Yumi Izutsu

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.

Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos

In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie‐2, a Xenopus homolog of the tie‐2 receptor tyrosine kinase, was examined. Whole‐mount in situ hybridization analysis revealed that Xtie‐2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie‐2‐positive cells are predominantly present just outside the boundary of α‐globin‐positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie‐2 and α‐globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a ‘dorsal determinant’ from the vegetal cytoplasm at the 1‐cell stage. Second, expression of both Xtie‐2 and α‐globin were markedly induced in ectodermal explants (animal caps) from embryos co‐injected with activin and bone morphogenetic protein (BMP)‐4 RNA. Furthermore, both Xtie‐2 and α‐globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP‐4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP‐4‐dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.

The keratin-related Ouroboros proteins function as immune antigens mediating tail regression in Xenopus metamorphosis

Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.

Characterization of myeloid cells derived from the anterior ventral mesoderm in the Xenopus laevis embryo

A recent study revealed the presence of a unique population of myeloid cells in the anterior ventral (AV) mesoderm of Xenopus laevis embryo, as characterized by the expression of peroxidase 2 (POX2), which encodes for a leukocyte‐specific enzyme. The current report further characterized the POX2‐positive cells in terms of their contribution to hematopoiesis in tadpole and regulatory mechanism in differentiation. Grafting experiments with cytogenetically labeled tissues revealed that AV‐derived mesoderm supplies a transient population of migrating leukocytes in the mesenchyme of early tadpole. These cells were rarely found in blood vessels at any stages. Using a ventral marginal zone explant system, we demonstrated that dkk1, shown as a heart inducer in this system, has a strong ability to induce the expression of POX2. Injection of a high dose dkk1 RNA induced a heart marker while a low dose of dkk1 preferentially induced the expression of POX2, suggesting that dkk1 works as a morphogen to determine the different lineages. Overall results indicate that wnt signal inhibitors induce leukocytes at the early neurula stage and that these cells spread to the entire body and exist until the ventral blood island‐derived leukocytes appear in the body.

Distinct mechanisms control the timing of differentiation of two myeloid populations in Xenopus ventral blood islands.

Previous study has suggested that distinct populations of myeloid cells exist in the anterior ventral blood islands (aVBI) and posterior ventral blood islands (pVBI) in Xenopus neurula embryo. However, details for differentiation programs of these two populations have not been elucidated. In the present study, we examined the role of Wnt, vascular endothelial growth factor (VEGF) and fibroblast growth factor signals in the regulation of myeloid cell differentiation in the dorsal marginal zone and ventral marginal zone explants that are the sources of myeloid cells in the aVBI and pVBI. We found that regulation of Wnt activity is essential for the differentiation of myeloid cells in the aVBI but is not required for the differentiation of myeloid cells in the pVBI. Endogenous activity of the VEGF signal is necessary for differentiation of myeloid cells in the pVBI but is not involved in the differentiation of myeloid cells in the aVBI. Overall results reveal that distinct mechanisms are involved in the myeloid, erythroid and endothelial cell differentiation in the aVBI and pVBI.

The immune system is involved in Xenopus metamorphosis.

Amphibian metamorphosis provides a model to elucidate the mechanisms underlying how vertebrates reconstitute a body plan and how the immune system develops during ontogeny. In Xenopus, T cells are expanded from the early developmental stages just after hatching. These T cells switch from larval-type in an easily tolerizable state into an adult-type having a potent immune responsiveness comparable to that of mammals. During metamorphosis, tadpoles exhibit morphological changes in skin that completely transforms from larval-type to adult-type. Only tail tissue behaves differently; it remains a larval-type tissue until it disappears at the end of metamorphosis. Thus, at metamorphic climax, four different types of cells co-exist in a tadpole body: larval tissue cells; adult tissue cells; larval immune cells; and adult immune cells. Based on the results showing that tadpole tail skin is rejected by syngeneic adult, it is proposed that the elimination of the larval tissue cells by the adult T cells that occurs during metamorphosis is immunologically mediated. Recent results indicate that the antigenic proteins expressed in the metamorphosing skin cells participate in the process of tail regression. This chapter describes how animals adjust and survive through such crises associated with large scale replacement of entire body cells.

A role of D domain-related proteins in differentiation and migration of embryonic cells in Xenopus laevis.

We have characterized a cDNA clone, rdd (repeated D domain-like), that encodes for a secretory protein consisting of repeated domains of cysteine-rich sequence. Whole-mount in situ hybridization analysis revealed that rdd2, rdd3 and rdd4 are transiently expressed in the ventral and lateral mesoderm and the overlying ectoderm at the late gastrula and tailbud stages. Morpholino oligonucleotide (MO) was used to inhibit the translation of endogenous rdd3 and rdd4, and we found that the circulation of red blood cells completely disappears in the MO-injected tadpoles. Histological analysis showed that formation of the ventral aorta, dorsal aorta and posterior cardinal vein in the trunk region was severely disorganized in these animals. Injection of MO affected the expression of alpha-globin, a terminal differentiation marker of red blood cells, but did not affect the expression of scl, flk-1 or tie-2, suggesting that angiopoietic and hematopoietic precursor cells differentiate normally in the rdd-depleted embryo. The transplantation of labeled tissues followed by tracing of the donor cells revealed a role of rdds in migration of the embryonic angioblasts and myeloid cells. These observations first demonstrate the role of the novel cysteine-rich proteins in migration of the embryonic cells.

Analyses of immune responses to ontogeny-specific antigens using an inbred strain of Xenopus laevis (J strain).

In this chapter, the procedures for specific detection of ontogenic emerging antigens during animal development are described. Anuran metamorphosis has provided us with a good experimental model for investigation of the mechanisms of tissue remodeling. The establishment of a syngeneic strain of Xenopus laevis described in this chapter has enabled us to perform a unique experiment to develop antibodies that specifically react to ontogenic antigens by immunizing syngeneic animals. This strategy was successful because the antibody repertoires produced in the adult frog serum were well subtracted by a number of common antigens expressed in syngeneic larvae. Here we show, using results of immunohistochemical and T-cell proliferation analyses that adult frogs exhibit humoral and cell-mediated immune responses to larva- or metamorphosis-specific antigen molecules in epidermal cells.

Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as ‘non‐self’. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose‐dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult‐type immune system.

Involvement of Neptune in induction of the hatching gland and neural crest in the Xenopus embryo.

Neptune, a Krüppel-like transcription factor, is expressed in various regions of the developing Xenopus embryo and it has multiple functions in the process of development in various organs. In situ hybridization analysis showed that Neptune is expressed in the boundary region between neural and non-neural tissues at the neurula stage, but little is known about the function of Neptune in this region. Here, we examined the expression and function of Neptune in the neural plate border (NPB) in the Xenopus embryo. Depletion of Neptune protein in developing embryos by using antisense MO caused loss of the hatching gland and otic vesicle as well as malformation of neural crest-derived cranial cartilages and melanocytes. Neptune MO also suppressed the expression of hatching gland and neural crest markers such as he, snail2, sox9 and msx1 at the neurula stage. Subsequent experiments showed that Neptune is necessary and sufficient for the differentiation of hatching gland cells and that it is located downstream of pax3 in the signal regulating the differentiation of these cells. Thus, Neptune is a new member of hatching gland specifier and plays a physiological role in determination and specification of multiple lineages derived from the NPB region.