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The origin of the notochord is a central issue in chordate evolution. This study examined the development of the acorn worm pygochord, a putative homologue of the notochord. Because the pygochord differentiates only after metamorphosis, the developmental was followed process by inducing regeneration after artificial amputation in Ptychodera flava. It was found that although the regeneration of the posterior part of the body did not proceed via formation of an obvious regeneration bud, pygochord regeneration was observed within a few weeks, possibly via trans-differentiation of endoderm cells. The expression of the fibrillary collagen gene (Fcol) and elav in the pygochord during regeneration was detected. This indicates that pygochord cells are not part of gut epithelial cells, but that they differentiated as a distinct cell type. Our gene expression analyses do not provide supporting evidence for the homology between the pygochord and notochord, but rather favored the convergent evolution between them., (© 2018 Japanese Society of Developmental Biologists.)

The stress distribution inside a Xenopus laevis tailbud embryo was estimated to examine the cause of the straightening and elongation. The embryos were cut in the middle, yielding a cross-section perpendicular to the body axis. The section was not flat, owing to the residual stress relief. The stress needed to restore the flatness corresponded to the stress inside the embryo and was calculated using the surface topography and Young's-moduli in the section. We found the areas of the notochord (Nc), neural tube (NT), and abdominal tissue (AT) bulged in the cross-section, which revealed that compressive forces acted in these tissues. The moduli of the Nc, NT, and AT were in the order of several thousand, hundred, and tens of pascals, respectively. In the Nc, the compressive force was largest and increased with the development, suggesting Nc playing a central role in the elongation. The bending moment generated by the AT was 10 times higher than that by the Nc in the early stages of the tailbud formation, and the two were similar in the latter stages, suggesting that the compressive force in the AT was the major cause of the straightening during the early stage. The straightening and elongation could be orchestrated by changes in the compressive forces acting on the Nc, NT, and AT over time. For the sake of simplicity, we calculated the compressive force only and neglected the tensile force. Thus, it should be noted that the amount of the compressive force was somewhat overestimated., (© 2017 Japanese Society of Developmental Biologists.)

Pitx genes play important roles in a variety of developmental processes in vertebrates. In an ascidian species, Halocynthia roretzi, Hr-Pitx, the only Pitx gene of this species, has been reported to be expressed in the left epidermis at the tailbud stage. In the present study, first, we have shown that Hr-Pitx is also expressed in the papilla-forming region at the neurula to tailbud stages, and then we addressed transcription regulatory mechanisms for the expression of Hr-Pitx in the papilla-forming region. We have identified the genomic region ranging from 850 to 1211 bp upstream from the translation start site of the Hr-Pitx gene as an enhancer region that drives the transcription of Hr-Pitx in the papilla-forming region. Within the enhancer region, putative transcriptional factor binding sites for Otx as well as Fox were shown to be required for its activity. Finally, we carried out knocking down experiments of Hr-Otx function using an antisense morpholino oligonucleotide, in which the knocking down of Hr-Otx function resulted in reduction of the enhancer activity and loss of the expression of Hr-Pitx in the papilla-forming region. In Xenopus laevis, it has been reported that Pitx genes are expressed downstream of Otx function during development of the cement gland, an adhesive organ of its larva. Taken together, it is suggested that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates., (© 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.)

Muscle differentiation has been widely described in zebrafish and Xenopus, but nothing is known about this process in amphibian urodeles. Both anatomical features and locomotor activity in urodeles are known to show intermediate features between fish and anurans. Therefore, a better understanding of myogenesis in urodeles could be useful to clarify the evolutionary changes that led to the formation of skeletal muscle in the trunk of land vertebrates. We report here a detailed morphological and molecular investigation on several embryonic stages of Ambystoma mexicanum and show that the first differentiating muscle fibers are the slow ones, originating from a myoblast population initially localized close to the notochord that forms a superficial layer on the somitic surface afterwards. Subsequently, fast fibers differentiation ensues. We also identified and cloned A. mexicanum Myf5 as a muscle-specific transcriptional factor likely involved in urodele muscle differentiation., (© 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.)

Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo.

An immunoglobulin superfamily neuronal adhesion molecule, Contactin, has been implicated in axon guidance of spinal sensory neurons in Xenopus embryos. To identify the guidance signaling molecules that Contactin recognizes in tailbud embryos, an in situ binding assay was performed using recombinant Contactin-alkaline phosphatase fusion protein (Contactin-AP) as a probe. In the assay of whole-mount or sectioned embryos, Contactin-AP specifically bound to the notochord and its proximal regions. This binding was completely blocked by either digestion of embryo sections with chondroitinase ABC or pretreatment of Contactin-AP with chondroitin sulfate A. When the spinal cord and the notochord explants were co-cultured in collagen gel, growing Contactin-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Contactin antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Contactin on spinal sensory axons.

The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochord tissue, which did not undergo vacuolation. The XBtg2-depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5-D-4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.

Ascidian eggs develop into tadpole larvae. They have a simple central nervous system (CNS) at the dorsal midline. The CNS is formed through neural tube formation at the neurula stage, as in vertebrates. The total number of cells in the CNS is approximately 300. In Halocynthia roretzi, the anterior part of the CNS, which consists of the brain (sensory vesicle) and the visceral ganglion, is exclusively derived from 10 blastomeres at the 110-cell stage. The anterior CNS is relatively complex and shows remarkable left-right asymmetry, with the lumen of the sensory vesicle, the otolith, and the ocellus on the right side, and the presumed hydrostatic pressure organ on the left side. We labeled these 10 precursor blastomeres - six in the animal hemisphere (a-line) and four in the vegetal hemisphere (A-line) - with lineage tracer, and examined the fates in swimming larvae. The clonal organization of the anterior CNS is essentially invariant among individuals, although slight variation in the clonal boundary was observed. There was no extensive mixing between descendants of each precursor. We observed no evidence of cell migration except for two neuronal cells derived from a8.25 blastomeres. The eventual fates of the bilateral blastomeres produced extensive left-right asymmetry. The results suggest that the anterior neural tube rotates in a clockwise direction when viewed from the posterior pole. Staged observation indicated that this rotation takes place during the last 5 h of embryogenesis. We describe detailed positions of descendants of each precursor blastomere. In particular, specific cells of sensory structures were identified by their morphology and staining with specific antibodies and probes. The otolith and ocellus pigment cells were derived from left and right a8.25 blastomeres. Lens cells of the ocellus have a right A8.8 origin, and most of the photoreceptor cells originated from the right A8.7. The presumed pressure organ was formed by descendants of left and right a8.19 and left a8.17 blastomeres. The description of cell lineages of the CNS would facilitate future research to analyze the mechanisms of development of the simple CNS of ascidian tadpole larvae.

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole-mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior-posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm-inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.

Expression of the Brachyury (Ci-Bra) gene of the ascidian Ciona intestinalis is initiated at the 64-cell stage. Gene expression is restricted to notochord precursor cells, and Ci-Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci-Bra-downstream genes that are expressed in notochord cells were isolated. The present determination, by whole-mount in situ hybridization, of the temporal expression patterns of 19 notochord-specific and 20 notochord-predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord-specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.

Brachyury (T) is involved in mesoderm induction during early mouse development. T expression in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro, was studied. Endogenous T expression in P19 cells was transiently induced when the cells were allowed to form aggregates. This expression was enhanced by dimethyl sulfoxide. In situ hybridization showed that T expressing cells formed clusters on the aggregates. Transfection of plasmids encoding reporter genes under the control of the upstream region of T showed that the sequence up to -351 bp can resemble the differentiation-dependent expression of T in P19 cells. To define the promoter region regulating T expression, transgenic mice carrying LacZ under the control of the upstream region were prepared. The region up to -351 bp is sufficient to direct the expression in the primitive streak and tail-bud. The upstream region up to -2400 bp does not support expression in the notochord. The sequence between -987 and -585 bp enhances expression in the primitive streak and tail-bud. In the tail-bud where new cells for elongation of the anteroposterior axis were continuously supplied, the sequence up to -987 bp drove lacZ expression in gut endoderm and prospective neuroectoderm as well as in mesoderm derivatives.

Differentiation of notochord cells in the ascidian embryo requires cell-cell interactions and signal transduction pathways. Isolation and characterization of an ascidian gene (HrSH2) from Halocynthia roretzi is reported. Sequence analysis suggests that HrSH2 encodes a polypeptide with an SH2 domain and a tyrosine kinase phosphorylation site, that are implicated in signaling pathways through tyrosine phosphorylation. Zygotic expression of HrSH2 was transient. The gene expression began at the 110-cell stage but was downregulated by the larval stage. Whole-mount in situ hybridization, taking advantage of well-known lineage, revealed that the HrSH2 transcript first appeared in primordial notochord cells as well as a few endoderm cells of the 110-cell embryo. During gastrulation and neurulation, expression in the endoderm was downregulated, and instead HrSH2 transcript became evident in notochord cells, nerve cord cells, endodermal strand cells and epidermal cells of the tail. These results suggest the possibility that HrSH2 is involved in the signal transduction pathways required for notochord formation and for differentiation of other cells in conjunction with the notochord.

We examined the timing and mechanisms of mesodermal and neural determination in Cynops, using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops.