Biomedical subjects

Ken W Y Cho

Publications and source records attributed to Ken W Y Cho.

At least 19 recordsLinked to original sources

PubMed2025

Foxi2 and Sox3 are master transcription regulators that control ectoderm germ layer specification in Xenopus.

Germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomic analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification.

Animals↗

PubMed2025

Foxh1 is a locus-specific PRC2 recruiter governing germ layer silencing.

Polycomb Repressive Complex 2 (PRC2) establishes H3K27me3 marks to shape spatiotemporal gene expression during embryogenesis. While its dysregulation is linked to developmental disorders, cancer, and aging, the mechanisms guiding PRC2 to specific genomic loci remain a subject of ongoing debate. A prevailing model proposes that PRC2 recruitment occurs via its intrinsic affinity for chromatin rather than through sequence-specific transcription factors. Here, we provide evidence that the maternally deposited pioneer transcription factor Foxh1 plays a critical role in directing PRC2 to specific genomic loci during zygotic genome activation in Xenopus. Foxh1 is a critical transcription factor mediating Nodal signaling, but it also plays an earlier role by pre-binding enhancers prior to signaling activation. This pre-binding is essential for forming enhanceosome complexes that trigger mesendodermal gene expression and drive gastrulation, in cooperation with other maternal transcription factors. Using maternal Foxh1-null embryos, we demonstrate that Foxh1 directly recruits Ezh2, the catalytic subunit of PRC2, to Foxh1-bound loci. Loss of Foxh1 impairs Ezh2 recruitment, leading to a global reduction in H3K27me3. These findings support a dual-function model in which Foxh1 not only activates endodermal gene expression in endoderm, but also recruits PRC2 to silence the same genes in ectoderm. This dual activity of Foxh1 allows the spatially coordinated epigenetic states of the endodermal gene regulatory program during early embryogenesis.

CRISPR/Cas9↗

PubMed2025

Foxi2 and Sox3 are master regulators controlling ectoderm germ layer specification.

In vertebrates, germ layer specification represents a critical transition where pluripotent cells acquire lineage-specific identities. We identify the maternal transcription factors Foxi2 and Sox3 to be pivotal master regulators of ectodermal germ layer specification in Xenopus. Ectopic co-expression of Foxi2 and Sox3 in prospective endodermal tissue induces the expression of ectodermal markers while suppressing mesendodermal markers. Transcriptomics analyses reveal that Foxi2 and Sox3 jointly and independently regulate hundreds of ectodermal target genes. During early cleavage stages, Foxi2 and Sox3 pre-bind to key cis-regulatory modules (CRMs), marking sites that later recruit Ep300 and facilitate H3K27ac deposition, thereby shaping the epigenetic landscape of the ectodermal genome. These CRMs are highly enriched within ectoderm-specific super-enhancers (SEs). Our findings highlight the pivotal role of ectodermal SE-associated CRMs in precise and robust ectodermal gene activation, establishing Foxi2 and Sox3 as central architects of ectodermal lineage specification.

Ep300↗

PubMed2006

Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase.

Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.

Animals↗

PubMed2006

Grainyhead-like 3, a transcription factor identified in a microarray screen, promotes the specification of the superficial layer of the embryonic epidermis.

The Xenopus ectoderm consists of two populations of cells, superficial polarised epithelial cells and deep, non-epithelial cells. These two cell types differ in their developmental fate. In the neural ectoderm, primary neurons are derived only from the deep cells. In the epidermal ectoderm, superficial cells express high levels of differentiation markers, while most of the deep cells do not differentiate until later when they produce the stratified adult epidermis. However, few molecular differences are known between the deep and superficial cells. Here, we have undertaken a systematic approach to identify genes that show layer-restricted expression by microarray analysis of deep and superficial cells at the gastrula stage, followed by wholemount in situ hybridisation. We have identified 32 differentially expressed genes, of which 26 show higher expression in the superficial layer and 6 in the deep layer and describe their expression at the gastrula and neurula stage. One of the identified genes is the transcription factor Grhl3, which we found to be expressed in the superficial layer of the gastrula ectoderm and the neurula epidermis. By using markers identified in this work, we show that Grlh3 promotes superficial gene expression in the deep layer of the epidermis. Concomitantly, deep layer specific genes are switched off, showing that Grlh3 can promote deep cells to take on a superficial cell identity in the embryonic epidermis.

Animals↗

PubMed2006

The Xfeb gene is directly upregulated by Zic1 during early neural development.

The transcription factor Zic1 plays important roles in patterning the neural plate in early vertebrate development. However, few genes that are regulated by Zic1 are known. We have identified a new direct downstream target gene of Zic1 that we have named Xfeb. Xfeb is a member of the pathogenesis-related (PR) protein superfamily and contains five tandem SCP domains. The sequence of Xfeb suggests that it may possess serine protease activity. Xfeb is expressed in the presumptive hindbrain region during neurula stages and in somite tissues later in development. Xfeb represses the hindbrain gene hoxB1 and the anterior neural gene otx2, suggesting that Xfeb is involved in regionalizing the neural plate, possibly by ensuring a posterior expression limit for otx2.

Amino Acid Sequence↗

PubMed2006

Schnurri transcription factors from Drosophila and vertebrates can mediate Bmp signaling through a phylogenetically conserved mechanism.

Bone Morphogenetic Proteins (Bmps) are secreted growth factors that play crucial roles in animal development across the phylogenetic spectrum. Bmp signaling results in the phosphorylation and nuclear translocation of Smads, downstream signal transducers that bind DNA. In Drosophila, the zinc finger protein Schnurri (Shn) plays a key role in signaling by the Bmp2/Bmp4 homolog Decapentaplegic (Dpp), by forming a Shn/Smad repression complex on defined promoter elements in the brinker (brk) gene. Brk is a transcriptional repressor that downregulates Dpp target genes. Thus, brk inhibition by Shn results in the upregulation of Dpp-responsive genes. We present evidence that vertebrate Shn homologs can also mediate Bmp responsiveness through a mechanism similar to Drosophila Shn. We find that a Bmp response element (BRE) from the Xenopus Vent2 promoter drives Dpp-dependent expression in Drosophila. However, in sharp contrast to its activating role in vertebrates, the frog BRE mediates repression in Drosophila. Remarkably, despite these opposite transcriptional polarities, sequence changes that abolish cis-element activity in Drosophila also affect BRE function in Xenopus. These similar cis requirements reflect conservation of trans-acting factors, as human Shn1 (hShn1; HIVEP1) can interact with Smad1/Smad4 and assemble an hShn1/Smad complex on the BRE. Furthermore, both Shn and hShn1 activate the BRE in Xenopus embryos, and both repress brk and rescue embryonic patterning defects in shn mutants. Our results suggest that vertebrate Shn proteins function in Bmp signal transduction, and that Shn proteins recruit coactivators and co-repressors in a context-dependent manner, rather than acting as dedicated activators or repressors.

Animals↗

PubMed2006

Two modes of action by which Xenopus hairy2b establishes tissue demarcation in the Spemann-Mangold organizer.

The Hairy and Enhancer-of-Split (HES) family of transcriptional repressors plays important roles in pattern formation during development throughout the animal kingdom. Generally, HES proteins repress the expression of genes specific for neighboring tissues to maintain the nature of cells expressing HES proteins, resulting in pattern formation. Xhairy2b, a Xenopus HES, establishes the prospective anterior prechordal mesoderm identity in the Spemann-Mangold organizer by both inducing specific genes and repressing the genes specific for neighboring tissues. Here we report that Xhairy2b has two modes of action, each of which corresponds to inductive and repressive functions. We show that the inductive function is independent of direct transcriptional regulation and is exhibited by the C-terminal WRPW tetrapeptide motif alone, although it induces the expression of a wide variety of the organizer genes that Xhairy2b represses. The transcriptional repression by Xhairy2b is responsible for only the repressive function. We propose that the activity of the WRPW motif intrinsically induces the expression of genes specific for the organizer in a rather non-specific manner to ensure the organizer environment. Then, the transcriptional repression selectively down-regulates the expression of some of these genes, resulting in the regionalization of the axial mesoderm. Our study provides new insight into how a region of the vertebrate embryo is demarcated by one dual-functional transcription factor in the early stages of development.

Animals↗

PubMed2005

Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes.

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.

Animals↗

PubMed2005

Xenopus as a model system to study transcriptional regulatory networks.

Development is controlled by a complex series of events requiring sequential gene activation. Understanding the logic of gene networks during development is necessary for a complete understanding of how genes contribute to phenotype. Pioneering work initiated in the sea urchin and Drosophila has demonstrated that reasonable transcriptional regulatory network diagrams representing early development in multicellular animals can be generated through use of appropriate genomic, genetic, and biochemical tools. Establishment of similar regulatory network diagrams for vertebrate development is a necessary step. The amphibian Xenopus has long been used as a model for vertebrate early development and has contributed greatly to the elucidation of gene regulation. Because the best and most extensively studied transcriptional regulatory network in Xenopus is that underlying the formation and function of Spemann's organizer, we describe the current status of our understanding of this gene regulatory network and its relationship to mesodermal patterning. Seventy-four transcription factors currently known to be expressed in the mesoendoderm of Xenopus gastrula were characterized according to their modes of action, DNA binding consensus sequences, and target genes. Among them, nineteen transcription factors were characterized sufficiently in detail, allowing us to generate a gene regulatory network diagram. Additionally, we discuss recent amphibian work using a combined DNA microarray and bioinformatics approach that promises to accelerate regulatory network studies.

Animals↗

PubMed2005

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.

Animals↗

PubMed2005

A Xenopus DNA microarray approach to identify novel direct BMP target genes involved in early embryonic development.

Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor-beta (TGF-beta) superfamily, were originally isolated from bone on the basis of their ability to induce ectopic bone development. Although BMPs are involved in a wide range of developmental and physiological functions, very few vertebrate target genes in this pathway have been identified. To identify target genes regulated by the BMP growth factor family in Xenopus, large-scale microarray analyses were conducted to discover genes directly activated by this factor in dissociated animal cap tissues treated with a combination of the protein synthesis inhibitor cycloheximide and BMP2. Consequent expression patterns and behaviors of the most highly induced genes were analyzed by in situ and reverse transcriptase-polymerase chain reaction analyses. Here, we describe two sets of the most highly induced direct BMP target genes identified using microarrays prepared from two different stages of early Xenopus development. A wide variety of genes are induced by BMP2, ranging from cell cycle controllers, enzymes, signal transduction cascade components, and components of the blood and vascular system. The finding reinforces the notion that BMP signals play important roles in a variety of biological processes.

Animals↗

PubMed2005

Global analysis of RAR-responsive genes in the Xenopus neurula using cDNA microarrays.

Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we used microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 up-regulated genes (92% of which were validated), including known RA-responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). Sixty-seven validated down-regulated genes were identified, including known RA-responsive genes and anterior marker genes. The expression patterns of selected up-regulated genes (n = 45) were examined at neurula stages using whole-mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros, and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways, and their further characterization will expand our understanding of RA signaling during development.

Animals↗

PubMed2005

Xenopus hairy2b specifies anterior prechordal mesoderm identity within Spemann's organizer.

Spemann's organizer is a region of the gastrula stage embryo that contains future anterior endodermal and dorsal mesodermal tissues. During gastrulation, the dorsal mesoderm is divided into the prechordal mesoderm and the chordamesoderm. However, little is known regarding how this division is established. We analyzed the role of the anterior prechordal mesoderm-specific gene Xhairy2b in the regionalization of the organizer. We found that mesoderm-inducing transforming growth factor-beta signaling induced Xhairy2b expression. On the other hand, the ectopic expression of Xhairy2b induced the expression of organizer-specific genes and resulted in the formation of a secondary dorsal axis lacking head and notochord structures. We also showed that Xhairy2b down-regulated the expression of ventral mesodermal, anterior endodermal, and chordamesodermal genes. In Xhairy2b-depleted embryos, defects in the specification of anterior prechordal mesoderm identity were observed as the border between the prechordal mesoderm and the chordamesoderm was anteriorly shifted. These results suggest that Xhairy2b establishes the identity of the anterior prechordal mesoderm within Spemann's organizer by inhibiting the formation of neighboring tissues.

Animals↗

PubMed2005

Microarray-based identification of VegT targets in Xenopus.

The Xenopus T box family member VegT is expressed maternally in the vegetal hemisphere of the embryo. Mis-expression of VegT in prospective ectodermal tissue causes ectopic activation of mesodermal and endodermal markers, and ablation of VegT transcripts prevents proper formation of the mesendoderm, with the entire embryo developing as epidermis. These observations define VegT as a key initiator of mesendodermal development in the Xenopus embryo, and in an effort to understand how it exerts its effects we have used microarray analysis to compare gene expression in control animal caps with that in ectodermal tissue expressing an activated form of VegT. This procedure allowed the identification of 99 potential VegT targets, and we went on to study the expression patterns of these genes and then to ask, for those that are expressed in mesoderm or endoderm, which are direct targets of VegT. The putative regulatory regions of the resulting 14 genes were examined for T domain binding sites, and we also asked whether their expression is down-regulated in embryos in which VegT RNA is ablated. Finally, the functions of these genes were assayed by both over-expression and by use of antisense morpholino oligonucleotides. Our results provide new insights into the function of VegT during early Xenopus development.

Animals↗

PubMed2005

Regulatory targets for transcription factor AP2 in Xenopus embryos.

The transcription factor AP2 (TFAP2) has an important role in regulating gene expression in both epidermis and neural crest cells. In order to further characterize these functions we have used a hormone inducible TFAP2alpha fusion protein in a Xenopus animal cap assay to identify downstream targets of this factor. The most common pattern comprised genes predominantly expressed in the epidermis. A second group was expressed at high levels in the neural crest, but all of these were also expressed in the epidermis as well as in other tissues in which TFAP2alpha has not been detected, suggesting modular control involving both TFAP2-dependent and TFAP2-independent components. In addition, a few strongly induced genes did not overlap at all in expression pattern with that of TFAP2alpha in the early embryo, and were also activated precociously in the experimentally manipulated ectoderm, and thus likely represent inappropriate regulatory interactions. A final group was identified that were repressed by TFAP2alpha and were expressed in the neural plate. These results provide further support for the importance of TFAP2alpha in ectoderm development, and also highlight the molecular linkage between the epidermis and neural crest in the Xenopus embryo.

Animals↗

PubMed2004

Cloning and expression of an SH3 domain-containing protein (Xchef-1), a novel downstream target of activin/nodal signaling.

The activity of the activin/nodal signaling cascade is essential for the proper specification of germ layers during gastrulation. Many of the components of this signaling pathway have been identified, but relatively few downstream targets have been discovered. Using cDNA microarrays, we have identified a novel SH3-domain-containing gene we have named Xchef-1 that is upregulated in response to activin/nodal signaling. Xchef-1 is a direct downstream target of activin and is expressed in the marginal zones of gastrulating Xenopus embryos in a dynamic pattern reminiscent of nodal expression. At neurula stages, Xchef-1 is expressed in neural crest of the head and trunk as well as in the anterior neural plate. These domains of expression are then restricted at tailbud stages to the branchial arches, and the region of the future gall bladder.

Activin Receptors↗

PubMed2003

Expression pattern of a basic helix-loop-helix transcription factor Xhairy2b during Xenopus laevis development.

We report on the temporal and spatial expression pattern of the Xenopus laevis hairy2b ( Xhairy2b) transcription factor. Xhairy2b transcripts are present maternally, and expressed throughout the prospective ectoderm prior to the gastrula stage. During gastrulation, Xhairy2b expression is restricted to the deep layer of the Spemann organizer and three distinct regions in the prospective neuroectoderm, neural plate border, notoplate and anterior neural plate. At later stages, Xhairy2b expression is localized to prechordal plate, presomitic mesoderm, neural tube, neural crest derivatives and several tissue territories of the central nervous system. The analyses of Xhairy2b and several other hairy-related genes suggest potential roles for Xhairy2b in the formation of boundaries in neural tissue territories.

Animals↗