Control of intercalation is cell-autonomous in the notochord of Ciona intestinalis

Dishevelled signaling plays a critical role in the control of cell intercalation during convergent extension in vertebrates. This study presents evidence that Dishevelled serves a similar function in the Ciona notochord. Embryos transgenic for mutant Dishevelled fail to elongate their tails, and notochord cells fail to intercalate, though notochord cell fates are unaffected. Analysis of mosaic transgenics revealed that the effects of mutant Dishevelled on notochord intercalation are cell-autonomous in Ciona, though such defects have nonautonomous effects in Xenopus. Furthermore, our data indicate that notochord cell intercalation in Ciona does not require the progressive signals which coordinate cell intercalation in the Xenopus notochord, highlighting an important difference in how mediolateral cell intercalation is controlled in the two animals. Finally, this study establishes the Ciona embryo as an effective in vivo system for the study of the molecular control of morphogenetic cell movements in chordates.     

Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, beta4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos.     

Tail morphogenesis in the ascidian, Ciona intestinalis, requires cooperation between notochord and muscle

We present evidence that notochord and muscle differentiation are crucial for morphogenesis of the ascidian tail. We developed a novel approach for embryological manipulation of the developing larval tissues using a simple method to introduce DNA into Ciona intestinalis and the several available tissue-specific promoters. With such promoters, we misexpressed the Xenopus homeobox gene bix in notochord or muscle of Ciona embryos as a means of interfering with development of these tissues. Ciona embryos expressing bix in the notochord from the 64-cell stage develop into larvae with very short tails, in which the notochord precursors fail to intercalate and differentiate. Larvae with mosaic expression of bix have intermediate phenotypes, in which a partial notochord is formed by the precursor cells that did not receive the transgene while the precursors that express the transgene cluster together and fail to undergo any of the cell-shape changes associated with notochord differentiation. Muscle cells adjacent to differentiated notochord cells are properly patterned, while those next to the notochord precursor cells transformed by bix exhibit various patterning defects. In these embryos, the neural tube extends in the tail to form a nerve cord, while the endodermal strand fails to enter the tail region. Similarly, expression of bix in muscle progenitors impairs differentiation of muscle cells, and as a result, notochord cells fail to undergo normal extension movements. Hence, these larvae have a shorter tail, due to a block in the elongation of the notochord. Taken together, these observations suggest that tail formation in ascidian larvae requires not only signaling from notochord to muscle cells, but also a "retrograde" signal from muscle cells to notochord.     

Ciona intestinalis Noto4 contains a phosphotyrosine interaction domain and is involved in the midline intercalation of notochord cells

Brachyury plays a pivotal role in the notochord formation in ascidian embryos. Ciona intestinalis Noto4 (Ci-Noto4) was isolated as a gene downstream of Ci-Bra. This gene encodes a 307 amino-acid protein with a C-terminal phosphotyrosine interaction domain (PTB/PID). Expression of Ci-Noto4 commences at the neural plate stage and is specific to notochord cells. Suppression of Ci-Noto4 levels with specific antisense morpholino oligonucleotides resulted in the formation of two rows of notochord cells owing to a lack of midline intercalation between the bilateral populations of progenitor cells. In contrast, overexpression of Ci-Noto4 by injection of a Ci-Bra(promoter):Ci-Noto4-EGFP construct into fertilized eggs disrupted the localization of notochord cells. Ci-Noto4 overexpression did not affect cellular differentiation in the notochord, muscle, mesenchyme, or nervous system. Analysis of Ci-Noto4 regions that are responsible for its function suggested significant roles for the PTB/PID and a central region, an area with no obvious sequence similarity to other known proteins. These results suggested that PTB/PID-containing Ci-Noto4 is essential for midline intercalation of notochord cells in chordate embryos.     

Ciona intestinalis notochord as a new model to investigate the cellular and molecular mechanisms of tubulogenesis

Biological tubes are a prevalent structural design across living organisms. They provide essential functions during the development and adult life of an organism. Increasing progress has been made recently in delineating the cellular and molecular mechanisms underlying tubulogenesis. This review aims to introduce ascidian notochord morphogenesis as an interesting model system to study the cell biology of tube formation, to a wider cell and developmental biology community. We present fundamental morphological and cellular events involved in notochord morphogenesis, compare and contrast them with other more established tubulogenesis model systems, and point out some unique features, including bipolarity of the notochord cells, and using cell shape changes and cell rearrangement to connect lumens. We highlight some initial findings in the molecular mechanisms of notochord morphogenesis. Based on these findings, we present intriguing problems and put forth hypotheses that can be addressed in future studies.