Skip to main content
NCBI home page
Integrative and Comparative Biology logoLink to Integrative and Comparative Biology
. 2023 Jul 4;63(5):990–998. doi: 10.1093/icb/icad071

Loss of collagen gene expression in the notochord of the tailless tunicate Molgula occulta

Sydney Popsuj 1, Anna Di Gregorio 2,✉, Billie J Swalla 3,✉, Alberto Stolfi 4,✉
PMCID: PMC10714901  PMID: 37403333

Synopsis

In tunicates, several species in the Molgulidae family have convergently lost the tailed, swimming larval body plan, including the morphogenesis of the notochord, a major chordate-defining trait. Through the comparison of tailless M. occulta and a close relative, the tailed species M. oculata, we show that notochord-specific expression of the Collagen Type I/II Alpha (Col1/2a) gene appears to have been lost specifically in the tailless species. Using CRISPR/Cas9-mediated mutagenesis in the tailed laboratory model tunicate Ciona robusta, we demonstrate that Col1/2a plays a crucial role in the convergent extension of notochord cells during tail elongation. Our results suggest that the expression of Col1/2a in the notochord, although necessary for its morphogenesis in tailed species, is dispensable for tailless species. This loss is likely a result of the accumulation of cis-regulatory mutations in the absence of purifying selective pressure. More importantly, the gene itself is not lost, likely due to its roles in other developmental processes, including during the adult stage. Our study further confirms the Molgulidae as an interesting family in which to study the evolutionary loss of tissue-specific expression of indispensable genes.

Introduction

Tunicates are non-vertebrate chordates and comprise the sister group to vertebrates (Delsuc et al. 2006). Most species have a distinct biphasic life cycle divided between a tailed, swimming larva and a sessile, filter-feeding adult (Fodor et al. 2021a). They are key organisms amenable to studies of the evolution and the development of chordate traits (Lemaire 2009, 2011). Their tailed, tadpole-like larvae swim in search of a place to settle and undergo metamorphosis thanks to key traits shared with vertebrates, such as a dorsal central nervous system and a notochord flanked by paraxial muscles (Nishino 2018). However, they develop distinct body plans for the swimming larval phase and the sessile adult phase. The larval body plan is eventually degraded when larvae settle and undergo metamorphosis to give rise to a new adult body plan (Karaiskou et al. 2015).

In some tunicate species, mostly ascidians of the family Molgulidae, the swimming larval phase has been lost, and instead, development proceeds through tailless, non-swimming larvae before metamorphosis begins (Lacaze-Duthiers 1874; Maliska et al. 2013; Fodor et al. 2021b). The evolutionary loss of the swimming larval body plan posits an interesting case for the study of developmental trait loss. In some cases, a gene exclusively required for the larval stage can become pseudogenized in non-swimming species, as it is the case with Tyrosinase, which is required for the melanization of the sensory organ pigment cells of swimming larvae (Racioppi et al. 2017). However, many other genes cannot be entirely lost, due to their roles in forming the adult body plan, yet appear downregulated during the development of tailless larvae relative to their tailed counterparts (Fodor et al. 2021b).

When gene expression is evolutionarily lost in a specific tissue or cell type in one species, but the gene is not lost from its genome, an interesting set of questions emerge regarding the molecular mechanisms underlying this change. Evolutionary loss of tissue-specific gene expression might also indicate that the gene plays an important role in a developmental process that is no longer needed. One of the key chordate traits is the presence of a notochord, a group of mesodermal cells that intercalate to form a rod-like single-file row of cells during embryogenesis. In the larvae of most tailed ascidian species, the notochord is formed by exactly 40 cells (Fig. 1) (Satoh 2013). These cells, much like in vertebrates, undergo convergent extension to form a rod-shaped notochord that will later become a hollow tube through extensive vacuolization, serving ultimately as a hydrostatic skeleton for larval swimming (Miyamoto 1985; Jiang and Smith 2007; Denker and Jiang 2012 ). In the tailless species Molgula occulta, only 20 notochord cells are specified and they fail to undergo convergent extension (Fig. 1) (Berrill 1931; Swalla and Jeffery 1990). Notochord development is partially rescued in interspecific hybrids formed by the fertilization of M. occulta eggs with sperm from the closely related, tailed species M. oculata (Swalla and Jeffery 1990). While there are still only 20 notochord cells in the hybrid, these cells undergo convergent extension, suggesting that zygotic gene expression is sufficient to drive this process in development (Swalla and Jeffery 1990). Ultimately, M. occulta provides a unique opportunity for studying notochord development and evolutionary loss of gene expression. The purpose of the present study is two-fold (1) To identify the molecular mechanisms underlying evolutionary loss of tissue-specific gene expression in M. occulta, and (2) to use M. occulta as a “natural” genetic screen for genes that may be essential for notochord morphogenesis in tailed species.

Fig. 1.

Fig. 1.

Open in a new tab

Notochord development in Molgula. Diagram comparing notochord morphogenesis in tailed swimming (M. oculata) and tailless non-swimming (M. occulta) species and their interspecific hybrids (M. occulta eggs fertilized with M. oculata sperm). “h” = hours post-fertilization.

Previously analyzed transcriptomic data from M. occulta, M. oculata, and their hybrids, identified a list of notochord-expressed genes whose expression is lost or reduced in the tailless embryos (Lowe et al. 2014; Lowe et al. 2021; Fodor et al. 2021b). Many appeared to be involved in the production or processing of collagen, a major component of the notochord extracellular matrix in both tunicates and vertebrates (Miller and Mathews 1974; Zhang and Cohn 2006; Kugler et al. 2011; Yasuoka 2020). In this study, we set out to investigate the connection between loss of expression of the Collagen Type I/II Alpha (Col1/2a) gene, and the lack of notochord convergent extension in M. occulta. We show that M. occulta embryos lack distinct expression of Col1/2a in the notochord, but not in other tissues. This was consistent with transcriptomic data from hybrid embryos, which showed enrichment of transcripts from the M. oculata (tailed) parental Col1/2a allele relative to the M. occulta (tailless) allele. Due to the difficulty of DNA transfection and gene knockout approaches in Molgula, we carried out functional tests in the laboratory model tunicate Ciona robusta (formerly intestinalis Type A) instead. CRISPR/Cas9-mediated-disruption of Col1/2a in the Ciona notochord revealed a key role for this gene in notochord intercalation in tailed larvae. However, cis-regulatory analyses of Molgula Col1/2a genes were inconclusive, likely due to the well-documented phenomenon of developmental system drift between Molgula and Ciona (Stolfi et al. 2014b; Coulcher et al. 2020).

Methods

Molgula embryo collection

Molgula occulta and M. oculata can be collected simultaneously in the waters surrounding the Station Biologique de Roscoff, France. Gravid during July and August, these animals were collected and dissected to obtain the eggs and sperm necessary for their fertilization, and for the generation of interspecific (M. oculata sperm × M. occulta eggs) hybrids. Embryos were raised, dechorionated, and fixed as previously described (Swalla and Jeffery 1990). Images were then acquired on an inverted fluorescence DM IL LED or DMI8 microscopes (Leica). RNAseq data were generated and previously published (Lowe et al. 2014; Fodor et al. 2021b). In situ hybridizations were performed as previously described (Lowe et al. 2021).

Electroporations for Ciona

Ciona robusta (intestinalis Type A) adults were collected in San Diego, CA, USA, by M-REP. Animals were then dissected to isolate gametes for in vitro fertilization as previously described (Christiaen et al. 2009b). Dechorionated zygotes were then transfected with plasmids via electroporation as described (Christiaen et al. 2009a). Embryos were raised at 20°C and fixed for 15 min in MEM-FA fixative (3.7% formaldehyde, 0.1 M MOPS pH 7.4, 0.5 M NaCl, 1 mM EGTA, 2 mM MgSO4, and 0.1% Triton-X100), rinsed first in 1X PBS, 0.4% Triton-X100, 50 mM NH4Cl, then in 1X PBS, 0.1% Triton-X100, before being mounted in 2% DABCO/1X PBS/50% glycerol mounting solution. Images were then acquired as described above for Molgula.

CRISPR/Cas9-mediated genome editing

Single-chain guide RNA (sgRNA) plasmids were designed with the assistance of the algorithm CRISPOR (Haeussler et al. 2016). Cas9 and sgRNA (“F + E”) scaffold sequences used and cloning procedures were previously published (Stolfi et al. 2014a; Gandhi et al. 2018). Col1/2a sgRNAs were tested by co-electroporating 50 μg of each individual sgRNA expression plasmid in combination with 30 μg Foxa.a > Cas9 (Di Gregorio et al. 2001; Gibboney et al. 2020), 45 μg Ebf > Unc-76:: mCherry (Stolfi and Levine 2011), and 45 μg Ebf > TESTR1:: GFP per 700 μl of total electroporation volume. The “TESTR1” sequence in this case contained target sites for all Col1/2a sgRNAs tested. Loss of GFP signal in Ebf + cells (labeled by Unc-76:: mCherry) was interpreted as efficient mutagenesis by the particular sgRNA tested. Using this method, we settled on U6 > Col1/2a.658 and U6 > Col1/2a.694 sgRNAs (Supplementary Fig. 1). All sequences, published and unpublished, and electroporation protocols can be found in the Supplementary Sequences File.

Results

Loss of Col1/2a expression in the notochord of tailless M. occulta

Previous analysis of RNA sequencing (RNAseq) data from M. occulta, M. oculata, and their interspecific hybrids revealed the reduced expression of several putative notochord-expressed genes (Lowe et al. 2014; Lowe et al. 2021; Fodor et al. 2021b). The orthologs of these genes are known to be expressed in the notochord of Ciona, thanks to the extensive characterization of the notochord gene regulatory network in this species (Kugler et al. 2008; Kobayashi et al. 2013; Di Gregorio 2020). Many of these genes with reduced expression in M. occulta appeared to be involved in collagen production or processing, including Col1/2a, previously called Fibrillar collagen 1 (Ci-FCol1) in Ciona (Katikala et al. 2013). According to the tunicate sequence database ANISEED (Dardaillon et al. 2020), tunicate Col1/2a is equally similar to human COL1A1, COL1A2, and COL2A1, all of which, especially COL2A1, have been reported as markers of the notochord or notochord-derived nucleus pulposus in humans and other vertebrates (Su et al. 1991; Yan et al. 1995; Peck et al. 2017). Because of the enrichment for collagen pathway genes in our RNAseq and the homology to vertebrate notochord-expressed collagen genes, we reasoned that Col1/2a would be a good candidate gene to study further.

Our RNAseq analysis indicated that Col1/2a expression is reduced in M. occulta during tail elongation, in comparison to M. oculata (Fig. 2A). While Col1/2a expression is significantly upregulated in the tailed M. oculata between neurula and tailbud stage, when tail extension is happening, the expression in M. occulta did not significantly change. Similarly, interspecific hybrids between M. occulta and M. oculata showed enriched expression of their tailed M. oculata parental allele relative to the M. occulta parental allele at 6 hpf, indicating that changes in cis could be contributing to the divergence in the level of Col1/2a gene expression between the two species.

Fig. 2.

Fig. 2.

Open in a new tab

Loss of Col1/2a expression in the notochord of M. occulta. (A) RNAseq data from Fodor et al. 2021b, focusing on the Col1/2a gene in M. oculata, M. occulta, and M. oculata × M. occulta hybrids. LogFC = Log2 fold-change of reads measured between different stages of development: Gastrula (“Gast,” 3 hours post-fertilization), Neurula (“Neur,” 4 hpf), and Tailbud (“Tb,” 6 hpf). Positive values indicate upregulation at the later stage indicated (neurula relative to gastrula, or tailbud relative to neurula). Negative values indicate downregulation. P = p-value. For allele-specific data, LogFC = Log2 fold-change of reads mapped specifically to one species’ genome or the other in hybrid embyos at the indicated stage (neurula or tailbud). Positive values = more M. oculata reads than M. occulta reads. (B) Two-color, whole-mount fluorescent mRNA in situ hybridization of Col1/2 and Islet in M. oculata tailbud embryo showing expression of Col1/2a in notochord (“noto.,” dashed outline) and muscles (“mu.”). Embryo positioned laterally, showing tail bud extending posteriorly and ventrally. (C) In situ hybridization of Col1/2a in M. occulta, showing expression in muscles but not notochord (dashed outline). Nearly spherical M. occulta embryo positioned dorsally, with anterior (A) to the top and posterior (P) to the bottom. Embryonic midline shown as yellow line. Compare to Islet expression in Supplementary Fig. 2.

Because it is known that Col1/2a is also expressed in tail muscles in Ciona (Kugler et al. 2010), we next performed whole-mount in situ hybridizations to determine whether Col1/2a expression was reduced in these tissues. As expected, tailed M. oculata embryos showed expression of Col1/2a in both tail muscles and notochord, the latter of which also expressed the notochord marker Islet (Fig. 2B). However, tailless M. occulta clearly showed expression only in muscle cells, while notochord cells in the center of the embryo were negative for Col1/2a expression (Fig. 2C), though positive for Islet (Supplementary Fig. 2). Taken together, these results confirmed that Col1/2a expression has been specifically lost in the notochord of tailless M. occulta embryos.

Divergence between putative M. occulta and M. oculata Col1/2a cis-regulatory sequences

The evolutionary loss of expression of a given gene in a specific tissue or cell type can be due to the loss of regulatory sequences in cis (e.g., mutations in cis-regulatory sequences), or to the loss of expression or function of regulatory proteins in trans (e.g., loss of an upstream transcription factor). To further decipher the molecular basis of reduced Col1/2a expression in the notochord, we used published genome assemblies (Stolfi et al. 2014b) to align M. occulta and M. oculata sequences upstream of Col1/2a (Fig. 3A). We focused on a ∼630 bp fragment upstream of the Col1/2a gene, which in C. robusta contained the cis-regulatory modules (CRMs, or enhancers) that are sufficient to drive expression separately in notochord and muscle (Kugler et al. 2010; Katikala et al. 2013). By aligning Col1/2a upstream sequences from M. occulta/oculata using the online service MAFFT (Katoh et al. 2019), we found a high sequence identity through much of the sequence, except for a gap of high divergence from roughly −475 bp to −298 bp upstream of the start of exon 1 (Fig. 3A and B), which prompted us to further analyze this region both manually and using the transcription factor binding site search algorithm JASPAR (Castro-Mondragon et al. 2022) (see Supplementary Sequence File). We found two predicted Brachyury binding sites arranged in tandem in tailed M. oculata but absent in tailless M. occulta, which were identified using the mouse Tbxt (Bra) position weight matrix (Fig. 3C). This was of potential interest because Brachyury is the key transcription factor regulating notochord specification in Ciona (Di Gregorio 2020). These predicted binding sites were rearranged and divergent in M. occulta to the point where JASPAR predicted for them a much lower affinity score (see Supplementary Sequence File). Interestingly, a roughly equivalent region in the Col1/2a upstream sequence of C. robusta comprises the notochord-specific CRM and its required Brachyury binding sites (Fig. 3D) (Katikala et al. 2013). There were additional predicted Brachyury binding sites in conserved regions both proximal to and distal from the divergent region. However, some of these are also potential binding sites for Tbx6, which has been shown to regulate Col1/2a in the tail muscles of Ciona (Kugler et al. 2010).

Fig. 3.

Fig. 3.

Open in a new tab

Col1/2a cis-regulatory sequence analysis. (A) Alignment plot from MAFFT, indicating loss of alignment (red line) between M. oculata and M. occulta Col1/2a upstream sequences roughly between −475 and −298 upstream of the start of exon 1. (B) MAFFT alignment of the Col1/2 upstream sequences, with the −475/−298 region of low identity indicated by the purple outline box. (C) Magnified view of smaller region in pink dotted outline showing low sequence identity between M. oculata and M. occulta, with predicted mouse Brachyury (Bra) sites and their affinity scores in the M. oculata sequences highlighted. (D) Diagram of Col1/2a upstream region in C. robusta, showing known CRM previously characterized, including a minimal notochord CRM with functionally validated Ciona Brachyury binding sites highlighted.

Unfortunately, we were not able to test these sequences in Molgula, due to technical limitations (see in section “Discussion”). Thus, we attempted to test these sequences in much more tractable C. robusta embryos. Electroporation in Ciona embryos revealed Molgula Col1/2a reporter expression only in tail muscles, regardless of whether the sequence was from M. oculata (−643/−1 upstream of the start codon) or M. occulta (−632/−1 upstream) (Fig. 4). This suggests that the cis-regulatory logic of Col1/2a expression in the notochord is highly divergent between these distantly related genera due to developmental system drift, as we have previously shown for other CRMs (Stolfi et al. 2014b; Lowe and Stolfi 2018). Alternatively, the full cis-regulatory sequences required for notochord expression may not be contained in our reporters. However, we find this explanation less likely, as the C. robusta notochord CRM is located between 302 and 237 bp upstream of Col1/2a (Fig. 3D), and Molgula genomes are even more compact than that of Ciona (Stolfi et al. 2014b).

Fig. 4.

Fig. 4.

Open in a new tab

Molgula Col1/2a reporter activity in Ciona larvae. Images of the tails of C. robusta larvae electroporated with M. oculata (top) and M. occulta (bottom) Col1/2a H2B:: GFP reporter plasmids. H2B:: GFP expression (green nuclei) was observed in tail muscles with both M. oculata or M. occulta reporters, but no notochord labeling was seen with either construct. This suggests that the notochord CRM might be “unintelligible” between these distantly related tunicate genera (i.e., Molgula vs. Ciona). Tail outlines are indicated by dashed lines.

A role for Col1/2a in notochord morphogenesis revealed by CRISPR in Ciona

Evolutionary loss of the larval tail in several Molgula species is thought to be due to a lack of any selective advantage for swimming larvae in certain habitats, especially for those species inhabiting mud or sand flats at northern latitudes (Huber et al. 2000). While species with swimming larvae such as Ciona swim upward to attach to the undersides of outcroppings or other suspended substrates, M. occulta larvae fall to the ocean floor and live buried in the sand as adults (Berrill 1931). As the hollow, rod-like notochord likely functions as a hydrostatic skeleton for the tail muscles during swimming (Stemple 2005), a convergent extension of the notochord appears to be dispensable for tunicate species whose larvae do not need to swim in order to disperse and settle. Therefore, M. occulta may have lost the expression of genes important for notochord morphogenesis simply due to a lack of purifying selection. To show that Col1/2a expression in the notochord is important for its morphogenesis in tailed species, we turned to tissue-specific CRISPR/Cas9-mediated mutagenesis in C. robusta.

Notochord-specific disruption of Col1/2a was performed by co-electroporating a Brachyury > Cas9:: Geminin-N plasmid (Corbo et al. 1997; Song et al. 2022) together with two Col1/2a-targeting guide RNA plasmids (Fig. 5A) and Brachyury > Venus(YFP) to assay notochord morphogenesis. Col1/2a disruption resulted in notochord intercalation defects compared to a negative control condition omitting the Col1/2a-specific guide RNAs (Fig. 5B and C). The severity of the notochord defects observed correlated with mosaic incorporation of plasmid DNA, as assayed by YFP fluorescence, consistent with the fact that Col1/2a protein secreted by unaffected notochord cells might be able to partially rescue Col1/2a mutations in neighboring cells (Fig. 5C). Taken together, these data suggest that Col1/2a is essential for proper notochord morphogenesis in tunicate species with tailed, swimming larvae.

Fig. 5.

Fig. 5.

Open in a new tab

CRISPR/Cas9-mediated disruption of Col1/2a in Ciona impairs notochord morphogenesis. (A) Diagram of part of the Col1/2a gene in C. robusta, indicating the target sites of sgRNAs used for CRISPR/Cas9-mediated mutagenesis in this study. (B) Embryos subjected to notochord-specific CRISPR/Cas9-mediated mutagenesis of Col1/2a, compared to a control embryo. Examples of mild and severe notochord morphogenesis defects are shown. Notochord cells labeled by Brachyury > Venus (green). (C) Scoring data of notochord defects observed in CRISPR and negative control embryos, one trial each. Left: all YFP + embryos in both conditions. Right: Col1/2a CRISPR scoring data broken down by left/right mosaic expression of YFP (due to mosaic incorporation of electroporated plasmids) and “full” YFP expression.

Discussion

We have presented evidence supporting the hypothesis that the expression of genes important for notochord morphogenesis has been lost over the course of the evolution of tunicates with non-swimming larvae such as M. occulta. RNAseq and in situ hybridization data show that expression of the extracellular matrix component Col1/2a is reduced in the notochord of M. occulta when compared to that in its close tailed relative M. oculata. Differential parental allele-specific expression of Col1/2a in M. occulta X M. oculata hybrids supports the hypothesis that this reduction is due to changes in cis-regulatory sequences, which is further supported by bioinformatic alignment and transcription factor binding sequence analysis. Using CRISPR/Cas9 in the tailed laboratory model tunicate Ciona, we show that Col1/2a is likely involved in the morphogenesis of the notochord.

However, we did not test the requirement of Col1/2a in tailed Molgula species, as CRISPR/Cas9 has not been adapted to these species yet. Furthermore, we were unable to test differential cis-regulatory sequence activity, due to our inability to access Molgula embryos for electroporation, and due to cis-regulatory incompatibility between Molgula and Ciona (Lowe and Stolfi 2018). We were not able to routinely obtain M. oculata/occulta specimens, as these species overlap in an extremely limited geographic region and spawn only during one month of the year (Berrill 1931; Huber et al. 2000). Furthermore, 2022 was the hottest European summer in recorded history (van Daalen et al. 2022), and this has likely affected the timing and duration of gamete production (Bates 2005). In the future, it will be important to overcome these technical hurdles to rigorously investigate the cis-regulatory basis of tail loss in Molgula.

It is important to note that, unlike other genes such as Tyrosinase (Racioppi et al. 2017), the Col1/2a gene itself has not been lost from M. occulta. This is probably due to the important roles of collagens in the adult tissues (Rychel and Swalla 2007). One interesting observation is that Col1/2a expression is still observed in the otherwise non-functional larval tail muscles of M. occulta (Fig. 2C). This might be due to a requirement for Col1/2a in adult muscles. If the same cis-regulatory element was required to drive the expression of Col1/2a in both larval and adult muscles, then that would preclude the loss of expression in larval tail muscles. In contrast, the notochord is a tissue that is not present in any adult ascidian (being lost at metamorphosis), and thus notochord-specific cis-regulatory elements are not expected to be under the same evolutionary constraints. In sum, the tailless larvae of M. occulta remain an intriguing model in which to study molecular and cellular principles of evolutionary losses. Future progress will depend greatly on the ability to further adapt electroporation and CRISPR techniques to directly test cis-regulatory and protein-coding sequences in Molgula.

Supplementary Material

icad071_Supplemental_Files
Click here for additional data file. (1MB, zip)

Acknowledgement

We thank Elijah K. Lowe. and C. Titus Brown for their contributions to and continued support for Molgula transcriptomic studies. We thank Susanne Gibboney for assistance in generating and validating sgRNAs for CRISPR of Col1/2a. We thank Sophie Booker and Station Biologique de Roscoff staff for their assistance in organizing research visits and procuring Molgula specimens.

Notes

From the symposium “Genomics of marine larval evolution and development” presented at the annual meeting of the Society for Integrative and Comparative Biology virtual annual meeting, January 16–March 31, 2023.

Contributor Information

Sydney Popsuj, School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA.

Anna Di Gregorio, Department of Molecular Pathobiology, New York University College of Dentistry, New York, NY 10010, USA.

Billie J Swalla, Department of Biology, University of Washington, Seattle, WA 98195, USA.

Alberto Stolfi, School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332, USA.

Funding

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health, under awards number R03HD098395-02S1 and R03HD107314 to A.D.G.. This research was also supported by the Department of Molecular Pathobiology Accelerator Award B01 2020 to A.D.G., by a Royalty Research Fund grant from the University of Washington to B.J.S., and by award R01GM143326 from the National Institute of General Medical Sciences of the National Institutes of Health to A.S.

Conflict of interest

The authors declare that they have no competing financial interests.

Data availability

The data underlying this article will be shared upon request to the corresponding authors.

References

  1. Bates WR. 2005. Environmental factors affecting reproduction and development in ascidians and other protochordates. Can J Zool. 83:51–61. [Google Scholar]
  2. Berrill NJ. 1931. Studies in tunicate development. Part ii. Abbreviation of development in the molgulidae. Philos Trans R Soc Lond B Biol Sci. 219:281–346. [Google Scholar]
  3. Castro-Mondragon JA, Riudavets-Puig R, Rauluseviciute I, Berhanu Lemma R, Turchi L, Blanc-Mathieu R, Lucas J, Boddie P, Khan A, Manosalva Pérez Net al. 2022. Jaspar 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 50:D165–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christiaen L, Wagner E, Shi W, Levine M.. 2009a. Electroporation of transgenic dnas in the sea squirt ciona. Cold Spring Harb Protoc. 2009:pdb–rot5345. [DOI] [PubMed] [Google Scholar]
  5. Christiaen L, Wagner E, Shi W, Levine M.. 2009b. Isolation of sea squirt (Ciona) gametes, fertilization, dechorionation, and development. Cold Spring Harb Protoc. 2009:pdb–rot5344. [DOI] [PubMed] [Google Scholar]
  6. Corbo JC, Levine M, Zeller RW.. 1997. Characterization of a notochord-specific enhancer from the brachyury promoter region of the ascidian, Ciona intestinalis. Development. 124:589–602. [DOI] [PubMed] [Google Scholar]
  7. Coulcher, JF., Roure, A., Chowdhury, R., Robert, M., Lescat, L., Bouin, A., Carvajal Cadavid, J., Nishida, H.and Darras, S. (2020) Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos. Elife. 9, [[page]].e59157 33191918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dardaillon J, Dauga D, Simion P, Faure E, Onuma TA, DeBiasse MB, Louis A, Nitta KR, Naville M, Besnardeau Let al. 2020. Aniseed 2019: 4d exploration of genetic data for an extended range of tunicates. Nucleic Acids Res. 48:D668–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delsuc F, Brinkmann H, Chourrout D, Philippe H.. 2006. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature. 439:965–8. [DOI] [PubMed] [Google Scholar]
  10. Denker E, Jiang D. 2012. Ciona intestinalis notochord as a new model to investigate the cellular and molecular mechanisms of tubulogenesis. Semin Cell Dev Biol. 23:308–19. [DOI] [PubMed] [Google Scholar]
  11. Di Gregorio A, Corbo JC, Levine M.. 2001. The regulation of forkhead/hnf-3β expression in the ciona embryo. Dev Biol. 229:31–43. [DOI] [PubMed] [Google Scholar]
  12. Di Gregorio A. 2020. The notochord gene regulatory network in chordate evolution: conservation and divergence from Ciona to vertebrates. Curr Top Dev Biol. 139: 325–74. [DOI] [PubMed] [Google Scholar]
  13. Fodor ACA, Liu J, Turner L, Swalla BJ.. 2021a. Transitional chordates and vertebrate origins: tunicates. Curr Top Dev Biol. 139:325–74. [DOI] [PubMed] [Google Scholar]
  14. Fodor ACA, Powers M, Andrykovich K, Liu J, Lowe EK, Brown CT, Stolfi A, Swalla BJ.. 2021b. The degenerate taile of ascidian tails. Integr Comp Biol. 61:358–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gandhi S, Razy-Krajka F, Christiaen L, Stolfi A.. 2018. Crispr knockouts in Ciona embryos. In: Sasakura Y, editor. Transgenic Ascidians. Singapore: Springer. p.141–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gibboney S, Orvis J, Kim K, Johnson CJ, Martinez-Feduchi P, Lowe EK, Sharma S, Stolfi A.. 2020. Effector gene expression underlying neuron subtype-specific traits in the motor ganglion of ciona. Dev Biol. 458:52–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud J-B, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J.. 2016. Evaluation of off-target and on-target scoring algorithms and integration into the guide rna selection tool crispor. Genome Biol. 17:1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huber JL, da Silva KB, Bates WR, Swalla BJ.. 2000. The evolution of anural larvae in molgulid ascidians. Semin Cell Dev Biol. 11:419–26. [DOI] [PubMed] [Google Scholar]
  19. Jiang D, Smith WC. 2007. Ascidian notochord morphogenesis. Dev Dyn. 236:1748–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karaiskou A, Swalla BJ, Sasakura Y, Chambon JP.. 2015. Metamorphosis in solitary ascidians. Genesis. 53:34–47. [DOI] [PubMed] [Google Scholar]
  21. Katikala L, Aihara H, Passamaneck YJ, Gazdoiu S, José-Edwards DS, Kugler JE, Oda-Ishii I, Imai JH, Nibu Y, Di Gregorio A.. 2013. Functional brachyury binding sites establish a temporal read-out of gene expression in the Ciona notochord. PLoS Biol. 11:e1001697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Katoh K, Rozewicki J, Yamada KD.. 2019. Mafft online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings Bioinf. 20:1160–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kobayashi K, Yamada L, Satou Y, Satoh N.. 2013. Differential gene expression in notochord and nerve cord fate segregation in the Ciona intestinalis embryo. Genesis. 51:647–59. [DOI] [PubMed] [Google Scholar]
  24. Kugler JE, Gazdoiu S, Oda-Ishii I, Passamaneck YJ, Erives AJ, Di Gregorio A.. 2010. Temporal regulation of the muscle gene cascade by macho1 and tbx6 transcription factors in Ciona intestinalis. J Cell Sci. 123:2453–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kugler JE, Kerner P, Bouquet J-M, Jiang D, Di Gregorio A.. 2011. Evolutionary changes in the notochord genetic toolkit: a comparative analysis of notochord genes in the ascidian Ciona and the larvacean Oikopleura. BMC Evol Biol. 11:1–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kugler JE, Passamaneck YJ, Feldman TG, Beh J, Regnier TW, Di Gregorio A.. 2008. Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis. Genesis. 46:697–710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lacaze-Duthiers H. 1874. Histoire des ascidies simples des cotes de France: 2: etudes des especes. 3, Arch Zool Exp Gen, 119–656. [Google Scholar]
  28. Lemaire P. 2009. Unfolding a chordate developmental program, one cell at a time: invariant cell lineages, short-range inductions and evolutionary plasticity in ascidians. Dev Biol. 332:48–60. [DOI] [PubMed] [Google Scholar]
  29. Lemaire P. 2011. Evolutionary crossroads in developmental biology: the tunicates. Development. 138:2143–52. [DOI] [PubMed] [Google Scholar]
  30. Lowe EK, Racioppi C, Peyriéras N, Ristoratore F, Christiaen L, Swalla BJ, Stolfi A.. 2021. A cis-regulatory change underlying the motor neuron-specific loss of Ebf expression in immotile tunicate larvae. Evol Dev. 23:72–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lowe EK, Stolfi A.. 2018. Developmental system drift in motor ganglion patterning between distantly related tunicates. EvoDevo. 9:18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lowe EK, Swalla BJ, Brown CT.. 2014. Evaluating a lightweight transcriptome assembly pipeline on two closely related ascidian species. PeerJ PrePrints. 2: e505v1. doi:10.7287/peerj.preprints.505v1. [Google Scholar]
  33. Maliska ME, Pennell MW, Swalla BJ.. 2013. Developmental mode influences diversification in ascidians. Biol Lett. 9:20130068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miller EJ, Mathews MB. 1974. Characterization of notochord collagen as a cartilage-type collagen. Biochem Biophys Res Commun. 60:424–30. [DOI] [PubMed] [Google Scholar]
  35. Miyamoto DM. 1985. Formation of the notochord in living ascidian embryos. [PubMed]
  36. Nishino A. 2018. Morphology and physiology of the ascidian nervous systems and the effectors. Transgenic ascidians. Springer: Singapore, p.179–96. [DOI] [PubMed] [Google Scholar]
  37. Peck SH, McKee KK, Tobias JW, Malhotra NR, Harfe BD, Smith LJ.. 2017. Whole transcriptome analysis of notochord-derived cells during embryonic formation of the nucleus pulposus. Sci Rep. 7:1–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Racioppi C, Valoroso MC, Coppola U, Lowe EK, Brown CT, Swalla BJ, Christiaen L, Stolfi A, Ristoratore F.. 2017. Evolutionary loss of melanogenesis in the tunicate molgula occulta. EvoDevo. 8:11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rychel AL, Swalla BJ.. 2007. Development and evolution of chordate cartilage. J Exp Zool. 308:325–35. [DOI] [PubMed] [Google Scholar]
  40. Satoh N. 2013. Developmental genomics of ascidians. Hoboken, NJ: John Wiley & Sons. [Google Scholar]
  41. Song M, Yuan X, Racioppi C, Leslie M, Stutt N, Aleksandrova A, Christiaen L, Wilson MD, Scott IC.. 2022. Gata4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate. Sci Adv. 8:eabg0834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stemple DL. 2005. Structure and function of the notochord: an essential organ for chordate development. [DOI] [PubMed]
  43. Stolfi A, Gandhi S, Salek F, Christiaen L.. 2014a. Tissue-specific genome editing in Ciona embryos by crispr/cas9. Development. 141:4115–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stolfi A, Levine M.. 2011. Neuronal subtype specification in the spinal cord of a protovertebrate. Development. 138:995–1004. [DOI] [PubMed] [Google Scholar]
  45. Stolfi A, Lowe EK, Racioppi C, Ristoratore F, Brown CT, Swalla BJ, Christiaen L.. 2014b. Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. Elife. 3: e03728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Su M-W, Suzuki HR, Bieker JJ, Solursh M, Ramirez F.. 1991. Expression of two nonallelic type ii procollagen genes during xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts. J Cell Biol. 115:565–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Swalla BJ, Jeffery WR.. 1990. Interspecific hybridization between an anural and urodele ascidian: differential expression of urodele features suggests multiple mechanisms control anural development. Dev Biol. 142:319–34. [DOI] [PubMed] [Google Scholar]
  48. van Daalen KR, Romanello M, Rocklöv J, Semenza JC, Tonne C, Markandya A, Dasandi N, Jankin S, Achebak H, Ballester J. 2022. The 2022 europe report of the lancet countdown on health and climate change: towards a climate resilient future. Lancet Pub Health. 7:e942–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yan YL, Hatta K, Riggleman B, Postlethwait JH.. 1995. Expression of a type ii collagen gene in the zebrafish embryonic axis. Dev Dyn. 203:363–76. [DOI] [PubMed] [Google Scholar]
  50. Yasuoka Y. 2020. Morphogenetic mechanisms forming the notochord rod: the turgor pressure-sheath strength model. Dev Growth Differ. 62:379–90. [DOI] [PubMed] [Google Scholar]
  51. Zhang G, Cohn MJ.. 2006. Hagfish and lancelet fibrillar collagens reveal that type ii collagen-based cartilage evolved in stem vertebrates. Proc Natl Acad Sci USA. 103:16829–33. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

icad071_Supplemental_Files
Click here for additional data file. (1MB, zip)

Data Availability Statement

The data underlying this article will be shared upon request to the corresponding authors.

Articles from Integrative and Comparative Biology are provided here courtesy of Oxford University Press

RESOURCES