Natural reversal of cavefish heart asymmetry is controlled by Sonic Hedgehog effects on the left-right organizer.

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's Vesicle, the teleost left-right organizer, is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.

Differentially expressed chaperone genes reveal a stress response required for unidirectional regeneration in the basal chordate Ciona.

BACKGROUND: Unidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments. RESULTS: We identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration. CONCLUSIONS: The chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates.

Progenitor targeting by adult stem cells in Ciona homeostasis, injury, and regeneration.

In the ascidian Ciona intestinalis, oral siphon amputation activates adult stem cell niches in the branchial sac to divide and dispatch migratory progenitor cells to a regeneration blastema at the site of injury. This study shows that progenitor cells derived from branchial sac stem cell niches have roles in homeostasis, wound repair, and regeneration of the siphons and neural complex (NC). During homeostasis, progenitor cells targeted the pharyngeal stigmata to replace ciliated cells involved in filter feeding. After individual or double siphon amputations, progenitor cells specifically targeted the oral or atrial siphons or both siphons, and were involved in the replacement of siphon circular muscle fibers. After oral siphon wounding, progenitor cells targeted the wound sites, and in some cases a supernumerary siphon was formed, although progenitor cell targeting did not predict the induction of supernumerary siphons. Following NC ablation, progenitor cells specifically targeted the regenerating NC, and supplied the precursors of new brain and neural gland cells. The tissues and organs targeted by branchial sac stem cells exhibited apoptosis during homeostasis and injury. It is concluded that branchial sac progenitor cells are multipotent and show targeting specificity that is correlated with apoptosis during homeostatic growth, tissue repair, and regeneration.

Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration.

Astyanax mexicanus consists of two forms, a sighted surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Embryonic eyes are initially formed in cavefish but they are subsequently arrested in growth and degenerate during larval development. Previous lens transplantation studies have shown that the lens plays a central role in cavefish eye loss. However, several lines of evidence suggest that additional factors, such as the retinal pigment epithelium (RPE), which is morphologically altered in cavefish, could also be involved in the eye regression process. To explore the role of the RPE in cavefish eye degeneration, we generated an albino eyed (AE) strain by artificial selection for hybrid individuals with large eyes and a depigmented RPE. The AE strain exhibited an RPE lacking pigment granules and showed reduced expression of the RPE specific enzyme retinol isomerase, allowing eye development to be studied by lens ablation in an RPE background resembling cavefish. We found that lens ablation in the AE strain had stronger negative effects on eye growth than in surface fish, suggesting that an intact RPE is required for normal eye development. We also found that the AE strain develops a cartilaginous sclera lacking boney ossicles, a trait similar to cavefish. Extrapolation of the results to cavefish suggests that the RPE and lens have dual roles in eye degeneration, and that deficiencies in the RPE may be associated with evolutionary changes in scleral ossification.

Maternal genetic effects in Astyanax cavefish development.

The role of maternal factors in the evolution of development is poorly understood. Here we describe the use of reciprocal hybridization between the surface dwelling (surface fish, SF) and cave dwelling (cavefish, CF) morphs of the teleost Astyanax mexicanus to investigate the roles of maternal genetic effects in cavefish development. Reciprocal hybridization, a procedure in which F1 hybrids are generated by fertilizing SF eggs with CF sperm (SF × CF hybrids) and CF eggs with SF sperm (CF × SF hybrids), revealed that the CF degenerative eye phenotype showed maternal genetic effects. The eyes of CF × SF hybrids resembled the degenerate eyes of CF in showing ventral reduction of the retina and corresponding displacement of the lens within the optic cup, a smaller lens and eyeball, more lens apoptosis, a smaller cartilaginous sclera, and lens-specific gene expression characteristics compared to SF × CF hybrids, which showed eye and lens gene expression phenotypes resembling SF. In contrast, reciprocal hybridization failed to support roles for maternal genetic effects in the CF regressive pigmentation phenotype or in CF constructive changes related to enhanced jaw development. Maternal transcripts encoded by the pou2f1b, runx2b, and axin1 genes, which are involved in determining ventral embryonic fates, were increased in unfertilized CF eggs. In contrast, maternal mRNAs encoded by the ß-catenin and syntabulin genes, which control dorsal embryonic fates, showed similar expression levels in unfertilized SF and CF eggs. Furthermore, maternal transcripts of a sonic hedgehog gene were detected in SF and CF eggs and early cleaving embryos. This study reveals that CF eye degeneration is controlled by changes in maternal factors produced during oogenesis and introduces A. mexicanus as a model system for studying the role of maternal changes in the evolution of development.

Behavioural changes controlled by catecholaminergic systems explain recurrent loss of pigmentation in cavefish.

Multiple cave populations of the teleost Astyanax mexicanus have repeatedly reduced or lost eye and body pigmentation during adaptation to dark caves. Albinism, the complete absence of melanin pigmentation, is controlled by loss-of-function mutations in the oca2 gene. The mutation is accompanied by an increase in the melanin synthesis precursor l-tyrosine, which is also a precursor for catecholamine synthesis. In this study, we show a relationship between pigmentation loss, enhanced catecholamine synthesis and responsiveness to anaesthesia, determined as a proxy for catecholamine-related behaviours. We demonstrate that anaesthesia resistance (AR) is enhanced in multiple depigmented and albino cavefish (CF), inversely proportional to the degree of pigmentation loss, controlled by the oca2 gene, and can be modulated by experimental manipulations of l-tyrosine or the catecholamine norepinephrine (NE). Moreover, NE is increased in the brains of multiple albino and depigmented CF relative to surface fish. The results provide new insights into the evolution of pigment modification because NE controls a suite of adaptive behaviours similar to AR that may represent a target of natural selection. Thus, understanding the relationship between loss of pigmentation and AR may provide insight into the role of natural selection in the evolution of albinism via a melanin-catecholamine trade-off.

The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus.

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution.

Regressive evolution in Astyanax cavefish.

A diverse group of animals, including members of most major phyla, have adapted to life in the perpetual darkness of caves. These animals are united by the convergence of two regressive phenotypes, loss of eyes and pigmentation. The mechanisms of regressive evolution are poorly understood. The teleost Astyanax mexicanus is of special significance in studies of regressive evolution in cave animals. This species includes an ancestral surface dwelling form and many con-specific cave-dwelling forms, some of which have evolved their recessive phenotypes independently. Recent advances in Astyanax development and genetics have provided new information about how eyes and pigment are lost during cavefish evolution; namely, they have revealed some of the molecular and cellular mechanisms involved in trait modification, the number and identity of the underlying genes and mutations, the molecular basis of parallel evolution, and the evolutionary forces driving adaptation to the cave environment.

Evolution of the chordate regeneration blastema: Differential gene expression and conserved role of notch signaling during siphon regeneration in the ascidian Ciona.

The regeneration of the oral siphon (OS) and other distal structures in the ascidian Ciona intestinalis occurs by epimorphosis involving the formation of a blastema of proliferating cells. Despite the longstanding use of Ciona as a model in molecular developmental biology, regeneration in this system has not been previously explored by molecular analysis. Here we have employed microarray analysis and quantitative real time RT-PCR to identify genes with differential expression profiles during OS regeneration. The majority of differentially expressed genes were downregulated during OS regeneration, suggesting roles in normal growth and homeostasis. However, a subset of differentially expressed genes was upregulated in the regenerating OS, suggesting functional roles during regeneration. Among the upregulated genes were key members of the Notch signaling pathway, including those encoding the delta and jagged ligands, two fringe modulators, and to a lesser extent the notch receptor. In situ hybridization showed a complementary pattern of delta1 and notch gene expression in the blastema of the regenerating OS. Chemical inhibition of the Notch signaling pathway reduced the levels of cell proliferation in the branchial sac, a stem cell niche that contributes progenitor cells to the regenerating OS, and in the OS regeneration blastema, where siphon muscle fibers eventually re-differentiate. Chemical inhibition also prevented the replacement of oral siphon pigment organs, sensory receptors rimming the entrance of the OS, and siphon muscle fibers, but had no effects on the formation of the wound epidermis. Since Notch signaling is involved in the maintenance of proliferative activity in both the Ciona and vertebrate regeneration blastema, the results suggest a conserved evolutionary role of this signaling pathway in chordate regeneration. The genes identified in this investigation provide the foundation for future molecular analysis of OS regeneration.

Convergence in feeding posture occurs through different genetic loci in independently evolved cave populations of Astyanax mexicanus.

When an organism colonizes a new environment, it needs to adapt both morphologically and behaviorally to survive and thrive. Although recent progress has been made in understanding the genetic architecture underlying morphological evolution, behavioral evolution is poorly understood. Here, we use the Mexican cavefish, Astyanax mexicanus, to study the genetic basis for convergent evolution of feeding posture. When river-dwelling surface fish became entrapped in the caves, they were confronted with dramatic changes in the availability and type of food source and in their ability to perceive it. In this setting, multiple independent populations of cavefish exhibit an altered feeding posture compared with their ancestral surface forms. We determined that this behavioral change in feeding posture is not due to changes in cranial facial morphology, body depth, or to take advantage of the expansion in the number of taste buds. Quantitative genetic analysis demonstrates that two different cave populations have evolved similar feeding postures through a small number of genetic changes, some of which appear to be distinct. This work indicates that independently evolved populations of cavefish can evolve the same behavioral traits to adapt to similar environmental challenges by modifying different sets of genes.

Distinct genetic architecture underlies the emergence of sleep loss and prey-seeking behavior in the Mexican cavefish.

BACKGROUND: Sleep is characterized by extended periods of quiescence and reduced responsiveness to sensory stimuli. Animals ranging from insects to mammals adapt to environments with limited food by suppressing sleep and enhancing their response to food cues, yet little is known about the genetic and evolutionary relationship between these processes. The blind Mexican cavefish, Astyanax mexicanus is a powerful model for elucidating the genetic mechanisms underlying behavioral evolution. A. mexicanus comprises an extant ancestral-type surface dwelling morph and at least five independently evolved cave populations. Evolutionary convergence on sleep loss and vibration attraction behavior, which is involved in prey seeking, have been documented in cavefish raising the possibility that enhanced sensory responsiveness underlies changes in sleep. RESULTS: We established a system to study sleep and vibration attraction behavior in adult A. mexicanus and used high coverage quantitative trait loci (QTL) mapping to investigate the functional and evolutionary relationship between these traits. Analysis of surface-cave F2 hybrid fish and an outbred cave population indicates that independent genetic factors underlie changes in sleep/locomotor activity and vibration attraction behavior. High-coverage QTL mapping with genotyping-by-sequencing technology identify two novel QTL intervals that associate with locomotor activity and include the narcolepsy-associated tp53 regulating kinase. These QTLs represent the first genomic localization of locomotor activity in cavefish and are distinct from two QTLs previously identified as associating with vibration attraction behavior. CONCLUSIONS: Taken together, these results localize genomic regions underlying sleep/locomotor and sensory changes in cavefish populations and provide evidence that sleep loss evolved independently from enhanced sensory responsiveness.

Closing the wounds: one hundred and twenty five years of regenerative biology in the ascidian Ciona intestinalis.

This year marks the 125th anniversary of the beginning of regeneration research in the ascidian Ciona intestinalis. A brief note was published in 1891, reporting the regeneration of the Ciona neural complex and siphons. This launched an active period of Ciona regeneration research culminating in the demonstration of partial body regeneration: the ability of proximal body parts to regenerate distal ones, but not vice versa. In a process resembling regeneration, wounds in the siphon tube were discovered to result in the formation of an ectopic siphon. Ciona regeneration research then lapsed into a period of relative inactivity after the purported demonstration of the inheritance of acquired characters using siphon regeneration as a model. Around the turn of the present century, Ciona regeneration research experienced a new blossoming. The current studies established the morphological and physiological integrity of the regeneration process and its resemblance to ontogeny. They also determined some of the cell types responsible for tissue and organ replacement and their sources in the body. Finally, they showed that regenerative capacity is reduced with age. Many other aspects of regeneration now can be studied at the mechanistic level because of the extensive molecular tools available in Ciona.

Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism.

The genetic basis of vertebrate morphological evolution has traditionally been very difficult to examine in naturally occurring populations. Here we describe the generation of a genome-wide linkage map to allow quantitative trait analysis of evolutionarily derived morphologies in the Mexican cave tetra, a species that has, in a series of independent caves, repeatedly evolved specialized characteristics adapted to a unique and well-studied ecological environment. We focused on the trait of albinism and discovered that it is linked to Oca2, a known pigmentation gene, in two cave populations. We found different deletions in Oca2 in each population and, using a cell-based assay, showed that both cause loss of function of the corresponding protein, OCA2. Thus, the two cave populations evolved albinism independently, through similar mutational events.

The Tunicate CIONA: A Model System for Understanding the Relationship Between Regeneration and Aging.

The use of the tunicate Ciona intestinalis as a model system to study the relationship between regeneration and aging is reviewed. Ciona has powerful regeneration capacities, which fade with age. Some additional benefits are-, a relatively short life span, the ability to study regeneration in vitro, the close phylogenetic relationship between tunicates and vertebrates, and the host of molecular tools already established in this system. The neural complex (NC), the oral siphon (OS), and the oral siphon pigment organs (OPO) have high capacities for regeneration. However, these organs show an inverse relationship between rate of regeneration and age. The ability to regenerate a complete OS disappears in the oldest animals of a natural population, probably due to the inability to form a blastema at the wound site. Effects on blastema formation could also be involved in the reduction of NC regeneration capacity. The fidelity of OPO restoration is also compromised by excess differentiation of precursor cells in local siphon niches in the oldest animals. The Ciona model provides a pathway to understand the molecular basis of these phenomena.

The sensitivity of lateral line receptors and their role in the behavior of Mexican blind cavefish (Astyanax mexicanus).

The characid fish species Astyanax mexicanus offers a classic comparative model for the evolution of sensory systems. Populations of this species evolved in caves and became blind while others remained in streams (i.e. surface fish) and retained a functional visual system. The flow-sensitive lateral line receptors, called superficial neuromasts, are more numerous in cavefish than in surface fish, but it is unclear whether individual neuromasts differ in sensitivity between these populations. The aims of this study were to determine whether the neuromasts in cavefish impart enhanced sensitivity relative to surface fish and to test whether this aids their ability to sense flow in the absence of visual input. Sensitivity was assessed by modeling the mechanics and hydrodynamics of a flow stimulus. This model required that we measure the dimensions of the transparent cupula of a neuromast, which was visualized with fluorescent microspheres. We found that neuromasts within the eye orbit and in the suborbital region were larger and consequently about twice as sensitive in small adult cavefish as in surface fish. Behavioral experiments found that these cavefish, but not surface fish, were attracted to a 35 Hz flow stimulus. These results support the hypothesis that the large superficial neuromasts of small cavefish aid in flow sensing. We conclude that the morphology of the lateral line could have evolved in cavefish to permit foraging in a cave environment.

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish: response to Borowsky (2013).

Vibration attraction behavior (VAB) is the swimming of fish toward an oscillating object, a behavior that is likely adaptive because it increases foraging efficiency in darkness. VAB is seen in a small proportion of Astyanax surface-dwelling populations (surface fish) but is pronounced in cave-dwelling populations (cavefish). In a recent study, we identified two quantitative trait loci for VAB on Astyanax linkage groups 2 and 17. We also demonstrated that a small population of superficial neuromast sensors located within the eye orbit (EO SN) facilitate VAB, and two quantitative trait loci (QTL) were identified for EO SN that were congruent with those for VAB. Finally, we showed that both VAB and EO SN are negatively correlated with eye size, and that two (of several) QTL for eye size overlap VAB and EO SN QTLs. From these results, we concluded that the adaptive evolution of VAB and EO SN has contributed to the indirect loss of eyes in cavefish, either as a result of pleiotropy or tight physical linkage of the mutations underlying these traits. In a subsequent commentary, Borowsky argues that there is poor experimental support for our conclusions. Specifically, Borowsky states that: (1) linkage groups (LGs) 2 and 17 harbor QTL for many traits and, therefore, no evidence exists for an exclusive interaction among the overlapping VAB, EO SN and eye size QTL; (2) some of the QTL we identified are too broad (>20 cM) to support the hypothesis of correlated evolution due to pleiotropy or hitchhiking; and (3) VAB is unnecessary to explain the indirect evolution of eye-loss since the negative polarity of numerous eye QTL is consistent with direct selection against eyes. Borowsky further argues that (4) it is difficult to envision an evolutionary scenario whereby VAB and EO SN drive eye loss, since the eyes must first be reduced in order to increase the number of EO SN and, therefore, VAB. In this response, we explain why the evidence of one trait influencing eye reduction is stronger for VAB than other traits, and provide further support for a scenario whereby elaboration of VAB in surface fish may precede complete eye-loss.

Hypoxia-Sonic Hedgehog Axis as a Driver of Primitive Hematopoiesis Development and Evolution in Cavefish.

The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and show constructive and regressive phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by treatment with the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and the Shh signaling system. Lastly, we demonstrate that hypoxia treatment enhances expression of Shh signaling along the midline of surface fish embryos. Thus, we conclude that a hypoxia-Shh axis may drive the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish. Highlights: Hypoxia increases hematopoiesis and erythrocytes in surface fishShh upregulation increases hematopoiesis and erythrocytes in surface fishShh inhibition decreases hematopoiesis and erythrocytes in cavefishHypoxia upregulates Shh along the embryonic midline in surface fish.

Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish.

BACKGROUND: How and why animals lose eyesight during adaptation to the dark and food-limited cave environment has puzzled biologists since the time of Darwin. More recently, several different adaptive hypotheses have been proposed to explain eye degeneration based on studies in the teleost Astyanax mexicanus, which consists of blind cave-dwelling (cavefish) and sighted surface-dwelling (surface fish) forms. One of these hypotheses is that eye regression is the result of indirect selection for constructive characters that are negatively linked to eye development through the pleiotropic effects of Sonic Hedgehog (SHH) signaling. However, subsequent genetic analyses suggested that other mechanisms also contribute to eye regression in Astyanax cavefish. Here, we introduce a new approach to this problem by investigating the phenotypic and genetic relationships between a suite of non-visual constructive traits and eye regression. RESULTS: Using quantitative genetic analysis of crosses between surface fish, the Pachón cavefish population and their hybrid progeny, we show that the adaptive vibration attraction behavior (VAB) and its sensory receptors, superficial neuromasts (SN) specifically found within the cavefish eye orbit (EO), are genetically correlated with reduced eye size. The quantitative trait loci (QTL) for these three traits form two clusters of congruent or overlapping QTL on Astyanax linkage groups (LG) 2 and 17, but not at the shh locus on LG 13. Ablation of EO SN in cavefish demonstrated a major role for these sensory receptors in VAB expression. Furthermore, experimental induction of eye regression in surface fish via shh overexpression showed that the absence of eyes was insufficient to promote the appearance of VAB or EO SN. CONCLUSIONS: We conclude that natural selection for the enhancement of VAB and EO SN indirectly promotes eye regression in the Pachón cavefish population through an antagonistic relationship involving genetic linkage or pleiotropy among the genetic factors underlying these traits. This study demonstrates a trade-off between the evolution of a non-visual sensory system and eye regression during the adaptive evolution of Astyanax to the cave environment.

Cymric, a Maternal and Zygotic HTK-16-Like SHARK Family Tyrosine Kinase Gene, Is Disrupted in Molgula occulta, a Tailless Ascidian.

AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, cymric (Uro-1), a HTK-16-like (HydraTyrosineKinase-16) gene, identified in a subtractive screen for maternal ascidian cDNAs in Molgula oculata, an ascidian species with a tadpole larva. The cymric gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed M. oculata but is not detected in eggs or embryos of the closely related tailless species Molgula occulta. There is a large insertion in cymric in the M. occulta genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The cymric amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the HTK-16-like gene found in other ascidians. Thus, the ascidian cymric genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that cymric is lacking the tyrosine kinase domain in the tailless M. occulta, although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic HTK-16-like tyrosine kinase is another described pseudogene from M. occulta and appears not to be necessary for adult development.