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W H Klein

Publications and source records attributed to W H Klein.

At least 19 recordsLinked to original sources
PubMed2001

Gene expression in the developing mouse retina by EST sequencing and microarray analysis.

Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.

Animals↗
PubMed2001

Correct Expression of spec2a in the sea urchin embryo requires both Otx and other cis-regulatory elements.

Strongylocentrotus purpuratus Otx (SpOtx) is required simultaneously in sea urchin development for the activation of endo16 in the vegetal plate and for the activation of spec2a in the aboral ectoderm. Because Otx binding sites alone do not appear to be responsible for the spatially restricted expression of spec2a, additional DNA elements were sought. We show here that consensus Otx binding sites fused to basal promoters are sufficient to activate CAT reporter gene expression in all cell types, although expression in endomesoderm progenitors is enhanced. On the other hand, three non-Otx elements derived from the spec2a enhancer are needed together with Otx sites for specifically aboral ectoderm expression. A DNA element termed Y/CBF, lying just downstream from an Otx site within the spec2a enhancer, mediates general activation in the ectoderm. A second element lying between the Otx and Y/CBF sites, called OER, functions to prevent expression in the oral ectoderm. A third site, called ENR, overlapping another Otx site, is required to repress endoderm expression. Three distinct DNA binding proteins interact sequence specifically at the Y/CBF, OER, and ENR elements. The spec2a enhancer thus consists of closely linked activator and repressor elements that function collectively to cause expression of the spec2a gene in the aboral ectoderm.

Animals↗
PubMed2001

MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells.

myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.

Animals↗
PubMed2001

Requirement for math5 in the development of retinal ganglion cells.

math5 is a murine orthologue of atonal, a bHLH proneural gene essential for the formation of photoreceptors and chordotonal organs in Drosophila. The expression of math5 coincides with the onset of retinal ganglion cell (RGC) differentiation. Targeted deletion of math5 blocks the initial differentiation of 80% of RGCs and results in an increase in differentiated amacrine cells. Furthermore, the absence of math5 abolishes the retinal expression of brn-3b and the formation of virtually all brn-3b-expressing RGCs. These results imply that math5 is a proneural gene essential for RGC differentiation and that math5 acts upstream to activate brn-3b-dependent differentiation processes in RGCs.

Animals↗
PubMed2001

Similar myogenic functions for myogenin and MRF4 but not MyoD in differentiated murine embryonic stem cells.

The emergence of four genes encoding myogenic bHLH transcription factors in the stem vertebrate is a unique feature of vertebrate evolution that led to the acquisition of new target genes and novel regulatory circuits for skeletal muscle formation. One unresolved question is the extent to which each factor has evolved specialized functions for regulating muscle gene transcription. We have developed a system using differentiated myogenin (-/-) embryonic stem (ES) cells to determine whether other myogenic factors can replace myogenin's function in muscle differentiation. Previously, we showed that constitutively expressed myogenin restores myofiber formation in differentiated myogenin (-/-) ES cells, but constitutively expressed MyoD does not. Here, we confirm the distinction between myogenin and MyoD using another expression vector and show that constitutively expressed MRF4 leads to myofiber formation in myogenin's absence. Our analysis reveals a correlation between the levels of myogenin plus MRF4 and the extent of myofiber formation, suggesting a synergy between these factors. The results indicate that unlike MyoD, MRF4 plays a role similar to myogenin in skeletal muscle differentiation.

Animals↗
PubMed2001

Sea urchin goosecoid function links fate specification along the animal-vegetal and oral-aboral embryonic axes.

We have identified a single homolog of goosecoid, SpGsc, that regulates cell fates along both the animal-vegetal and oral-aboral axes of sea urchin embryos. SpGsc mRNA is expressed briefly in presumptive mesenchyme cells of the approximately 200-cell blastula and, beginning at about the same time, accumulates in the presumptive oral ectoderm through pluteus stage. Loss-of-function assays with morpholine-substituted antisense oligonucleotides show that SpGsc is required for endoderm and pigment cell differentiation and for gastrulation. These experiments and gain-of-function tests by mRNA injection show that SpGsc is a repressor that antagonizes aboral ectoderm fate specification and promotes oral ectoderm differentiation. We show that SpGsc competes for binding to specific cis elements with SpOtx, a ubiquitous transcription activator that promotes aboral ectoderm differentiation. Moreover, SpGsc represses transcription in vivo from an artificial promoter driven by SpOtx. As SpOtx appears long before SpGsc transcription is activated, we propose that SpGsc diverts ectoderm towards oral fate by repressing SpOtx target genes. Based on the SpGsc-SpOtx example and other available data, we propose that ectoderm is first specified as aboral by broadly expressed activators, including SpOtx, and that the oral region is subsequently respecified by the action of negative regulators, including SpGsc. Accumulation of SpGsc in oral ectoderm depends on cell-cell interactions initiated by nuclear beta-catenin function, which is known to be required for specification of vegetal tissues, because transcripts are undetectable in dissociated or in cadherin mRNA-injected embryos. This is the first identified molecular mechanism underlying the known dependence of oral-aboral ectoderm polarity on intercellular signaling.

Amino Acid Sequence↗
PubMed2000

Thoracic skeletal defects in myogenin- and MRF4-deficient mice correlate with early defects in myotome and intercostal musculature.

Myogenin and MRF4 are skeletal muscle-specific bHLH transcription factors critical for muscle development. In addition to a variety of skeletal muscle defects, embryos homozygous for mutations in myogenin or MRF4 display phenotypes in the thoracic skeleton, including rib fusions and sternal defects. These skeletal defects are likely to be secondary because myogenin and MRF4 are not expressed in the rib cartilage or sternum. In this study, the requirement for myogenin and MRF4 in thoracic skeletal development was further examined. When a hypomorphic allele of myogenin and an MRF4-null mutation were placed together, the severity of the thoracic skeletal defects was greatly increased and included extensive rib cartilage fusion and fused sternebrae. Additionally, new rib defects were observed in myogenin/MRF4 compound mutants, including a failure of the rib cartilage to contact the sternum. These results suggested that myogenin and MRF4 share overlapping functions in thoracic skeletal formation. Spatial expression patterns of skeletal muscle-specific markers in myogenin- and MRF4-mutant embryos revealed early skeletal muscle defects not previously reported. MRF4-/- mice displayed abnormal intercostal muscle morphology, including bifurcation and fusion of adjacent intercostals. myogenin/MRF4-mutant combinations displayed ventral myotome defects, including a failure to express normal levels of myf5. The results suggested that the early muscle defects observed in myogenin and MRF4 mutants may cause subsequent thoracic skeletal defects, and that myogenin and MRF4 have overlapping functions in ventral myotome differentiation and intercostal muscle morphogenesis.

Alleles↗
PubMed2000

Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4.

The basic helix-loop-helix (bHLH) transcription factors-MyoD, Myf5, myogenin, and MRF4-can each activate the skeletal muscle-differentiation program in transfection assays. However, their functions during embryogenesis, as revealed by gene-knockout studies in mice, are distinct. MyoD and Myf5 have redundant functions in myoblast specification, whereas myogenin and either MyoD or MRF4 are required for differentiation. Paradoxically, myoblasts from myogenin mutant or MyoD/MRF4 double-mutant neonates differentiate normally in vitro, despite their inability to differentiate in vivo, suggesting that the functions of the myogenic bHLH factors are influenced by the cellular environment and that the specific myogenic defects observed in mutant mice do not necessarily reflect essential functions of these factors. Understanding the individual roles of these factors is further complicated by their ability to cross-regulate one another's expression. To investigate the functions of Myf5 in the absence of contributions from other myogenic bHLH factors, we generated triple-mutant mice lacking myogenin, MyoD, and MRF4. These mice appear to contain a normal number of myoblasts, but in contrast to myogenin or MyoD/MRF4 mutants, differentiated muscle fibers fail to form in vivo and myoblasts from neonates of this triple-mutant genotype are unable to differentiate in vitro. These results suggest that physiological levels of Myf5 are insufficient to activate the myogenic program in the absence of other myogenic factors and suggest that specialized functions have evolved for the myogenic bHLH factors to switch on the complete program of muscle gene expression.

Animals↗
PubMed2000

Abnormal polarization and axon outgrowth in retinal ganglion cells lacking the POU-domain transcription factor Brn-3b.

The POU domain transcription factor Brn-3b (also called Brn-3.2) is essential for the normal development of retinal ganglion cells (RGCs) in the mouse. Without Brn-3b, RGCs commit to their fate and migrate to the ganglion cell layer, but most cells die during fetal development. An earlier report (L. Gan et al., 1999, Dev. Biol. 210, 469-480) suggested that cell death was caused by abnormal axon formation. Here, we use retinal explants from wild-type and mutant embryos to show that brn-3b-deficient RGCs are not properly polarized and tend to form dendrites rather than axons. Compared with wild-type explants, neurites of RGCs from brn-3b-deficient retinal explants grew slower, were shorter, and did not fasciculate properly. Mutant neurites had more microtubules than wild-type controls, and the arrangement of microtubules and neurofilaments was characteristic of dendrites rather than axons. Neurites from individual mutant RGCs displayed abnormal polarity and had dendrite-like branches extending outward from their main axis. Most mutant RGCs exhibited abnormal migratory behavior, and their neurites labeled intensely with the dendrite marker MAP-2. A small number of mutant RGCs were not migratory, and their neurites were longer and labeled positively for the axon marker tau-1, suggesting that some RGCs were not as severely affected by the absence of Brn-3b as others. Although tau-1 was not observed in most mutant neurites, it did accumulate in mutant cell bodies, implying that the absence of Brn-3b caused a defect in axon transport. Thus, Brn-3b appears to control the activity of genes that function in establishing RGC polarity, and without Brn-3b, RGCs cannot extend normal axons.

Animals↗
PubMed2000

Involvement of Tcf/Lef in establishing cell types along the animal-vegetal axis of sea urchins.

Members of the Tcf/Lef family interact with beta-catenin to activate programs of gene expression during development. Recently beta-catenin was shown to be essential for establishing cell fate along the animal-vegetal axis of the sea urchin embryo. To examine the role of Tcf/Lef in sea urchins we cloned a Strongylocentrotus purpuratus Tcf/Lef homolog. Expression of SpTcf/Lef was maximal when beta-catenin became localized to nuclei of vegetal blastomeres, consistent with its acting in combination with beta-catenin to specify vegetal cell fates. Expression of a dominant-negative SpTcf/Lef inhibited primary and secondary mesenchyma, endoderm, and aboral ectoderm formation in a manner similar to that observed when nuclear accumulation of beta-catenin was prevented. Our results suggest that SpTcf/Lef functions by interacting with beta-catenin to specify cell fates along the sea urchin animal-vegetal axis.

Amino Acid Sequence↗
PubMed1999

Identification of a differentially expressed RNA helicase by gene trapping.

A mouse line was generated that expressed a gene trap reporter construct, betageo, in a dynamic pattern during embryonic development. Differential expression was seen within the developing eyes, limbs, heart, neural tube, and skeleton. Two transcripts were cloned that contained endogenous sequences fused to the gene trap vector sequence. Analysis of the endogenous sequences revealed that the reporter integrated within a gene belonging to a small group of eukaryotic superfamily I helicases. Unexpectedly, the majority of transcripts produced from the trapped locus were not affected by the insertion of the reporter. Although the function of the trapped helicase gene is unknown, its complex transcription patterns and widespread spatial-temporal distribution suggest that the gene product plays a role in RNA metabolism in multiple tissues and organs within the developing embryo.

Amino Acid Sequence↗
PubMed1999

Requirement of SpOtx in cell fate decisions in the sea urchin embryo and possible role as a mediator of beta-catenin signaling.

We show here that the homeodomain transcription factor SpOtx is required for endoderm and aboral ectoderm formation during sea urchin embryogenesis. SpOtx target genes were repressed by fusing the SpOtx homeodomain to an active repression domain of Drosophila Engrailed. The Engrailed-SpOtx fusion protein reduced the expression of endoderm- and aboral ectoderm-specific genes and inhibited the formation of endoderm and aboral ectoderm cell types. Coexpressing activated beta-catenin with Engrailed-SpOtx did not overcome the inhibition of endoderm and aboral ectoderm formation, suggesting that SpOtx functioned either downstream of or parallel to nuclear beta-catenin. Embryos expressing C-cadherin, which blocks nuclear translocation of beta-catenin, have defects in endoderm and aboral ectoderm formation. Coexpressing SpOtx with C-cadherin restored aboral ectoderm-specific gene expression and aboral ectoderm morphology, but with C-cadherin present, SpOtx was not sufficient for endoderm formation. Our results show that SpOtx plays a key role in the activation of aboral ectoderm- and endoderm-specific gene expression and, in addition, suggest that SpOtx mediates some of beta-catenin's functions in endoderm and aboral ectoderm formation.

Animals↗
PubMed1999

POU domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial cell fate specification.

While the mammalian retina is well understood at the anatomical and physiological levels, little is known about the mechanisms that give rise to the retina's highly ordered pattern or its diverse neuronal cell types. Previous investigations have shown that gene disruption of the POU-IV class transcription factor Brn-3b (Brn-3.2) resulted in the loss of most retinal ganglion cells in retinas of postnatal mice. Here, we used lacZ and human placental alkaline phosphatase genes knocked into the brn-3b locus to follow the fate of brn-3b-mutant cells in the developing retina. We found that Brn-3b was not required for the initial commitment of retinal ganglion cell fate or for the migration of ganglion cells to the ganglion cell layer. However, Brn-3b was essential for the normal differentiation of retinal ganglion cells; without it, the cells underwent enhanced apoptosis. Retinal ganglion cells lacking brn-3b extended processes at the appropriate time in development, but these processes were disorganized, resulting in a thinner optic nerve. Explanted retinas from brn-3b-null embryos also extended processes when cultured in vitro, but the processes were shorter and less bundled than in wild-type retinas. Ultrastructural and marker analyses showed that the processes of mutant ganglion cells had dendritic rather than axonal features, suggesting that mutant cells formed dendrites in place of axons. These results suggest that Brn-3b regulates the activity of genes whose products play essential roles in the formation of retinal ganglion cell axons.

Alkaline Phosphatase↗
PubMed1999

Function and evolution of Otx proteins.

Otx proteins comprise an important class of homeodomain-containing transcription factors known for their essential roles in anterior head formation. Here, we briefly review the basic structural features and functional diversity of Otx proteins and describe current views on the evolution of Otx genes in metazoans. A prominent feature of Otx homeodomains is a lysine residue at position 9 of the recognition helix, which confers high-affinity binding to TAATCC/T elements on DNA. Besides their DNA binding properties, surprisingly little is known about how Otx proteins function to activate target genes in selective regions of the embryo. While an essential and ancient role for Otx is to pattern the anterior regions of the head, drawing conclusions about primordial functions is difficult. This is because Otx proteins have been recruited for numerous developmental roles, and derived functions have often evolved to meet the specialized requirements of individual taxonomic groups. In sea urchin embryos, one form of Otx may have been co-opted by the Wnt--catenin signaling pathway. The consequence of such an evolutionary event would be to link a highly conserved signal transduction pathway to a set of novel downstream genes that make use of Otx for their transcription.

Animals↗
PubMed1999

A hypomorphic myogenin allele reveals distinct myogenin expression levels required for viability, skeletal muscle development, and sternum formation.

The myogenic basic helix-loop-helix transcription factor myogenin plays an essential role in the differentiation of skeletal muscle and, secondarily, in rib and sternum formation during mouse development. However, virtually nothing is known about the quantitative requirements for myogenin in these processes. Here, we describe the generation of mice carrying a hypomorphic allele of myogenin, which expresses myogenin transcripts at approximately one-fourth the level of the wild-type myogenin allele. The hypomorphic allele in combination with wild-type and myogenin-null alleles was used to create an allelic series. Embryos representing the complete range of genotypes from homozygous wild type to homozygous null were analyzed for their viability, ability to form normal ribs and sternum, and extent of skeletal muscle differentiation. Embryos carrying the hypomorphic myogenin allele over a wild-type allele were normal. In embryos bearing homozygous hypomorphic alleles, the sternum developed normally and extensive skeletal muscle differentiation occurred. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable as neonates. When the hypomorphic allele was placed over a myogenin-null allele, the resulting embryos had sternum defects resembling homozygous myogenin-null embryos, and there was severe muscle hypoplasia. Our results demonstrate that skeletal muscle formation is highly sensitive to the absolute levels of myogenin and that correct sternum formation, skeletal muscle differentiation, and viability each require distinct threshold levels of myogenin.

Alleles↗
PubMed1999

Temporal, spatial and tissue-specific expression of a myogenin-lacZ transgene targeted to the Hprt locus in mice.

A lacZ transgene, expressed by the myogenin promoter, was introduced into the mouse hypoxanthine phosphoribosyltransferase (Hprt) locus by gene targeting in embryonic stem cells. Embryos between E10.5-E18.5 days were analyzed for expression of the transgene after staining for beta-galactosidase activity. Transgene expression was restricted to the skeletal muscle lineages reflecting a similar temporal and spatial pattern previously demonstrated for the endogenous myogenin gene. Additionally, a second transgene, MC1tk, showed expression in 87% of the clones when targeted to Hprt. This strategy, called targeted transgenesis, provides control for analyzing promoter sequences and for comparing various transgenes expressed by the same promoter.

Animals↗
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↗