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Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis

Kugler, Jamie E; Gazdoiu, Stefan; Oda-Ishii, Izumi; Passamaneck, Yale J; Erives, Albert J; Di Gregorio, Anna
For over a century, muscle formation in the ascidian embryo has been representative of 'mosaic' development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos.
PMCID:2894658
PMID: 20592183
ISSN: 0021-9533
CID: 1345502

Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidian Ciona intestinalis

Passamaneck, Yale J; Katikala, Lavanya; Perrone, Lorena; Dunn, Matthew P; Oda-Ishii, Izumi; Di Gregorio, Anna
The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.
PMCID:2761113
PMID: 19820186
ISSN: 0950-1991
CID: 1345512

The evolutionarily conserved leprecan gene: its regulation by Brachyury and its role in the developing Ciona notochord

Dunn, Matthew P; Di Gregorio, Anna
In Ciona intestinalis, leprecan was identified as a target of the notochord-specific transcription factor Ciona Brachyury (Ci-Bra) (Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R.W., Levine, M., Satoh, N., 1999. Brachyury downstream notochord differentiation in the ascidian embryo. Genes Dev. 13, 1519-1523). By screening approximately 14 kb of the Ci-leprecan locus for cis-regulatory activity, we have identified a 581-bp minimal notochord-specific cis-regulatory module (CRM) whose activity depends upon T-box binding sites located at the 3'-end of its sequence. These sites are specifically bound in vitro by a GST-Ci-Bra fusion protein, and mutations that abolish binding in vitro result in loss or decrease of regulatory activity in vivo. Serial deletions of the 581-bp notochord CRM revealed that this sequence is also able to direct expression in muscle cells through the same T-box sites that are utilized by Ci-Bra in the notochord, which are also bound in vitro by the muscle-specific T-box activators Ci-Tbx6b and Ci-Tbx6c. Additionally, we created plasmids aimed to interfere with the function of Ci-leprecan and categorized the resulting phenotypes, which consist of variable dislocations of notochord cells along the anterior-posterior axis. Together, these observations provide mechanistic insights generally applicable to T-box transcription factors and their target sequences, as well as a first set of clues on the function of Leprecan in early chordate development.
PMCID:2666983
PMID: 19217895
ISSN: 0012-1606
CID: 1345522

Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis

Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna
To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription.
PMCID:3066068
PMID: 18802963
ISSN: 1526-954x
CID: 1345532

Conservation of notochord gene expression across chordates: insights from the Leprecan gene family

Capellini, Terence D; Dunn, Matthew P; Passamaneck, Yale J; Selleri, Licia; Di Gregorio, Anna
The notochord is a defining character of the chordates, and the T-box transcription factor Brachyury has been shown to be required for notochord development in all chordates examined. In the ascidian Ciona intestinalis, at least 44 notochord genes have been identified as bona fide transcriptional targets of Brachyury. We examined the embryonic expression of a subset of murine orthologs of Ciona Brachyury target genes in the notochord to assess its conservation throughout chordate evolution. We focused on analyzing the Leprecan gene family, which in mouse is composed of three genes, as opposed to the single-copy Ciona gene. We found that all three mouse Leprecan genes are expressed in the notochord. Additionally, while Leprecan expression in C. intestinalis is confined to the notochord, expression of its mouse orthologs includes dorsal root ganglia, limb buds, branchial arches, and developing kidneys. These results have interesting implications for the evolution and development of chordates.
PMCID:3065379
PMID: 18798549
ISSN: 1526-954x
CID: 1345542

Pbx1/Pbx2 govern axial skeletal development by controlling Polycomb and Hox in mesoderm and Pax1/Pax9 in sclerotome

Capellini, Terence D; Zewdu, Rediet; Di Giacomo, Giuseppina; Asciutti, Stefania; Kugler, Jamie E; Di Gregorio, Anna; Selleri, Licia
The post-cranial axial skeleton consists of a metameric series of vertebral bodies and intervertebral discs, as well as adjoining ribs and sternum. Patterning of individual vertebrae and distinct regions of the vertebral column is accomplished by Polycomb and Hox proteins in the paraxial mesoderm, while their subsequent morphogenesis depends partially on Pax1/Pax9 in the sclerotome. In this study, we uncover that Pbx1/Pbx2 are co-expressed during successive stages of vertebral and rib development. Next, by exploiting a Pbx1/Pbx2 loss-of-function mouse, we show that decreasing Pbx2 dosage in the absence of Pbx1 affects axial development more severely than single loss of Pbx1. Pbx1/Pbx2 mutants exhibit a homogeneous vertebral column, with loss of vertebral identity, rudimentary ribs, and rostral hindlimb shifts. Of note, these axial defects do not arise from perturbed notochord function, as cellular proliferation, apoptosis, and expression of regulators of notochord signaling are normal in Pbx1/Pbx2 mutants. While the observed defects are consistent with loss of Pbx activity as a Hox-cofactor in the mesoderm, we additionally establish that axial skeletal patterning and hindlimb positioning are governed by Pbx1/Pbx2 through their genetic control of Polycomb and Hox expression and spatial distribution in the mesoderm, as well as of Pax1/Pax9 in the sclerotome.
PMCID:5918304
PMID: 18691704
ISSN: 0012-1606
CID: 1345552

Simple chordates exhibit a proton-independent function of acid-sensing ion channels

Coric, Tatjana; Passamaneck, Yale J; Zhang, Ping; Di Gregorio, Anna; Canessa, Cecilia M
Acid-sensing ion channels (ASICs) constitute a family of neuron-specific voltage-insensitive sodium channels gated by extracellular protons. Functions of ASICs in mammals include nociception, mechanosensation, and modulation of synaptic transmission. However, the role protons play in mediating the effects of ASICs remains elusive. We have examined ASICs from the ascidian Ciona intestinalis, a simple chordate organism whose nervous system in the larval stage exhibits high similarity to that of higher vertebrates. We found that the ascidian genome contains a single ASIC gene that gives rise to two splice forms analogous to the mammalian ASIC1 and ASIC2. CiASIC is expressed in most neurons of the larva but is absent in the adult. Despite high sequence similarity with mammalian counterparts, CiASIC is proton-insensitive when examined in heterologous systems or in larval neurons; the latter rules out the possibility that proton sensitivity is conferred by accessory proteins or particular factors present only in Ciona neurons. Down-regulation of the CiASIC transcript by double-stranded RNA disrupted the regular pattern of larval swimming, implying that proton-independent mechanisms mediate the effects of ASIC in vivo. Together the data identify ASIC as a highly conserved channel distinctive of chordate nervous systems and show that protons are not essential for ASIC function.
PMID: 18211956
ISSN: 0892-6638
CID: 1345562

Dynamic and polarized muscle cell behaviors accompany tail morphogenesis in the ascidian Ciona intestinalis

Passamaneck, Yale J; Hadjantonakis, Anna-Katerina; Di Gregorio, Anna
BACKGROUND: Axial elongation is a key morphogenetic process that serves to shape developing organisms. Tail extension in the ascidian larva represents a striking example of this process, wherein paraxially positioned muscle cells undergo elongation and differentiation independent of the segmentation process that characterizes the formation of paraxial mesoderm in vertebrates. Investigating the cell behaviors underlying the morphogenesis of muscle in ascidians may therefore reveal the evolutionarily conserved mechanisms operating during this process. METHODOLOGY/PRINCIPLE FINDINGS: A live cell imaging approach utilizing subcellularly-localized fluorescent proteins was employed to investigate muscle cell behaviors during tail extension in the ascidian Ciona intestinalis. Changes in the position and morphology of individual muscle cells were analyzed in vivo in wild type embryos undergoing tail extension and in embryos in which muscle development was perturbed. Muscle cells were observed to undergo elongation in the absence of positional reorganization. Furthermore, high-speed high-resolution live imaging revealed that the onset and progression of tail extension were characterized by the presence of dynamic and polarized actin-based protrusive activity at the plasma membrane of individual muscle cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that in the Ciona muscle, tissue elongation resulted from gradual and coordinated changes in cell geometry and not from changes in cell topology. Proper formation of muscle cells was found to be necessary not only for muscle tissue elongation, but also more generally for completion of tail extension. Based upon the characterized dynamic changes in cell morphology and plasma membrane protrusive activity, a three-phase model is proposed to describe the cell behavior operating during muscle morphogenesis in the ascidian embryo.
PMCID:1934933
PMID: 17684560
ISSN: 1932-6203
CID: 1345572

Lineage-independent mosaic expression and regulation of the Ciona multidom gene in the ancestral notochord

Oda-Ishii, Izumi; Di Gregorio, Anna
The transcription factor Ciona Brachyury (Ci-Bra) plays an essential role in notochord development in the ascidian Ciona intestinalis. We characterized a putative Ci-Bra target gene, which we named Ci-multidom, and analyzed in detail its expression pattern in normal embryos and in embryos where Ci-Bra was misexpressed. Ci-multidom encodes a novel protein, which contains eight CCP domains and a partial VWFA domain. We show that an EGFP-multidom fusion protein localizes preferentially to the endoplasmic reticulum (ER), and is excluded from the nucleus. In situ hybridization experiments demonstrate that Ci-multidom is expressed in the notochord and in the anterior neural boundary (ANB). We found that the expression in the ANB is fully recapitulated by an enhancer element located upstream of Ci-multidom. By means of misexpression experiments, we provide evidence that Ci-Bra controls transcription of Ci-multidom in the notochord; however, while Ci-Bra is homogeneously expressed throughout this structure, Ci-multidom is transcribed at detectable levels only in a random subset of notochord cells. The number of notochord cells expressing Ci-multidom varies among different embryos and is independent of developmental stage, lineage, and position along the anterior-posterior axis. These results suggest that despite its morphological simplicity and invariant cell-lineage, the ancestral notochord is a mosaic of cells in which the gene cascade downstream of Brachyury is differentially modulated.
PMID: 17576134
ISSN: 1058-8388
CID: 1345582

The multidimensionality of cell behaviors underlying morphogenesis: a case study in ascidians

Di Gregorio, Anna; Hadjantonakis, Anna-Katerina
Databases where different types of information from different sources can be integrated, cross-referenced and interactively accessed are necessary for building a quantitative understanding of the molecular and cell biology intrinsic to the morphogenesis of an embryo. Tassy and colleagues recently reported the development of software tailor-made to perform such a task, along with the generation and integration of three-dimensional anatomical models of embryos. They convincingly illustrated the utility of their approach by applying it to the early ascidian embryo.
PMID: 16937345
ISSN: 0265-9247
CID: 1345592