Distinct stem-like cell populations facilitate functional regeneration of the Cladonema medusa tentacle.

Blastema formation is a crucial process that provides a cellular source for regenerating tissues and organs. While bilaterians have diversified blastema formation methods, its mechanisms in non-bilaterians remain poorly understood. Cnidarian jellyfish, or medusae, represent early-branching metazoans that exhibit complex morphology and possess defined appendage structures highlighted by tentacles with stinging cells (nematocytes). Here, we investigate the mechanisms of tentacle regeneration, using the hydrozoan jellyfish Cladonema pacificum. We show that proliferative cells accumulate at the tentacle amputation site and form a blastema composed of cells with stem cell morphology. Nucleoside pulse-chase experiments indicate that most repair-specific proliferative cells (RSPCs) in the blastema are distinct from resident stem cells. We further demonstrate that resident stem cells control nematogenesis and tentacle elongation during both homeostasis and regeneration as homeostatic stem cells, while RSPCs preferentially differentiate into epithelial cells in the newly formed tentacle, analogous to lineage-restricted stem/progenitor cells observed in salamander limbs. Taken together, our findings propose a regeneration mechanism that utilizes both resident homeostatic stem cells (RHSCs) and RSPCs, which in conjunction efficiently enable functional appendage regeneration, and provide novel insight into the diversification of blastema formation across animal evolution.

Repetitive accumulation of interstitial cells generates the branched structure of Cladonema medusa tentacles.

The shaping of tissues and organs in many animals relies on interactions between the epithelial cell layer and its underlying mesoderm-derived tissues. Inductive signals, such as receptor tyrosine kinase (RTK) signaling emanating from mesoderm, act on cells of the epithelium to initiate three-dimensional changes. However, how tissues are shaped in a diploblastic animal with no mesoderm remains largely unknown. In this study, the jellyfish Cladonema pacificum was used to investigate branch formation. The tentacles on its medusa stage undergo branching, which increases the epithelial surface area available for carrying nematocytes, thereby maximizing prey capture. Pharmacological and cellular analyses of the branching process suggest a two-step model for tentacle branch formation, in which mitogen-activated protein kinase kinase signaling accumulates interstitial cells in the future branch-forming region, and fibroblast growth factor signaling regulates branch elongation. This study highlights an essential role for these pluripotent stem cells in the tissue-shaping morphogenesis of a diploblastic animal. In addition, it identifies a mechanism involving RTK signaling and cell proliferative activity at the branch tip for branching morphogenesis that is apparently conserved across the animal kingdom.

Expression and Functional Analyses of Ectodermal Transcription Factors FoxJ-r, SoxF, and SP8/9 in Early Embryos of the Ascidian Halocynthia roretzi.

The spatiotemporal expression of zygotic genes is regulated by transcription factors, which mediate cell fate decision and morphogenesis. Investigation of the expression patterns and their transcriptional regulatory relationships is crucial to understand embryonic development. Staged RNA-seq of the ascidian Halocynthia roretzi has previously shown that nine genes encoding transcription factors are transiently expressed at the blastula stage, which is the stage at which cell fates are specified and differentiation starts. Six of these transcription factors have already been found to play important roles during early development. However, the functions of the other transcription factors (FoxJ-r, SoxF, and SP8/9) remain unknown. The study of the spatial and temporal expression patterns showed that all three genes were expressed in the animal hemisphere as early as the 16-cell stage. This is likely due to transcription factor genes that are expressed in the vegetal hemisphere, which have been extensively and comprehensively analyzed in previous studies of ascidians. Functional analyses using FoxJ-r morphants showed that they resulted in the disruption of laterality and the absence of epidermal mono-cilia, suggesting FoxJ-r functions in cilia formation and, consequently, in the generation of left-right asymmetry, as observed in vertebrates. SoxF knockdown resulted in incomplete epiboly by the ectoderm during gastrulation, while SP8/9 knockdown showed no phenotype until the tailbud stage in the present study, although it was expressed during blastula stages. Our results indicate that transcription factor genes expressed at the cleavage stages play roles in diverse functions, and are not limited to cell fate specification.

Dynein-Mediated Regional Cell Division Reorientation Shapes a Tailbud Embryo.

Regulation of cell division orientation controls the spatial distribution of cells during development and is essential for one-directional tissue transformation, such as elongation. However, little is known about whether it plays a role in other types of tissue morphogenesis. Using an ascidian Halocynthia roretzi, we found that differently oriented cell divisions in the epidermis of the future trunk (anterior) and tail (posterior) regions create an hourglass-like epithelial bending between the two regions to shape the tailbud embryo. Our results show that posterior epidermal cells are polarized with dynein protein anteriorly localized, undergo dynein-dependent spindle rotation, and divide along the anteroposterior axis. This cell division facilitates constriction around the embryo's circumference only in the posterior region and epithelial bending formation. Our findings, therefore, provide an important insight into the role of oriented cell division in tissue morphogenesis.

H3K27me3 suppresses sister-lineage somatic gene expression in late embryonic germline cells of the ascidian, Halocynthia roretzi.

Protection of the germline from somatic differentiation programs is crucial for germ cell development. In many animals, whose germline development relies on the maternally inherited germ plasm, such protection in particular at early stages of embryogenesis is achieved by maternally localized global transcriptional repressors, such as PIE-1 of Caenorhabditis elegans, Pgc of Drosophila melanogaster and Pem of ascidians. However, zygotic gene expression starts in later germline cells eventually and mechanisms by which somatic gene expression is selectively kept under repression in the transcriptionally active cells are poorly understood. By using the ascidian species Halocynthia roretzi, we found that H3K27me3, a repressive transcription-related chromatin mark, became enriched in germline cells starting at the 64-cell stage when Pem protein level and its contribution to transcriptional repression decrease. Interestingly, inhibition of H3K27me3 together with Pem knockdown resulted in ectopic expression in germline cells of muscle developmental genes Muscle actin (MA4) and Snail, and of Clone 22 (which is expressed in all somatic but not germline cells), but not of other tissue-specific genes such as the notochord gene Brachyury, the nerve cord marker ETR-1 and a heart precursor gene Mesp, at the 110-cell stage. Importantly, these ectopically expressed genes are normally expressed in the germline sister cells (B7.5), the last somatic lineage separated from the germline. Also, the ectopic expression of MA4 was dependent on a maternally localized muscle determinant Macho-1. Taken together, we propose that H3K27me3 may be responsible for selective transcriptional repression for somatic genes in later germline cells in Halocynthia embryos and that the preferential repression of germline sister-lineage genes may be related to the mechanism of germline segregation in ascidian embryos, where the germline is segregated progressively by successive asymmetric cell divisions during cell cleavage stages. Together with findings from C. elegans and D. melanogaster, our data for this urochordate animal support the proposal for a mechanism, conserved widely throughout the animal kingdom, where germline transcriptional repression is mediated initially by maternally localized factors and subsequently by a chromatin-based mechanism.

Branching pattern and morphogenesis of medusa tentacles in the jellyfish Cladonema pacificum (Hydrozoa, Cnidaria).

BACKGROUND: Branched structures are found in many natural settings, and the molecular and cellular mechanisms underlying their formation in animal development have extensively studied in recent years. Despite their importance and the accumulated knowledge from studies on several organs of Drosophila and mammals, much remains unknown about branching mechanisms in other animal species. We chose to study the jellyfish species Cladonema pacificum. Unlike many other jellyfish, this species has branched medusa tentacles, and its basal phylogenetic position in animal evolution makes it an ideal organism for studying and understanding branching morphogenesis more broadly. Branched tentacles are unique compared to other well-studied branched structures in that they have two functionally distinct identities: one with adhesive organs for attaching to a substratum, and another with nematocyst clusters for capturing prey. RESULTS: We began our analyses on C. pacificum tentacles by observing their branching during growth. We found that tentacle branches form through repeated addition of new branches to the proximal region of the main tentacle while it is elongating. At the site of branch bud formation, we observed apical thickening of the epidermal epithelial layer, possibly caused by extension of the epithelial cells along the apico-basal axis. Interestingly, tentacle branch formation required receptor tyrosine kinase signaling, which is an essential factor for branching morphogenesis in Drosophila and mammals. We also found that new branches form adhesive organs first, and then are transformed into branches with nematocyst clusters as they develop. CONCLUSIONS: These results highlight unique features in branch generation in C. pacificum medusa tentacles and illuminate conserved and fundamental mechanisms by which branched structures are created across a variety of animal species.

Control of Pem protein level by localized maternal factors for transcriptional regulation in the germline of the ascidian, Halocynthia roretzi.

Localized maternal mRNAs play important roles in embryogenesis, e.g. the establishment of embryonic axes and the developmental cell fate specification, in various animal species. In ascidians, a group of maternal mRNAs, called postplasmic/PEM RNAs, is localized to a subcellular structure, called the Centrosome-Attracting Body (CAB), which contains the ascidian germ plasm, and is inherited by the germline cells during embryogenesis. Posterior end mark (Pem), a postplasmic/PEM RNAs member, represses somatic gene expression in the germline during cleavage stages by inhibition of RNA polymerase II activity. However, the functions of other postplasmic/ PEM RNAs members in germline formation are largely unknown. In this study, we analyzed the functions of two postplasmic/PEM RNAs, Popk-1 and Zf-1, in transcriptional regulation in the germline cells. We show that Popk-1 contributes to transcriptional quiescence by controlling the size of the CAB and amount of Pem protein translated at the CAB. Our studies also indicated that zygotic expression of a germline gene starts around the onset of gastrulation and that the decrease of Pem protein is necessary and sufficient for the zygotic germline gene expression. Finally, further studies showed that the decrease of the Pem protein level is facilitated by Zf-1. Taken together, we propose that postplasmic/PEM RNAs such as Popk-1 and Zf-1 control the protein level of the transcriptional repressor Pem and regulate its transcriptional state in the ascidian germline.

Microinjection of Exogenous DNA into Eggs of Halocynthia roretzi.

Exogenous gene expression assays during development, including reporters under the control of 5' upstream enhancer regions of genes, constitute a powerful technique for understanding the mechanisms of tissue-specific gene expression regulation and determining the characteristics, behaviors, and functions of cells that express these genes. The simple marine chordate Halocynthia roretzi has been used for these transgenic analyses for a long time and is an excellent model system for such studies, especially in comparative analyses with other ascidians. In this study, I describe simple methods for microinjecting H. roretzi eggs with exogenous DNA, such as a promoter construct consisting of a 5' upstream region and a reporter gene, which are prerequisites for transgenic analyses. I also describe basic knowledge regarding this ascidian species, providing reasons why it is an ideal subject for developmental biology studies.

Redundant mechanisms are involved in suppression of default cell fates during embryonic mesenchyme and notochord induction in ascidians.

During embryonic induction, the responding cells invoke an induced developmental program, whereas in the absence of an inducing signal, they assume a default uninduced cell fate. Suppression of the default fate during the inductive event is crucial for choice of the binary cell fate. In contrast to the mechanisms that promote an induced cell fate, those that suppress the default fate have been overlooked. Upon induction, intracellular signal transduction results in activation of genes encoding key transcription factors for induced tissue differentiation. It is elusive whether an induced key transcription factor has dual functions involving suppression of the default fates and promotion of the induced fate, or whether suppression of the default fate is independently regulated by other factors that are also downstream of the signaling cascade. We show that during ascidian embryonic induction, default fates were suppressed by multifold redundant mechanisms. The key transcription factor, Twist-related.a, which is required for mesenchyme differentiation, and another independent transcription factor, Lhx3, which is dispensable for mesenchyme differentiation, sequentially and redundantly suppress the default muscle fate in induced mesenchyme cells. Similarly in notochord induction, Brachyury, which is required for notochord differentiation, and other factors, Lhx3 and Mnx, are likely to suppress the default nerve cord fate redundantly. Lhx3 commonly suppresses the default fates in two kinds of induction. Mis-activation of the autonomously executed default program in induced cells is detrimental to choice of the binary cell fate. Multifold redundant mechanisms would be required for suppression of the default fate to be secure.

Evolution of germline segregation processes in animal development.

Germline segregation is a complex process by which germline cells are separated from somatic tissues during development. Recent animal studies on germline segregation allow for comparisons of the mechanisms used by different species and to propose evolutionary scenarios underlying the diversification of these processes. In this review, several proposed models of germline segregation are presented, and in addition, recent findings from the increasing number of studies are discussed, particularly concerning the need to reconsider these models based on available data.

Transcription factor Tbx6 plays a central role in fate determination between mesenchyme and muscle in embryos of the ascidian, Halocynthia roretzi.

Developmental fates are determined in relation to other fates in surrounding cells within the embryo, creating diversified tissue or cell types in the course of development. In ascidian embryos, maternally localized factors and inductive signals play essential and cooperative roles in fate determination at appropriate spatial and temporal positions during embryogenesis. Here, to clarify fate determination mechanisms of mesenchyme and muscle during mesenchyme induction by the fibroblast growth factor (FGF) signal in Halocynthia roretzi, we examined the function of transcription factor Tbx6, which acts as a pivotal mediator of the maternally localized muscle differentiation determinant Macho-1. Our results suggest that the level of Tbx6 expression increases in muscle lineage cells through positive feedback regulation that promotes muscle differentiation as well as mesenchymal fate suppression. In addition, the FGF signal inactivated Tbx6 transcriptional activity and positive feedback, leading to induction of the mesenchymal lineage. Taken together, our finding suggests that Tbx6 is an important factor for determining mesenchyme and muscle fates.

Regulation of the number of cell division rounds by tissue-specific transcription factors and Cdk inhibitor during ascidian embryogenesis.

Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a cyclin-dependent kinase inhibitor, CKI-b, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. CKI-b gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination.

Neurula rotation determines left-right asymmetry in ascidian tadpole larvae.

Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left-sided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anterior-posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species.

Polarizing animal cells via mRNA localization in oogenesis and early development.

The localization of mRNAs in developing animal cells is essential for establishing cellular polarity and setting up the body plan for subsequent development. Cellular and molecular mechanisms by which maternal mRNAs are localized during oogenesis have been extensively studied in Drosophila and Xenopus. In contrast, evidence for mechanisms used in the localization of mRNAs encoded by developmentally important genes has also been accumulating in several other organisms. This offers the opportunity to unravel the fundamental mechanisms of mRNA localization shared among many species, as well as unique mechanisms specifically acquired or retained by animals based on their developmental needs. In addition to maternal mRNAs, the localization of zygotically expressed mRNAs in the cells of cleaving embryos is also important for early development. In this review, mRNA localization dynamics in the oocytes/eggs of Drosophila and Xenopus are first summarized, and evidence for localized mRNAs in the oocytes/eggs and cleaving embryos of other organisms is then presented.

A maternal factor unique to ascidians silences the germline via binding to P-TEFb and RNAP II regulation.

Suppression of zygotic transcription in early embryonic germline cells is tightly linked to their separation from the somatic lineage. Many invertebrate embryos utilize localized maternal factors that are successively inherited by the germline cells for silencing the germline. Germline quiescence has also been associated with the underphosphorylation of Ser2 of the C-terminal domain (CTD-Ser2) of RNA polymerase II [1-3]. Here, using the ascidian Halocynthia roretzi, we identified a first deuterostome example of a maternally localized factor, posterior end mark (PEM), which globally represses germline transcription. PEM knockdown resulted in ectopic transcription and ectopic phosphorylation of CTD-Ser2 in the germline. Overexpression of PEM abolished all transcription and led to the underphosphorylation of CTD-Ser2 in the somatic cells. PEM protein was reiteratively detected in the nucleus of the germline cells and coimmunoprecipitated with CDK9, a component of posterior transcription elongation factor b (P-TEFb). These results suggest that nonhomologous proteins, PEM and Pgc of Drosophila [3-5] and PIE-1 of C. elegans [1, 6, 7], repress germline gene expression through analogous functions: by keeping CTD-Ser2 underphosphorylated through binding to the P-TEFb complex. The present study is an interesting example of evolutionary constraint on how a mechanism of germline silencing can evolve in diverse animals.

The transcription factor FoxB mediates temporal loss of cellular competence for notochord induction in ascidian embryos.

In embryos of the ascidian Halocynthia roretzi, the competence of isolated presumptive notochord blastomeres to respond to fibroblast growth factor (FGF) for induction of the primary notochord decays by 1 hour after cleavage from the 32- to 64-cell stage. This study analyzes the molecular mechanisms responsible for this loss of competence and provides evidence for a novel mechanism. A forkhead family transcription factor, FoxB, plays a role in competence decay by preventing the induction of notochord-specific Brachyury (Bra) gene expression by the FGF/MAPK signaling pathway. Unlike the mechanisms reported previously in other animals, no component in the FGF signal transduction cascade appeared to be lost or inactivated at the time of competence loss. Knockdown of FoxB functions allowed the isolated cells to retain their competence for a longer period, and to respond to FGF with expression of Bra beyond the stage at which competence was normally lost. FoxB acts as a transcription repressor by directly binding to the cis-regulatory element of the Bra gene. Our results suggest that FoxB prevents ectopic induction of the notochord fate within the cells that assume a default nerve cord fate, after the stage when notochord induction has been completed. The merit of this system is that embryos can use the same FGF signaling cascade again for another purpose in the same cell lineage at later stages by keeping the signaling cascade itself available. Temporally and spatially regulated FoxB expression in nerve cord cells was promoted by the ZicN transcription factor and absence of FGF/MAPK signaling.

Polo-like kinase 1 is required for localization of Posterior End Mark protein to the centrosome-attracting body and unequal cleavages in ascidian embryos.

In ascidian embryos, the posterior-localized maternal factor Posterior End Mark (PEM) is responsible for patterning embryos along the anterior-posterior axis with regard to both cleavage pattern involving unequal cell divisions and gene expression. Although PEM plays important roles in embryogenesis, its mechanism of action is still unclear because PEM has no known functional domain. In the present study, we explored the candidate of PEM partner proteins in Halocynthia roretzi using yeast two-hybrid screening. We isolated a homologue of Polo-like kinase 1 (Plk1), a key regulator of cell division and highly conserved in eukaryotes, as the first potential binding partner of PEM. We biochemically confirmed that interaction occurred between the Plk1 and PEM proteins. Immunostaining showed that Plk1 protein concentrates in the centrosome-attracting body (CAB) at the posterior pole, where PEM protein is also localized. The CAB is a subcellular structure that plays an important role in generating the posterior cleavage pattern. Plk1 localization to the CAB was dependent on the cell cycle phases during unequal cleavage. Inhibition of Plk1 with specific drugs resulted in failure of the nucleus to migrate towards the posterior pole and formation of a microtubule bundle between the CAB and a centrosome, similarly to inhibition of PEM function, suggesting that both proteins are involved in the same process of unequal cleavages. This interrupted nuclear migration was rescued by overexpression of PEM. In Plk1-inhibited embryos, the localization of PEM protein to the CAB was impaired, indicating that Plk1 is required for appropriate localization of PEM.

Segregation of germ layer fates by nuclear migration-dependent localization of Not mRNA.

An important step in early embryonic development is the allocation and segregation of germ layer fates into distinct embryonic regions. However, the mechanism that segregates the mesendoderm into mesoderm and endoderm fates remains largely unknown in most animals. Here, using ascidians, a primitive chordate, we show that these fates are segregated by partitioning of asymmetrically localized Not mRNA from the mesendoderm cell to its mesodermal daughter. Migration of the mesendoderm cell nucleus to the future mesoderm-forming region, release of Not mRNA from the nucleus, Wnt5α-dependent local retention of the mRNA, and subsequent repositioning of the mitotic spindle to the center of the cell are each required for the asymmetric localization and partitioning of Not mRNA. Our results show that nuclear migration plays an unexpected role in asymmetric cell divisions that segregate germ layer fates in chordate embryos.

Macho-1 regulates unequal cell divisions independently of its function as a muscle determinant.

The anterior-posterior (A-P) axis in ascidian embryos is established through the posteriorizing activities of a localized egg region known as the posterior vegetal cortex/cytoplasm (PVC). Here we describe a novel function of macho-1, a maternally-localized muscle determinant, in establishment of the A-P axis in the Halocynthia roretzi embryo. Macho-1, in addition to its known function in the formation of posterior tissue such as muscle and mesenchyme, and suppression of the anterior-derived notochord fate, acts independently of its transcriptional activity as a regulator of posterior-specific unequal cell divisions, in cooperation with beta-catenin. Our results suggest that macho-1 and beta-catenin regulate the formation of a microtubule bundle that shortens and pulls the centrosome toward a sub-cellular cortical structure known as centrosome-attracting body (CAB), which is located at the posterior pole of the embryo during unequal cell divisions, and act upstream of PEM, a recently-identified regulator of unequal cell divisions. We also present data that suggest that PEM localization to the CAB may not be required for unequal cleavage regulation. The present study provides an important and novel insight into the role of the zinc-finger-containing transcription factor and indicates that it constitutes a major part of the PVC activity.

Spatial and temporal expression of two transcriptional isoforms of Lhx3, a LIM class homeobox gene, during embryogenesis of two phylogenetically remote ascidians, Halocynthia roretzi and Ciona intestinalis.

Lhx3 genes are members of one of the six subfamilies of the LIM class homeobox genes. In ascidians, Lhx3 is known to play a critical role in endoderm differentiation, while in vertebrates Lhx3 is involved in the development of pituitary and subsets of motor neurons. It has been shown recently, using RT-PCR analysis, that two transcriptional isoforms a and b are differentially expressed during the larval development of Ciona intestinalis (Christiaen et al., 2009). The present study provides an in-depth description of Lhx3 gene expression during the development of the two remote ascidian species, C. intestinalis and Halocynthia roretzi; for this, 5'RACE and whole-mount in situ hybridization (WISH) were employed. In both species, maternal expression of Lhx3a, but not Lhx3b, is evident. In H. roretzi, the maternal Lhx3a transcripts have been detected by WISH in the animal half of early cleavage stage embryos. In both species, transcriptional isoform a is also zygotically expressed in the sensory vesicle and the visceral ganglion lineages from the neurula stage onward. By contrast, Lhx3b transcripts are expressed only zygotically and localized in the endoderm, notochord and mesenchyme lineages during cleavage stage. Lhx3a, but not Lhx3b, transcripts are subjected to trans-splicing. Additionally, in C. intestinalis, other variations in the 5' region have been identified among Lhx3a transcripts. Although some differences are present, over-all developmental expression of Lhx3 is rather well conserved between the two ascidian species, which is quite different from that of vertebrate counterparts.