Yoshikazu Haramoto

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

The Conserved Rieske Oxygenase DAF-36/Neverland Is a Novel Cholesterol-metabolizing Enzyme

Steroid hormones play essential roles in a wide variety of biological processes in multicellular organisms. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which are synthesized from cholesterol as an indispensable precursor. The first critical catalytic step in the biosynthesis of these ecdysozoan steroids is the conversion of cholesterol to 7-dehydrocholesterol. However, the enzymes responsible for cholesterol 7,8-dehydrogenation remain unclear at the molecular level. Here we report that the Rieske oxygenase DAF-36/Neverland (Nvd) is a cholesterol 7,8-dehydrogenase. The daf-36/nvd genes are evolutionarily conserved, not only in nematodes and insects but also in deuterostome species that do not produce dafachronic acids or ecdysteroids, including the sea urchin Hemicentrotus pulcherrimus, the sea squirt Ciona intestinalis, the fish Danio rerio, and the frog Xenopus laevis. An in vitro enzymatic assay system reveals that all DAF-36/Nvd proteins cloned so far have the ability to convert cholesterol to 7-dehydrocholesterol. Moreover, the lethality of loss of nvd function in the fruit fly Drosophila melanogaster is rescued by the expression of daf-36/nvd genes from the nematode Caenorhabditis elegans, the insect Bombyx mori, or the vertebrates D. rerio and X. laevis. These data suggest that daf-36/nvd genes are functionally orthologous across the bilaterian phylogeny. We propose that the daf-36/nvd family of proteins is a novel conserved player in cholesterol metabolism across the animal phyla.

FRL-1, a member of the EGF-CFC family, is essential for neural differentiation in Xenopus early development.

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.

Occupancy of Tissue-Specific cis-Regulatory Modules by Otx2 and TLE/Groucho for Embryonic Head Specification

Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

Zygotic VegT is required for Xenopus paraxial mesoderm formation and is regulated by Nodal signaling and Eomesodermin.

The T-box gene VegT plays a crucial role during mesendoderm specification of the amphibian embryo. While the function of maternal VegT (mVegT) has been extensively investigated, little is known about the function and transcriptional regulation of zygotic VegT (zVegT). In the present study, we used comparative genomics and a knockdown experiment to demonstrate that zVegT is the orthologous gene of zebrafish Spadetail/Tbx16 and chick Tbx6L/Tbx6, and has an essential role in paraxial mesodermal formation. zVegT knockdown embryos show several defects in the patterning of trunk mesoderm, such as abnormal segmentation of somites, a reduction in muscle, and the formation of an abnormal mass of cells at the tail tip. We also identified the cis-regulatory elements of zVegT that are necessary and sufficient for mesoderm-specific expression. These cis-regulatory elements are located in two separate upstream regions of zVegT, corresponding to the first intron of mVegT. The results of in vitro binding and functional assays indicate that Forkhead box H1 (FoxH1) and Eomesodermin (Eomes) are the trans-acting factors required for zVegT expression. Our results highlight the essential role of zVegT in organization of paraxial mesoderm, and reveal that zVegT is regulated by a coherent feedforward loop of Nodal signaling via Eomes.

Xnr2 and Xnr5 Unprocessed Proteins Inhibit Wnt Signaling Upstream of Dishevelled

Nodal and Nodal‐related proteins activate the Activin‐like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal‐related proteins apart from the canonical Activin‐like signal pathway. Here we report a novel function of Nodal‐related proteins using cleavage mutants of Xenopus nodal‐related genes (cmXnr2 and cmXnr5), which are known to be dominant‐negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin‐like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin‐Xnr2 and cmActivin‐Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced. Developmental Dynamics 234:900–910, 2005.

Hippo signaling components, Mst1 and Mst2, act as a switch between self-renewal and differentiation in Xenopus hematopoietic and endothelial progenitors.

Hippo signaling is a conserved pathway that regulates cell proliferation and organ size control. Mst1 and Mst2 were identified as homologs of hippo and as core kinases of the Hippo pathway in mammals. Here, we have characterized the role of Mst1 and Mst2 during Xenopus primitive hematopoiesis. We showed that Mst1 and Mst2 were strongly expressed in the Xenopus ventral blood island, where primitive hematopoiesis is initiated. Loss-of-function analysis of Mst1/2 revealed morphogenetic defects, including short axis, smaller eyes and abnormal epidermis, and decreased phosphorylation of Yap. Mst1/2 morphants did not exhibit any change in the expression of hematopoietic and endothelial progenitor markers in early hematopoietic development. In addition, we have shown that such progenitor markers were continuously expressed through to the late hematopoietic development stage. As a result, the expression of erythroid, myeloid and endothelial differentiation markers were decreased in Mst1/2 morphants. Our results indicate that Mst1/2 act as a differentiation switch in Xenopus hematopoietc and endothelial progenitors.

The transcriptional coactivators Yap and TAZ are expressed during early Xenopus development

The Yap and TAZ genes encode highly conserved domains which bind various transcription factors. Yap and TAZ act as transcriptional coactivators to modulate transcriptional activity. The activities of Yap and TAZ are negatively regulated by Hippo signaling via direct phosphorylation. In this study, we describe the expression patterns of Yap and TAZ during the development of Xenopus tropicalis. The Xenopus tropicalis Yap (xtYap) and Xenopus tropicalis TAZ (xtTAZ) genes are expressed maternally. xtYap is widely expressed throughout embryogenesis, particularly in the facial connective tissues, branchial arch, midbrain-hindbrain boundary, otic vesicle, pronephros, notochord, hindgut and tailbud. xtTAZ expression occurs predominantly in the presomitic mesoderm, facial connective tissues, brain, branchial arch, trunk neural crest cells and migrating hypaxial myoblasts. In the muscle lineage, xtTAZ expression is transient and restricted to proliferating cells, the presomitic mesoderm and the edges of the hypaxial myoblasts, with no expression detected in mature muscle cells. These results provide insights into the functions of Yap and TAZ and their regulation by Hippo signaling during early development in Xenopus.

Tracing of Xenopus tropicalis germ plasm and presumptive primordial germ cells with the Xenopus tropicalis DAZ‐like gene

A gamete is derived initially from a presumptive primordial germ cell (pPGC) and transmits genetic potential to the next generation. Xenopus tropicalis, which is a close relative of Xenopus laevis, has a diploid genome and advantages for genetic and genomic research; however, little is known about the developmental mechanism of its germinal lineage. Here, we identified the Xenopus tropicalis DAZ‐like gene (Xtdazl), which encodes RNA‐binding proteins homologous to Xdazl in Xenopus laevis and examined the expression patterns of Xtdazl transcripts during embryogenesis. In this work, we showed that Xtdazl mRNA was localized in the germ plasm and was expressed from the previtellogenic oocyte to early tadpole, in testis and ovary. The same localization patterns have been reported in Xenopus laevis germ plasm and pPGCs. These results indicate that Xtdazl mRNA is the first specific marker of germ plasm and pPGCs in Xenopus tropicalis and is very useful to trace Xenopus tropicalis pPGCs, including germ plasm until the early tadpole stage. Developmental Dynamics 229:367–372, 2004.

XRASGRP2 is essential for blood vessel formation during Xenopus development.

Ras guanyl nucleotide-releasing protein 2 (RASGRP2), one of the Ras guanine exchange factors, is implicated as a critical regulator of inside-out integrin activation in human lymphocytes, neutrophils and platelets. However, the activities of this protein in endothelial cells remain unclear. In the current study, we identify a physiological function in blood vessel formation for XRASGRP2, which is the Xenopus ortholog of mammalian RASGRP2. XRASGRP2 over-expression induced ectopic vascular formation, and XRASGRP2-knockdown embryos showed delayed vascular development. We also investigated the upstream signaling of XRASGRP2 in endothelium formation. XRASGRP2 expression was up-regulated in the presence of VEGF-A and down-regulated following VEGF-A depletion. XRASGRP2 knockdown abolished the ectopic induction of endothelial cells by VEGF-A in the posterior ventral blood island. These results suggest that XRASGRP2 is essential for vascular formation during Xenopus development.