Vertebrate Morphogenesis Research Articles

Cloning and expression of <I>Tbx3</I> gene in Siberian sturgeon, <I>Acipenser baerii</I>

Tbx3, a member of the TBX2 subfamily of T-box gene family, encodes a transcription factor with a highly conserved DNA-binding domain, which called T-domain. Tbx3 is involved in morphogenesis and organogenesis in vertebrates, such as limb development, heart remodeling, and neural placode differentiation. In the present study, a full-length 2 908 bp Tbx3 cDNA from Acipenser baerii (AbTbx3) was obtained using RT-PCR and RACE technique, which includes a 2 166 bp complete open reading frame encoding a putative peptide of 721 amino acids. AbTbx3 shares 73.5% identity with its human homolog. Particularly, the DNA-binding domain of AbTbx3 shared 95.2% identity with human Tbx3. Phylogenetic analysis revealed that AbTbx3 was grouped with Tbx3s in other vertebrates, which were clustered with Tbx2s and separated from Tbx4/5s. The predicted secondary and three-dimensional structures of the T-domain of AbTbx3 were remarkably similar to human Tbx3. Through semi-quantity RT-PCR, the expression of AbTbx3 was first detected at blastula stage during Siberian sturgeon embryonic development, increased gradually, reached its peak at early tail-bud stage and then decreased slightly. In adult sturgeon, AbTbx3 was strongly expressed in eye, brain, gill, intestines, pectoral fin and pelvic fin, but not in liver, blood, heart, kidney and muscle. The whole mount in situ hybridization showed that AbTbx3 was mainly expressed in the otic vesicle, hindbrain, dorsal notochord, pineal organ and dorsal fin bud in the larvae of stage 37 and 43.

Studies on thePinctada fucataBMP-2 Gene: Structural Similarity and Functional Conservation of Its Osteogenic Potential within the Animal Kingdom

Bone morphogenetic protein (BMP)-2 plays an important role in morphogenesis in both vertebrates and invertebrates. BMP-2 is one of the most powerful bioactive substances known to induce the osteogenic differentiation of mesenchymal cells. We examined the structural and functional conservation ofPinctada fucataBMP-2 in inducing osteogenesis in the murine mesenchymal stem cells, C3H10T1/2. Exposure of C3H10T1/2 cells to the recombinant mature fragment ofPinctada fucataBMP-2 resulted in osteoblastic differentiation. The sequence, SVPKPCCVPTELSSL, within the C-terminal portion ofPinctada fucataBMP-2, is homologous to the knuckle epitope of human BMP-2. This synthetic polypeptide was able to induce differentiation of C3H10T1/2 along the osteoblastic lineage, as confirmed by an increase in alkaline phosphatase activity, and the accumulation of calcium, as determined by von Kossa staining. Furthermore, using immunohistochemical staining, we observed an increased expression of collagen type I, osteopontin, and osteocalcin, which are known markers of osteogenesis. These results show that BMP-2 is conserved, not only in terms of its homology at the amino acid sequence, but also in terms of driving the formation of hard tissues in vertebrates and invertebrates.

Evaluation of copy number variations reveals novel candidate genes in autism spectrum disorder-associated pathways

Autism spectrum disorders (ASDs) are highly heritable, yet relatively few associated genetic loci have been replicated. Copy number variations (CNVs) have been implicated in autism; however, the majority of loci contribute to <1% of the disease population. Therefore, independent studies are important to refine associated CNV regions and discover novel susceptibility genes. In this study, a genome-wide SNP array was utilized for CNV detection by two distinct algorithms in a European ancestry case-control data set. We identify a significantly higher burden in the number and size of deletions, and disrupting more genes in ASD cases. Moreover, 18 deletions larger than 1 Mb were detected exclusively in cases, implicating novel regions at 2q22.1, 3p26.3, 4q12 and 14q23. Case-specific CNVs provided further evidence for pathways previously implicated in ASDs, revealing new candidate genes within the GABAergic signaling and neural development pathways. These include DBI, an allosteric binder of GABA receptors, GABARAPL1, the GABA receptor-associated protein, and SLC6A11, a postsynaptic GABA transporter. We also identified CNVs in COBL, deletions of which cause defects in neuronal cytoskeleton morphogenesis in model vertebrates, and DNER, a neuron-specific Notch ligand required for cerebellar development. Moreover, we found evidence of genetic overlap between ASDs and other neurodevelopmental and neuropsychiatric diseases. These genes include glutamate receptors (GRID1, GRIK2 and GRIK4), synaptic regulators (NRXN3, SLC6A8 and SYN3), transcription factor (ZNF804A) and RNA-binding protein FMR1. Taken together, these CNVs may be a few of the missing pieces of ASD heritability and lead to discovering novel etiological mechanisms.

Use of Synteny Conversion in Identification of Candidate Genes for Somitogenesis in Humans

Understanding the genetic component of scoliosis in humans has relied on the assumption that spine development is conserved across species. Since evolutionary conserved genes tend to lie within synteny blocks (HSBs) and genes which are not conserved lie within evolutionary breakpoint regions (EBRs), HSB analysis may be used to determine if spine development is conserved across species. We hypothesized that vertebral patterning genes are conserved in amniotes and their location is within stable or “syntenic” regions of chromosomes. Seventy seven patterning genes involved in Fgf, Wnt and Notch signaling pathways were analyzed to determine their location within HSBs or EBRs in the genomes of several amniotic species. The human genome was divided into 1 Mbp intervals and a comparison was made to determine whether these genes were preferentially localized within HSBs or EBRs associated with rapid evolution. The results indicate that genes associated with somite development in humans are preferentially located away from the EBRs: 0.014 genes in EBRs on genome average vs. 0.030 on average in other parts of the genome (p-value = 0.01). The concentration of vertebral patterning genes in HSBs, provides evidence that developmental pathways involved in vertebral morphogenesis are likely conserved across amniotes, consistent with their known function. These data support prior observations indicating that gene networks associated with major developmental processes such as neuronal, central nervous system, bone and blood vessel development, some mediated by Wnt and Notch signaling pathways, were less likely to be localized at EBRs.

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Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., (1, 2, 3, 4)). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick (5) or mouse-chick (6) chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues (7). Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras (8). The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

Hypoxia is like an ethiological factor in vertebral column deformity of salmon ( Salmo salar)

In Chilean salmonicultures, vertebral column deformity (VCD) is a common pathology that lowers the quality and commercialization of the product. Hypoxia has been related to other musculoskeletal deformities in fishes, but not to VCD. The notochord has a structural and functional role in vertebral morphogenesis and in mature fish resulting in mechanical support in swimming motility. This work analyzes the morphology of the vertebral column and notochord of Salmo salar alevins starting free life (950 Accumulated Temperature Units, ATU) cultured under hypoxia (60% O 2 saturation in the water tanks) for different time periods after hatching (2, 4, 6 and 8 days). These are compared with their normoxic control (100% O 2 saturation). Using histological, histochemical and morphometric techniques, it was found that there is an association between VCD and the time of exposure to hypoxia (Pearson X 2 (5) = 60.1050 Pr = 0.0001). The time of exposure to hypoxia is inversely proportional to the body length, mineralization to the vertebral body, notochord diameter and thickness of its sheath. Hypoxia decreases collagen type II in the notochord sheath and up regulation of collagen type I. The organic response to hypoxia was quantified by immunohistochemistry for HIF-1α as the sensor of hypoxia. Its expression increased significantly (p < 0.05) in the experimental groups that exhibit VCD. Although ethiology for VCD is probably multifactorial, this study allows to conclude that hypoxia by itself, produces VCD.

Coordinated gene expression during gilthead sea bream skeletogenesis and its disruption by nutritional hypervitaminosis A

BackgroundVitamin A (VA) has a key role in vertebrate morphogenesis, determining body patterning and growth through the control of cell proliferation and differentiation processes. VA regulates primary molecular pathways of those processes by the binding of its active metabolite (retinoic acid) to two types of specific nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which promote transcription of downstream target genes. This process is well known in most of higher vertebrates; however, scarce information is available regarding fishes. Therefore, in order to gain further knowledge of fish larval development and its disruption by nutritional VA imbalance, the relative expression of some RARs and RXRs, as well as several genes involved in morpho- and skeletogenesis such as peroxisome proliferator-activated receptors (PPARA, PPARB and PPARG); retinol-binding protein (RBP); insulin-like growth factors I and II (IGF1 and IGF2, respectively); bone morphogenetic protein 2 (Bmp2); transforming growth factor β-1 (TGFB1); and genes encoding different extracellular matrix (ECM) proteins such as matrix Gla protein (mgp), osteocalcin (bglap), osteopontin (SPP1), secreted protein acidic and rich in cysteine (SPARC) and type I collagen α1 chain (COL1A1) have been studied in gilthead sea bream.ResultsDuring gilthead sea bream larval development, specific expression profiles for each gene were tightly regulated during fish morphogenesis and correlated with specific morphogenetic events and tissue development. Dietary hypervitaminosis A during early larval development disrupted the normal gene expression profile for genes involved in RA signalling (RARA), VA homeostasis (RBP) and several genes encoding ECM proteins that are linked to skeletogenesis, such as bglap and mgp.ConclusionsPresent data reflects the specific gene expression patterns of several genes involved in larval fish RA signalling and skeletogenesis; and how specific gene disruption induced by a nutritional VA imbalance underlie the skeletal deformities. Our results are of basic interest for fish VA signalling and point out some of the potential molecular players involved in fish skeletogenesis. Increased incidences of skeletal deformities in gilthead sea bream fed with hypervitaminosis A were the likely ultimate consequence of specific gene expression disruption at critical development stages.

Expression of Iroquois genes is up-regulated during early lung development in the nitrofen-induced pulmonary hypoplasia

Background/Purpose Iroquois homeobox ( Irx) genes have been implicated in the early lung morphogenesis of vertebrates. Irx1-3 and Irx5 gene expression is seen in fetal lung in rodents up to day (D) 18.5 of gestation. Fetal lung in Irx knockdown mice shows loss of mesenchyme and dilated airspaces, whereas nitrofen-induced hypoplastic lung displays thickened mesenchyme and diminished airspaces. We hypothesized that the Irx genes are up-regulated during early lung morphogenesis in the nitrofen-induced hypoplastic lung. Methods Pregnant rats were exposed either to olive oil or nitrofen on D9. Fetal lungs harvested on D15 were divided into control and nitrofen groups; and the lungs harvested on D18 were divided into control, nitrofen without congenital diaphragmatic hernia (CDH[−]), and nitrofen with CDH (CDH[+]). Irx gene expression levels were analyzed by reverse transcriptase polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression of Irx family. Results Pulmonary Irx1-3 and Irx5 messenger RNA expression levels were significantly up-regulated in nitrofen group compared with controls at D15. On D15, Irx immunoreactivity was increased in nitrofen-induced hypoplastic lung compared with controls. Conclusion Overexpression of Irx genes in the early lung development may cause pulmonary hypoplasia in the nitrofen CDH model by inducing lung dysmorphogenesis with thickened mesenchyme and diminished airspaces.

Disabled is a bona fide component of the Abl signaling network

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.

Disabled is a bona fide component of the Abl signaling network

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.

Planar cell polarity breaks bilateral symmetry by controlling ciliary positioning

Defining the three body axes is a central event of vertebrate morphogenesis. Establishment of left-right (L-R) asymmetry in development follows the determination of dorsal-ventral (D-V) and anterior-posterior (A-P) body axes1,2, though the molecular mechanism underlying precise L-R symmetry breaking in reference to the other two axes is still poorly understood. Here by removing both Vangl1 and Vangl2, the two mouse homologues of a Drosophila core planar cell polarity (PCP) gene Van Gogh (Vang), we have uncovered a previously unappreciated function of PCP in initial breaking of lateral symmetry. The leftward nodal flow across the posterior notochord (PNC, also referred to as “the node”) has been identified as the earliest event in the de novo formation of L-R asymmetry3,4.We found that PCP is essential in interpreting the A-P patterning information and linking it to L-R asymmetry. In the absence of Vangl1 and Vangl2, cilia are positioned randomly around the center of the PNC cells and nodal flow is turbulent, which results in disrupted L-R asymmetry. Importantly, PCP in mouse, unlike what has been implicated in other vertebrate species, is not required for ciliogenesis, cilium motility, Sonic hedgehog (Shh) signaling or apical docking of basal bodies in ciliated tracheal epithelial cells. Our data suggest that PCP acts earlier than the unidirectional nodal flow during bilateral symmetry breaking in vertebrates and provide insight into the functional mechanism of PCP in organizing the vertebrate tissues in development.

Cell‐ and Tissue‐mechanics of Apical Contraction in Embryonic Epithelia

Apical contraction is one of the principle cellular mechanisms that sculpt epithelial sheets into structures such as tubes, pits, and furrows. In order to investigate the mechanics of apical contraction and epithelial morphogenesis in vertebrates we have developed three methods (chemical‐, electrical‐, and laser‐activation) to induce contraction in embryonic epithelia of the frog Xenopus laevis. We use these methods together with a biomechanical analytical framework to investigate the molecular signaling pathways regulating contraction and the mechanical coupling of these pathways to tissue movements. Thus far, we find pathways that control exogenously induced contractions are similar to the programs that regulate native epithelial morphogenesis. For instance, laser‐activation induces a Ca++ wave, activates RhoGTPase, and drives F‐actin remodeling. Epithelial stiffness and force‐generation measured during electrically induced contraction suggests these two properties are mechanically independent. In contrast to single‐cell studies we have found no evidence that mechanical‐stimulation can induce apical contraction. In addition to our results on the molecular and mechanical regulation of apical contraction our studies suggest morphogenetic processes can be co‐opted by tissue engineers to build replacement tissues and construct artificial organs.

Morphogenesis of the Medaka Cerebellum, with Special Reference to the Mesencephalic Sheet, a Structure Homologous to the Rostrolateral Part of Mammalian Anterior Medullary Velum

We have examined cerebellar morphogenesis after neural tube stage in medaka (Oryzias latipes), a ray-finned fish, by conventional histology and immunohistochemistry using anti-proliferating cell nuclear antigen (PCNA) and anti-acetylated tubulin antibodies. Our results indicate that the medaka cerebellum is formed in 4 successive stages: (1) formation and enlargement of the cerebellar primordia; (2) rostral midline fusion of the left/right halves of the cerebellar primordia; (3) formation of the cerebellar matrix zones in the midline and caudalmost regions of the primitive cerebellum, and (4) growth and differentiation of the cerebellum. Our results also show that cerebellar morphogenesis is different from that in mammals in 3 important points: the developmental origins of the primordia, directions along which cerebellar fusion proceeds, and number, locations and duration of the cerebellar matrix zones. During the course of this study, an alar-derived membranous structure between the cerebellum and the midbrain in the adult medaka brain was identified as the structure homologous to the rostrolateral part of the mammalian anterior medullary velum. We have named this structure in the adult teleostean brains as the ‘mesencephalic sheet’. The present study indicates that there exists both conserved and divergent patterns in cerebellar morphogenesis in vertebrates.

Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut

Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates.

Nusap1 is essential for neural crest cell migration in zebrafish

Microtubules play important roles in mitotic spindle assembly and chromosome segregation to maintain normal cell cycle progression. A number of microtubule-associated proteins have been identified in epithelial and neural cell cultures; however, their physiological significance is not well characterized due to the lack of appropriate in vivo animal models. Nucleolar spindle-associated protein (NuSAP) is a microtubule-binding protein and is reported to be involved in mitosis by cell culture studies. In this report, we identified the zebrafish homologue of human NuSAP and investigated its expression profile and functions. Using in situ hybridization, we demonstrated that transcripts of zebrafish nusap1 are specifically expressed in the retina, forebrain, hindbrain and neural crest. When the in vivo expression of nusap1 was knocked down through antisense oligonucleotide morpholino technology, the morphants of nusap1 showed impaired morphogenesis in the trunk and yolk extension, implying the involvement of Nusap1 in cell migration. Mechanistic studies revealed that nusap1 morphants have an altered expression pattern of neural crest markers crestin and sox9b, but normal expression of blood vessel and notochord markers gata1 and shh. In addition, nusap1 mRNA injection caused serious apoptosis in retina and hindbrain tissue, and these phenotypes can be rescued by co-injection of morpholino against nusap1. These observations not only suggest a role for Nusap1 in connecting apoptosis with cell migration, but also provide strong evidences that Nusap1 is potentially involved in morphogenesis in vertebrates.

Sculpturing digit shape by cell death

Physiological cell death is a key mechanism that ensures appropriate development and maintenance of tissues and organs in multicellular organisms. Most structures in the vertebrate embryo exhibit defined areas of cell death at precise stages of development. In this regard the areas of interdigital cell death during limb development provide a paradigmatic model of massive cell death with an evident morphogenetic role in digit morphogenesis. Physiological cell death has been proposed to occur by apoptosis, cellular phenomena genetically controlled to orchestrate cell suicide following two main pathways, cytochrome C liberation from the mitochondria or activation of death receptors. Such pathways converge in the activation of cysteine proteases known as caspases, which execute the cell death program, leading to typical morphologic changes within the cell, termed apoptosis. According to these findings it would be expected that caspases loss of function experiments could cause inhibition of interdigital cell death promoting syndactyly phenotypes. A syndactyly phenotype is characterized by absence of digit freeing during development that, when caused by absence of interdigital cell death, is accompanied by the persistence of an interdigital membrane. However this situation has not been reported in any of the KO mice or chicken loss of function experiments ever performed. Moreover histological analysis of dying cells within the interdigit reveals the synchronic occurrence of different types of cell death. All these findings are indicative of caspase alternative and/or complementary mechanisms responsible for physiological interdigital cell death. Characterization of alternative cell death pathways is required to explain vertebrate morphogenesis. Today there is great interest in cell death via autophagy, which could substitute or act synergistically to the apoptotic pathway. Here we discuss what is known about physiological cell death in the developing interdigital tissue of vertebrate embryos, paying special attention to the avian species.

An Hh-Dependent Pathway in Lateral Plate Mesoderm Enables the Generation of Left/Right Asymmetry

Breaking bilateral symmetry is critical for vertebrate morphogenesis. In the mouse, directional looping of the heart and rotation of the embryo, the first overt evidence of left/right asymmetry (L/R), are observed at early somite stages ( approximately E8.5) [1, 2]. Activation of a Nodal-Pitx2 regulatory pathway specifically within the left lateral plate mesoderm (LPM) is critical for these events [3-10]. Asymmetric expression of Nodal is thought to be triggered by left-oriented, cilia-generated flow within the ventral, midline node [11, 12]. Genetic removal of Hedgehog (Hh) signaling in the mouse demonstrates a requirement for Hedgehog signals in the symmetry-breaking process [13], and analysis of node trafficking has suggested a mechanism of directional transport in the node that might relate to symmetry breaking in the LPM [14]. Here we provide evidence that Hedgehog signaling in the node is not essential for breaking bilateral symmetry. In contrast, direct Hh signaling in the LPM is critical. Evidence is presented that Sonic and Indian hedgehog signals act together, through a Foxf1/Bmp4 pathway, to enable the initiation and propagation of Nodal signaling within the LPM, regulating the competence of that tissue to respond to the Nodal pathway.

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.