Biomedical subjects

Heithem M El-Hodiri

Publications and source records attributed to Heithem M El-Hodiri.

14 recordsLinked to original sources

PubMed2006

PP2A:B56epsilon is required for eye induction and eye field separation.

Eye induction and eye field separation are the earliest events during vertebrate eye development. Both of these processes occur much earlier than the formation of optic vesicles. The insulin-like growth factor (IGF) pathway appears to be essential for eye induction, yet it remains unclear how IGF downstream pathways are involved in eye induction. As a consequence of eye induction, a single eye anlage is specified in the anterior neural plate. Subsequently, this single eye anlage is divided into two symmetric eye fields in response to Sonic Hedgehog (Shh) secreted from the prechordal mesoderm. Here, we report that B56epsilon regulatory subunit of protein phosphatase 2A (PP2A) is involved in Xenopus eye induction and subsequent eye field separation. We provide evidence that B56epsilon is required for the IGF/PI3K/Akt pathway and that interfering with the PI3K/Akt pathway inhibits eye induction. In addition, we show that B56epsilon regulates the Hedgehog (Hh) pathway during eye field separation. Thus, B56epsilon is involved in multiple signaling pathways and plays critical roles during early development.

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PubMed2006

Expression of the forkhead transcription factor FoxN4 in progenitor cells in the developing Xenopus laevis retina and brain.

Forkhead proteins are involved in gene regulation in a large variety of developmental situations. Several forkhead gene products are expressed in the developing eye and brain. Here we characterize the expression of FoxN4 during Xenopus development. We report that FoxN4 is expressed in the eye from the earliest stages of specification through retinal maturation. FoxN4 is also expressed in the pallium, optic tectum, isthmus, reticular formation, and in cells lining the ventricle of the tadpole brain.

Amino Acid Sequence↗

PubMed2006

Pbx1 and Meis1 regulate activity of the Xenopus laevis Zic3 promoter through a highly conserved region.

Xenopus Zic3 (zinc finger in the cerebellum-3) is expressed in the dorsal neural tube of tailbud embryos and tadpoles. We have isolated a 3.1kb DNA fragment from the Xenopus laevis Zic3 locus that drives proper expression of a GFP reporter in transgenic frog tailbud embryos and tadpoles. This fragment contains regions that are highly similar among frogs, mice, and humans. One extremely conserved region contains a predicted Pbx binding site. We found that the transcription factors Pbx1b and Meis1 can bind this site and synergistically transactivate expression of a reporter containing the conserved region. Finally, we found that an intact Pbx site is essential for normal Zic3 promoter activity in transgenic frog embryos. Our data strongly suggest that a highly conserved region of the Zic3 promoter functions by direct interaction with Pbx1b and Meis1.

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PubMed2006

Conserved regulatory elements establish the dynamic expression of Rpx/HesxI in early vertebrate development.

TheRpx/Hesx1 homeobox gene is expressed during gastrulation in the anterior visceral and definitive endoderm and the cephalic neural plate. At later stages of development, its expression is restricted to Rathke's pouch, the primordium of the pituitary gland. This expression pattern suggests the presence of at least two distinct regulatory regions that control early and late Rpx transcription. Using transgenic mice, we have demonstrated that regulatory sequences in the 5' upstream region of Rpx are important for early expression in the anterior endoderm and neural plate and regulatory elements in the 3' region are required for late expression in Rathke's pouch. We have found that the genetically required LIM homeodomain-containing proteins Lim1/Lhx1 and Lhx3 are directly involved in the regulation of Rpx transcription. They bind two LIM protein-binding sites in the 5' upstream region of Rpx, which are required for Rpx promoter activity in both mice and Xenopus. Furthermore, we have found that a conserved enhancer in the 3' regulatory sequences of Rpx is not only required, but is also sufficient for the expression of Rpx transgenes in the developing Rathke's pouch.

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PubMed2006

The Rx-like homeobox gene (Rx-L) is necessary for normal photoreceptor development.

PURPOSE: The retinal homeobox (Rx) gene plays an essential role in retinal development. An Rx-like (Rx-L) gene from Xenopus laevis has been identified. The purpose of this study was to analyze the function of Rx-L in the developing retina. METHODS: DNA-binding properties of Rx-L were analyzed by electrophoretic mobility shift assay (EMSA), with in vitro-translated proteins and radiolabeled oligonucleotide probe. The Rx-L expression pattern was analyzed by in situ hybridization using whole or sectioned embryos and digoxigenin-labeled antisense riboprobes. Rx-L loss of function was studied by using antisense morpholino oligonucleotides targeted to the Rx-L translation initiation site. Embryos injected with control or Rx-L morpholinos were analyzed at stage 41 or 45. RESULTS: Rx-L shares homology with Rx at the homeo-, OAR, and Rx domains, but lacks an octapeptide motif. Rx-L is expressed in the developing retina beginning in the early tailbud stage. In the maturing retina, Rx-L expression is restricted primarily to the developing photoreceptor layer and the ciliary marginal zone. Rx-L can bind a photoreceptor conserved element-1 (PCE-1) oligonucleotide, an element conserved among all known photoreceptor gene promoters. In a promoter activity assay, Rx-L functions as a stronger transcriptional activator than Rx. Antisense morpholino-mediated knockdown of Rx-L expression resulted in a decrease in rhodopsin and red cone opsin expression levels in Xenopus retinas. Injection of the Rx-L antisense morpholino oligonucleotide also resulted in a decrease in the length of both rod and cone outer segments. CONCLUSIONS: The results suggest that Rx-L functions to regulate rod and cone development by activating photoreceptor-specific gene expression.

Amino Acid Sequence↗

PubMed2005

Expression of a novel Ski-like gene in Xenopus development.

Members of the Ski/Sno family of gene products contain a characteristic peptide domain involved in protein-protein or protein-DNA interaction. Here, we characterize the developmental expression of xDawg, in Xenopus laevis, of a new gene, related to the Ski/Sno family of transcription regulators. The Ski/Sno domain of xDawg is predicted to present an electropositive surface, consistent with a role in DNA binding. This gene is expressed in the marginal zone of early gastrulae, and in the brain, sensory vesicles, and cranial neural crest of neurula and tailbud embryos.

Amino Acid Sequence↗

PubMed2005

Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain development.

Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx-injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development.

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PubMed2005

Recombineered Xenopus tropicalis BAC expresses a GFP reporter under the control of Arx transcriptional regulatory elements in transgenic Xenopus laevis embryos.

The aristaless-related homeobox (Arx) gene is expressed in a dynamic pattern in the developing vertebrate forebrain. We identified a bacterial artificial chromosome (BAC) containing the Xenopus tropicalis Arx gene and replaced a portion of the first coding exon with a green fluorescent protein (GFP) expression cassette by homologous recombination in bacteria (recombineering). Transgenic X. laevis embryos obtained by microinjecting the modified BAC expressed GFP in the developing forebrain in a pattern identical to that of the endogenous Arx gene. Thus, this BAC contains transcriptional regulatory elements necessary for regulating proper expression in transgenic frogs. This work demonstrates that transgenesis using recombineered BACs is a viable technique in Xenopus.

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PubMed2005

Smad1 and Smad8 function similarly in mammalian central nervous system development.

Smads 1, 5, and 8 are the intracellular mediators for the bone morphogenetic proteins (BMPs), which play crucial roles during mammalian development. Previous research has shown that Smad1 is important in the formation of the allantois, while Smad5 has been shown to be critical in the process of angiogenesis. To further analyze the BMP-responsive Smads, we disrupted the murine Smad8 gene utilizing the Cre/loxP system. A Smad8 hypomorphic allele (Smad8(Deltaexon3)) was constructed that contains an in-frame deletion of exon 3, removing one-third of the MH2 domain and a small portion of the linker region. Xenopus injection assays indicated that this Smad8 deletion allele is still functional but has reduced ventralizing capability compared to the wild type. Although Smad8(Deltaexon3/Deltaexon3) embryos are phenotypically normal, homozygotes of another hypomorphic allele of Smad8 (Smad8(3loxP)) containing a neomycin cassette within intron 3, phenocopy an embryonic brain defect observed in roughly 22% of Smad1(+/)(-) embryos analyzed at embryonic day 11.5. These observations suggest that BMP-responsive Smads have critical functions in the development of the mammalian central nervous system.

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PubMed2005

Xenopus laevis FoxE1 is primarily expressed in the developing pituitary and thyroid.

The members of the FoxE subfamily of Fox (forkhead) genes are expressed in the developing pituitary, thyroid and lens. Mammalian Foxe1 is expressed primarily in the developing pituitary and thyroid gland, Foxe3 is expressed in the developing lens, while Xenopus FoxE4 is expressed in the developing lens and thyroid. Here we report the identification of Xenopus FoxE1, a gene that is primarily expressed in the developing pituitary and thyroid.

Amino Acid Sequence↗

PubMed2004

Smad2 and Smad3 coordinately regulate craniofacial and endodermal development.

Ligands of the transforming growth factor-beta (TGF-beta) superfamily are involved in numerous developmental and disease processes. TGF-beta, activins, and nodal ligands operate through the highly homologous Smad2 and Smad3 intracellular mediators. Smad2 mutants exhibit early embryonic lethality, while Smad3 mutants are viable, but show a plethora of postnatal phenotypes, including immune dysfunction and skeletal abnormalities. Previously, we have shown that the Smad2 and Smad3 genes function cooperatively during liver morphogenesis. Here we show that Smad2 and Smad3 are required at a full dosage for normal embryonic development. Animals lacking one allele of each gene exhibit a variably penetrant phenotype in which structures in the anterior and ventral midline are reduced or lost; additionally, we demonstrate that this craniofacial defect and the previously reported hepatic phenotypes are both due to defects in the definitive endoderm. A reduction of endodermal gene expression as well as a failure to displace the visceral endoderm occurs despite the formation of a normal foregut pocket. This precedes any defects in anterior patterning and likely causes the abnormalities observed in craniofacial and midline development, as well as hepatogenesis.

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PubMed2003

Evidence for antagonism of BMP-4 signals by MAP kinase during Xenopus axis determination and neural specification.

We have previously shown that mitogen-activated protein (MAP) kinase activity is required for neural specification in Xenopus. In mammalian cells, the BMP-4 effector Smad1 is inhibited by phosphorylation at MAP kinase sites (Kretzschmar et al., 1997). To test the hypothesis that MAP kinase inhibits the BMP-4/Smad1 pathway during early Xenopus development, we have generated a Smad1 mutant lacking the MAP kinase phosphorylation sites (M4A-Smad1) and compared the effects of wild-type (WT)- and M4A-Smad1 on axial pattern and neural specification in Xenopus embryos. Although overexpression of either WT- or M4A-Smad1 produced ventralized embryos, at each mRNA concentration, M4A-Smad1 had a greater ventralizing effect than WT-Smad1. Interestingly, overexpression of either form of Smad1 in ventral blastomeres disrupted posterior pattern and morphogenesis; again, more severe defects were produced by expression of M4A-Smad1 than by equal amounts of WT-Smad1. Ectodermal expression of M4A-Smad1 disrupted expression of the anterior neural gene otx2 in vivo and inhibited neural specification in response to endogenous signals in mesoderm-ectoderm recombinates. In contrast, overexpression of WT-Smad1 at identical levels had little effect on either neural specification or otx2 expression. Comparisons of protein levels following overexpression of either WT- or M4A-Smad1 indicate that WT-Smad1 may be slightly more stable than M4A-Smad1; thus, differences in stability cannot account for the increased effectiveness of M4A-Smad1. Our results demonstrate that mutations disrupting the MAPK phosphorylation sites act collectively as a gain-of-function mutation in Smad1 and that inhibitory phosphorylation of Smad1 may be a significant mechanism for the regulation of BMP-4/Smad1 signals during Xenopus development.

Animals↗

PubMed2003

Targeted expression of the dominant-negative FGFR4a in the eye using Xrx1A regulatory sequences interferes with normal retinal development.

Molecular analysis of vertebrate eye development has been hampered by the availability of sequences that can selectively direct gene expression in the developing eye. We report the characterization of the regulatory sequences of the Xenopus laevis Rx1A gene that can direct gene expression in the retinal progenitor cells. We have used these sequences to investigate the role of Fibroblast Growth Factor (FGF) signaling in the development of retinal cell types. FGFs are signaling molecules that are crucial for correct patterning of the embryo and that play important roles in the development of several embryonic tissues. FGFs and their receptors are expressed in the developing retina, and FGF receptor-mediated signaling has been implicated to have a role in the specification and survival of retinal cell types. We investigated the role of FGF signaling mediated by FGF receptor 4a in the development of retinal cell types in Xenopus laevis. For this purpose, we have made transgenic Xenopus tadpoles in which the dominant-negative FGFR4a (Delta FGFR4a) coding region was linked to the newly characterized regulatory sequences of the Xrx1A gene. We found that the expression of Delta FGFR4a in retinal progenitor cells results in abnormal retinal development. The retinas of transgenic animals expressing Delta FGFR4a show disorganized cell layering and specifically lack photoreceptor cells. These experiments show that FGFR4a-mediated FGF signaling is necessary for the correct specification of retinal cell types. Furthermore, they demonstrate that constructs using Xrx1A regulatory sequences are excellent tools with which to study the developmental processes involved in retinal formation.

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PubMed2002

The Xenopus arx gene is expressed in the developing rostral forebrain.

The human aristaless-related homeobox ( ARX) gene is mutated in several patients with X-linked mental retardation and/or other neurologic pathologies. We report the isolation and expression pattern of a Xenopus arx gene. Similar to other vertebrate arx genes, Xenopus arx is expressed in the developing telencephalon, diencephalon, and floor plate.

Amino Acid Sequence↗