Hajime Ogino

The Genome of the Western Clawed Frog Xenopus tropicalis

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633, see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes. Assembly, annotation, and analysis of the frog genome compares gene content and synteny with the human and chicken genomes. The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Regulation of Lens Fiber Cell Differentiation by Transcription Factor c-Maf

To elucidate the regulatory mechanisms underlying lens development, we searched for members of the large Maf family, which are expressed in the mouse lens, and found three, c-Maf, MafB, and Nrl. Of these, the earliest factor expressed in the lens was c-Maf. The expression of c-Maf was most prominent in lens fiber cells and persisted throughout lens development. To examine the functional contribution of c-Maf to lens development, we isolated genomic clones encompassing the murine c-maf gene and carried out its targeted disruption. Insertion of the β-galactosidase (lacZ) gene into the c-maf locus allowed visualization of c-Maf accumulation in heterozygous mutant mice by staining for LacZ activity. Homozygous mutant embryos and newborns lacked normal lenses. Histological examination of these mice revealed defective differentiation of lens fiber cells. The expression of crystallin genes was severely impaired in the c-maf-null mutant mouse lens. These results demonstrate that c-Maf is an indispensable regulator of lens differentiation during murine development.

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.

L-Maf, a downstream target of Pax6, is essential for chick lens development

During lens development in vertebrates, the orchestration of multiple transcriptional regulators is essential for fate determination and terminal differentiation. In early development, Pax6, Sox2 and Six3 are expressed in the head ectoderm, while L-maf, Prox1 and crystallin genes are expressed at a later stage in the lens placode in a more restricted fashion. To uncover the genetic interactions among these factors during lens development, we examined the effects of dominant-negative molecules of Pax6 and L-Maf, which play decisive roles in lens formation. The two dominant-negative isoforms of Pax6 repress L-maf, Prox1 and delta-crystallin expression, resulting in failure of lens formation. These effects of dominant-negative Pax6 are fully rescued by co-expression with wild-type L-Maf. In addition, dominant-negative L-Maf inhibits the expression of Prox1 and delta-crystallin, while misexpression of L-Maf causes ectopic induction of these genes in a Sox-2-dependent fashion. Our results demonstrate that L-Maf is a downstream target of Pax6 and mediates Pax6 activity in developing lens cells.

Conserved expression of mouse Six1 in the pre-placodal region (PPR) and identification of an enhancer for the rostral PPR

All cranial sensory organs and sensory neurons of vertebrates develop from cranial placodes. In chick, amphibians and zebrafish, all placodes originate from a common precursor domain, the pre-placodal region (PPR), marked by the expression of Six1/4 and Eya1/2. However, the PPR has never been described in mammals and the mechanism involved in the formation of PPR is poorly defined. Here, we report the expression of Six1 in the horseshoe-shaped mouse ectoderm surrounding the anterior neural plate in a pattern broadly similar to that of non-mammalian vertebrates. To elucidate the identity of Six1-positive mouse ectoderm, we searched for enhancers responsible for Six1 expression by in vivo enhancer assays. One conserved non-coding sequence, Six1-14, showed specific enhancer activity in the rostral PPR of chick and Xenopus and in the mouse ectoderm. These results strongly suggest the presence of PPR in mouse and that it is conserved in vertebrates. Moreover, we show the importance of the homeodomain protein-binding sites of Six1-14, the Six1 rostral PPR enhancer, for enhancer activity, and that Dlx5, Msx1 and Pax7 are candidate binding factors that regulate the level and area of Six1 expression, and thereby the location of the PPR. Our findings provide critical information and tools to elucidate the molecular mechanism of early sensory development and have implications for the development of sensory precursor/stem cells.

Developmental regulation of the chicken βB1-crystallin promoter in transgenic mice

The cis-elements responsible for the high-level, lens-specific expression of the chicken beta B1-crystallin gene were investigated by generating mice harboring beta B1-crystallin promoter/chloramphenicol acetyl transferase (CAT) transgenes. Deletion of promoter sequences -434/-153 and -152/-127 as well as site-directed mutagenesis of the PL1 (-116/-102) and Pl2 (-90/-76) elements significantly decreased CAT gene expression in the lenses of adult transgenic mice. Transfection studies using multimerized PL1 and PL2 elements fused to the chicken beta-actin basal promoter indicated that PL1 is a general activating element while PL2 is involved in the lens-specificity of the chicken beta B1-crystallin promoter. CAT histochemistry demonstrated that the chicken beta B1-crystallin promoter (-434/+30) was active in both primary and secondary lens fiber cells from 12.5 days post coitum (dpc) until adulthood. Activity of the -152/+30/CAT transgene was relatively low and confined to the primary lens fiber cells of 16.5 dpc mice. Together, these data suggest that the reduced activity of this promoter in the adult lens is due both to this developmentally restricted expression pattern and a reduction in promoter activity. RNA hybridization studies demonstrated that the chicken beta B1-crystallin/CAT (-434/+30) transgene was expressed at similar levels in the same cells as the endogenous mouse beta B1-crystallin gene in 16.5 dpc transgenic mouse embryos. These data show a strict conservation of the lens-specific spatial and temporal regulation of the chicken and mouse beta B1-crystallin genes.

Transcription factors involved in lens development from the preplacodal ectoderm

Lens development is a stepwise process accompanied by the sequential activation of transcription factors. Transcription factor genes can be classified into three groups according to their functions: the first group comprises preplacodal genes, which are implicated in the formation of the preplacodal ectoderm that serves as a common primordium for cranial sensory tissues, including the lens. The second group comprises lens-specification genes, which establish the lens-field within the preplacodal ectoderm. The third group comprises lens-differentiation genes, which promote lens morphogenesis after the optic vesicle makes contact with the presumptive lens ectoderm. Analyses of the regulatory interactions between these genes have provided an overview of lens development, highlighting crucial roles for positive cross-regulation in fate specification and for feed-forward regulation in the execution of terminal differentiation. This overview also sheds light upon the mechanisms of how preplacodal gene activities lead to the activation of genes involved in lens-specification.

Dynamic in vivo binding of transcription factors to cis-regulatory modules of cer and gsc in the stepwise formation of the Spemann–Mangold organizer

How multiple developmental cues are integrated on cis-regulatory modules (CRMs) for cell fate decisions remains uncertain. The Spemann–Mangold organizer in Xenopus embryos expresses the transcription factors Lim1/Lhx1, Otx2, Mix1, Siamois (Sia) and VegT. Reporter analyses using sperm nuclear transplantation and DNA injection showed that cerberus (cer) and goosecoid (gsc) are activated by the aforementioned transcription factors through CRMs conserved between X. laevis and X. tropicalis. ChIP-qPCR analysis for the five transcription factors revealed that cer and gsc CRMs are initially bound by both Sia and VegT at the late blastula stage, and subsequently bound by all five factors at the gastrula stage. At the neurula stage, only binding of Lim1 and Otx2 to the gsc CRM, among others, persists, which corresponds to their co-expression in the prechordal plate. Based on these data, together with detailed expression pattern analysis, we propose a new model of stepwise formation of the organizer, in which (1) maternal VegT and Wnt-induced Sia first bind to CRMs at the blastula stage; then (2) Nodal-inducible Lim1, Otx2, Mix1 and zygotic VegT are bound to CRMs in the dorsal endodermal and mesodermal regions where all these genes are co-expressed; and (3) these two regions are combined at the gastrula stage to form the organizer. Thus, the in vivo dynamics of multiple transcription factors highlight their roles in the initiation and maintenance of gene expression, and also reveal the stepwise integration of maternal, Nodal and Wnt signaling on CRMs of organizer genes to generate the organizer.

Different Requirement for Wnt/β-Catenin Signaling in Limb Regeneration of Larval and Adult Xenopus

Background In limb regeneration of amphibians, the early steps leading to blastema formation are critical for the success of regeneration, and the initiation of regeneration in an adult limb requires the presence of nerves. Xenopus laevis tadpoles can completely regenerate an amputated limb at the early limb bud stage, and the metamorphosed young adult also regenerates a limb by a nerve-dependent process that results in a spike-like structure. Blockage of Wnt/β-catenin signaling inhibits the initiation of tadpole limb regeneration, but it remains unclear whether limb regeneration in young adults also requires Wnt/β-catenin signaling. Methodology/Principal Findings We expressed heat-shock-inducible (hs) Dkk1, a Wnt antagonist, in transgenic Xenopus to block Wnt/β-catenin signaling during forelimb regeneration in young adults. hsDkk1 did not inhibit limb regeneration in any of the young adult frogs, though it suppressed Wnt-dependent expression of genes (fgf-8 and cyclin D1). When nerve supply to the limbs was partially removed, however, hsDkk1 expression blocked limb regeneration in young adult frogs. Conversely, activation of Wnt/β-catenin signaling by a GSK-3 inhibitor rescued failure of limb-spike regeneration in young adult frogs after total removal of nerve supply. Conclusions/Significance In contrast to its essential role in tadpole limb regeneration, our results suggest that Wnt/β-catenin signaling is not absolutely essential for limb regeneration in young adults. The different requirement for Wnt/β-catenin signaling in tadpoles and young adults appears to be due to the projection of nerve axons into the limb field. Our observations suggest that nerve-derived signals and Wnt/β-catenin signaling have redundant roles in the initiation of limb regeneration. Our results demonstrate for the first time the different mechanisms of limb regeneration initiation in limb buds (tadpoles) and developed limbs (young adults) with reference to nerve-derived signals and Wnt/β-catenin signaling.

Transcriptional regulators in the Hippo signaling pathway control organ growth in Xenopus tadpole tail regeneration

The size and shape of tissues are tightly controlled by synchronized processes among cells and tissues to produce an integrated organ. The Hippo signaling pathway controls both cell proliferation and apoptosis by dual signal-transduction states regulated through a repressive kinase cascade. Yap1 and Tead, transcriptional regulators that act downstream of the Hippo signaling kinase cascade, have essential roles in regulating cell proliferation. In amphibian limb or tail regeneration, the local tissue outgrowth terminates when the correct size is reached, suggesting that organ size is strictly controlled during epimorphic organ-level regeneration. We recently demonstrated that Yap1 is required for the regeneration of Xenopus tadpole limb buds (Hayashi et al., 2014, Dev. Biol. 388, 57-67), but the molecular link between the Hippo pathway and organ size control in vertebrate epimorphic regeneration is not fully understood. To examine the requirement of Hippo pathway transcriptional regulators in epimorphic regeneration, including organ size control, we inhibited these regulators during Xenopus tadpole tail regeneration by overexpressing a dominant-negative form of Yap (dnYap) or Tead4 (dnTead4) under a heat-shock promoter in transgenic animal lines. Each inhibition resulted in regeneration defects accompanied by reduced cell mitosis and increased apoptosis. Single-cell gene manipulation experiments indicated that Tead4 cell-autonomously regulates the survival of neural progenitor cells in the regenerating tail. In amphibians, amputation at the proximal level of the tail (deep amputation) results in faster regeneration than that at the distal level (shallow amputation), to restore the original-sized tail with similar timing. However, dnTead4 overexpression abolished the position-dependent differential growth rate of tail regeneration. These results suggest that the transcriptional regulators in the Hippo pathway, Tead4 and Yap1, are required for general vertebrate epimorphic regeneration as well as for organ size control in appendage regeneration. In regenerative medicine, these findings should contribute to the development of three-dimensional organs with the correct size for a patients body.