those of MyoD or Myf-5 and support the view that myogenin acts in a genetic pathway downstream of MyoD and Myf-5.

Animals↗

PubMed1995

An orthodenticle-related protein from Strongylocentrotus purpuratus.

Orthodenticle-related proteins function as regulators of head formation and other developmental events in flies and mice. Here, we characterize a cDNA clone encoding an orthodenticle-related protein from the sea urchin Strongylocentrotus purpuratus. The cDNA, termed SpOtx, has a highly conserved orthodenticle homeobox but otherwise diverges in sequence from its fly and mouse counterparts. Orthodenticle-related proteins bind with high affinity to DNA containing the sequence motif TAATCC/T. The S. purpuratus aboral ectoderm-specific Spec2a gene has several TAATCC/T sites in its control region, and we provide evidence, using bandshift analysis, that Spec2a may be target gene for SpOtx. Two SpOtx transcripts accumulate during embryogenesis, an early transcript whose level peaks at blastula stage and a late transcript accumulating to highest concentrations at gastrula stage. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells. In contrast, SpOtx protein was found in nuclei of all cells at both blastula and pluteus stages. Our results suggest that SpOtx plays a role in the activation of the Spec2a gene and most likely has additional functions in the developing sea urchin embryo.

Amino Acid Sequence↗

PubMed1995

Myogenin is required for late but not early aspects of myogenesis during mouse development.

Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD-lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts.

Animals↗