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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., (© 2018 John Wiley & Sons Ltd.)

Evolutionary changes in Astyanax mexicanus cavefish with respect to conspecific surface fish, including the regression of eyes, loss of pigmentation, and modification of the cranial skeleton, involve derivatives of the neural crest. However, the role of neural crest cells in cavefish evolution and development is poorly understood. One of the reasons is that experimental methods for neural crest analysis are not well developed in the Astyanax system. Here we describe neural crest transplantation between Astyanax surface fish and cavefish embryos. We found differences in the migration of cranial neural crest cells transplanted from the surface fish anterior hindbrain to the same region of surface fish or cavefish hosts. Cranial neural crest cells migrated extensively throughout the head, and to a lesser extent the trunk, in surface fish hosts but their migration was mostly restricted to the anterior and dorsal head regions in cavefish hosts. Cranial neural crest cells derived from the surface fish transplants invaded the degenerating eyes of cavefish hosts, resulting in increased eye size and suggesting that cavefish neural crest cells are defective in forming optic derivatives. We found that melanophores were formed in albino cavefish from grafts of surface fish trunk neural crest cells, showing that the cavefish tissue environment is conducive for pigment cell development, and implicating intrinsic changes in cavefish neural crest cells in loss of body pigmentation. It is concluded that changes in neural crest cells play key roles in the evolution of cavefish development.

Important contributions to evolutionary developmental biology have been made using the comparative organismal approach. As examples, I describe insights obtained from studies of Molgula ascidians and Astyanax cavefish., (© 2016 Elsevier Inc. All rights reserved.)

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.

Cave animals are excellent models to study the general principles of evolution as well as the mechanisms of adaptation to a novel environment: the perpetual darkness of caves. In this article, two of the major model systems used to study the evolution and development (evo-devo) of cave animals are described: the teleost fish Astyanax mexicanus and the isopod crustacean Asellus aquaticus. The ways in which these animals match the major attributes expected of an evo-devo cave animal model system are described. For both species, we enumerate the regressive and constructive troglomorphic traits that have evolved during their adaptation to cave life, the developmental and genetic basis of these traits, the possible evolutionary forces responsible for them, and potential new areas in which these model systems could be used for further exploration of the evolution of cave animals. Furthermore, we compare the two model cave animals to investigate the mechanisms of troglomorphic evolution. Finally, we propose a few other cave animal systems that would be suitable for development as additional models to obtain a more comprehensive understanding of the developmental and genetic mechanisms involved in troglomorphic evolution.

Epigenetic parental genetic effects are important in many biological processes but their roles in the evolution of adaptive traits and their consequences in naturally evolving populations remain to be addressed. By comparing two divergent blind cave-dwelling cavefish populations with a sighted surface-dwelling population (surface fish) of the teleost Astyanax mexicanus, we report here that convergences in vibration attraction behavior (VAB), the lateral line sensory receptors underlying this behavior, and the feeding benefits of this behavior are controlled by parental genetic effects, either maternal or paternal inheritance. From behavioral studies and mathematical evolutionary simulations, we further demonstrate that disparity in nuclear and mitochondrial DNA in one of these cavefish populations that has hybridized with surface fish can be explained by paternal inheritance of VAB. The results suggest that parental genetic effects in adaptive behaviors may be important factors in biasing mitochondrial DNA inheritance in natural populations that are subject to introgression., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave-dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L-tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L-DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L-DOPA and dopamine to DOPA-melanin and dopamine-melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave-adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave-adapted insects and teleosts., (© 2012 Wiley Periodicals, Inc.)

The relationship between growth rate and environmental space is an unresolved issue in teleosts. While it is known from aquaculture studies that stocking density has a negative relationship to growth, the underlying mechanisms have not been elucidated, primarily because the growth rate of populations rather than individual fish were the subject of all previous studies. Here we investigate this problem in the teleost Astyanax mexicanus, which consists of a sighted surface-dwelling form (surface fish) and several blind cave-dwelling (cavefish) forms. Surface fish and cavefish are distinguished by living in spatially contrasting environments and therefore are excellent models to study the effects of environmental size on growth. Multiple controlled growth experiments with individual fish raised in confined or unconfined spaces showed that environmental size has a major impact on growth rate in surface fish, a trait we have termed space dependent growth (SDG). In contrast, SDG has regressed to different degrees in the Pachón and Tinaja populations of cavefish. Mating experiments between surface and Pachón cavefish show that SDG is inherited as a dominant trait and is controlled by multiple genetic factors. Despite its regression in blind cavefish, SDG is not affected when sighted surface fish are raised in darkness, indicating that vision is not required to perceive and react to environmental space. Analysis of plasma cortisol levels showed that an elevation above basal levels occurred soon after surface fish were exposed to confined space. This initial cortisol peak was absent in Pachón cavefish, suggesting that the effects of confined space on growth may be mediated partly through a stress response. We conclude that Astyanax reacts to confined spaces by exhibiting SDG, which has a genetic component and shows evolutionary regression during adaptation of cavefish to confined environments.

How cave animals adapt to life in darkness is a poorly understood aspect of evolutionary biology [1]. Here we identify a behavioral shift and its morphological basis in Astyanax mexicanus, a teleost with a sighted surface-dwelling form (surface fish) and various blind cave-dwelling forms (cavefish) [2-4]. Vibration attraction behavior (VAB) is the ability of fish to swim toward the source of a water disturbance in darkness. VAB was typically seen in cavefish, rarely in surface fish, and was advantageous for feeding success in the dark. The potential for showing VAB has a genetic component and is linked to the mechanosensory function of the lateral line. VAB was evoked by vibration stimuli peaking at 35 Hz, blocked by lateral line inhibitors, first detected after developmental increases in superficial neuromast (SN) number and size [5-7], and significantly reduced by bilateral ablation of SN. We conclude that VAB and SN enhancement coevolved to compensate for loss of vision and to help blind cavefish find food in darkness., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study addresses the role of sonic hedgehog (shh) in increasing oral-pharyngeal constructive traits (jaws and taste buds) at the expense of eyes in the blind cavefish Astyanax mexicanus. In cavefish embryos, eye primordia degenerate under the influence of hyperactive Shh signaling. In concert, cavefish show amplified jaw size and taste bud numbers as part of a change in feeding behavior. To determine whether pleiotropic effects of hyperactive Shh signaling link these regressive and constructive traits, shh expression was compared during late development of the surface-dwelling (surface fish) and cave-dwelling (cavefish) forms of Astyanax. After an initial expansion along the midline of early embryos, shh was elevated in the oral-pharyngeal region in cavefish and later was confined to taste buds. The results of shh inhibition and overexpression experiments indicate that Shh signaling has an important role in oral and taste bud development. Conditional overexpression of an injected shh transgene at specific times in development showed that taste bud amplification and eye degeneration are sensitive to shh overexpression during the same early developmental period, although taste buds are not formed until much later. Genetic crosses between cavefish and surface fish revealed an inverse relationship between eye size and jaw size/taste bud number, supporting a link between oral-pharyngeal constructive traits and eye degeneration. The results suggest that hyperactive Shh signaling increases oral and taste bud amplification in cavefish at the expense of eyes. Therefore, selection for constructive oral-pharyngeal traits may be responsible for eye loss during cavefish evolution via pleiotropic function of the Shh signaling pathway.

The teleost Astyanax mexicanus is a single species consisting of two radically different forms: a sighted pigmented surface-dwelling form (surface fish) and a blind depigmented cave-dwelling form (cavefish). The two forms of Astyanax have favorable attributes, including descent from a common ancestor, ease of laboratory culture, and the ability to perform genetic analysis, permitting their use as a model system to explore questions in evolution and development. Here, we review current research on the molecular, cellular, and developmental mechanisms underlying the loss of eyes and pigmentation in Astyanax cavefish. Although functional eyes are lacking in adults, cavefish embryos begin to develop eye primordia, which subsequently degenerate. The major cause of eye degeneration appears to be apoptotic cell death of the lens, which prevents the growth of other optic tissues, including the retina. Ultimately, the loss of the eye is the cause of craniofacial differences between cavefish and surface fish. Lens apoptosis is induced by enhanced activity of the Hedgehog signaling system along the cavefish embryonic midline. The absence of melanin pigmentation in cavefish is due to a block in the ability of undifferentiated melanoblasts to accumulate L-tyrosine, the precursor of L-DOPA and melanin, in melanosomes. Genetic analysis has shown that this defect is caused by a hypomorphic mutation in the p/oca2 gene encoding an integral melanosomal membrane protein. We discuss how current studies of eye and pigment regression have revealed some of the mechanisms in which cavefish development has been changed during evolution.

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.

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.

Cavefish and their conspecific surface-dwelling ancestors (Astyanax mexicanus) are emerging as a model system to study the microevolution of development. Here we describe attributes that make this system highly promising for such studies. We review how the Astyanax system is being used to understand evolutionary forces underlying loss of eyes and pigmentation in cavefish. Pigment regression is probably explained by neutral mutations, whereas natural selection is a likely mechanism for loss of eyes. Finally, we discuss several research frontiers in which Astyanax is poised to make significant contributions in the future: evolution of constructive traits, the craniofacial skeleton, the central nervous system, and behavior.

This article reviews new insights from ascidians on the ancestry of vertebrate neural crest (NC) cells. Ascidians have neural crest-like cells (NCLC), which migrate from the dorsal midline, express some of the typical NC markers, and develop into body pigment cells. These characters suggest that primordial NC cells were already present in the common ancestor of the vertebrates and urochordates, which have been recently inferred as sister groups. The primitive role of NCLC may have been in pigment cell dispersal and development. Later, additional functions may have appeared in the vertebrate lineage, resulting in the evolution of definitive NC cells.

Ventral midline Sonic Hedgehog (Shh) signalling is crucial for growth and patterning of the embryonic forebrain. Here, we report how enhanced Shh midline signalling affects the evolution of telencephalic and diencephalic neuronal patterning in the blind cavefish Astyanax mexicanus, a teleost fish closely related to zebrafish. A comparison between cave- and surface-dwelling forms of Astyanax shows that cavefish display larger Shh expression in all anterior midline domains throughout development. This does not affect global forebrain regional patterning, but has several important consequences on specific regions and neuronal populations. First, we show expanded Nkx2.1a expression and higher levels of cell proliferation in the cavefish basal diencephalon and hypothalamus. Second, we uncover an Nkx2.1b-Lhx6-GABA-positive migratory pathway from the subpallium to the olfactory bulb, which is increased in size in cavefish. Finally, we observe heterochrony and enlarged Lhx7 expression in the cavefish basal forebrain. These specific increases in olfactory and hypothalamic forebrain components are Shh-dependent and therefore place the telencephalic midline organisers in a crucial position to modulate forebrain evolution through developmental events, and to generate diversity in forebrain neuronal patterning.

Pigmentation and eyes are often lost in cave-adapted animals. Although the mechanisms of eye degeneration are beginning to be understood, little is known about the evolutionary and developmental processes involved in pigment cell regression. In teleost embryos, a population of neural crest cells migrates into the body wall and differentiates into melanophores, xanthophores, and iridophores. All three pigment cell types are present in the eyed surface-dwelling form (surface fish) of the teleost Astyanax mexicanus. However, melanophores are absent or substantially reduced in number in various derived populations of the conspecific blind cave-dwelling form (cavefish). We show here that tyrosinase-positive melanoblasts are present in cavefish. DiI labeling revealed a population of trunk neural crest cells in cavefish embryos that migrate to locations normally occupied by differentiated melanophores. We also discovered a cell population in cavefish embryos and adults resembling melanoblasts in several features, including the ability to synthesize melanin when supplied with the tyrosinase substrate l-dopa. DiI-tyrosinase double-labeling and neural keel explant experiments showed that the tyrosinase-positive cells are derived from the neural crest. The number of melanoblasts varies in different adult cavefish populations relative to the extent of melanophore reduction. Although cavefish melanoblasts can synthesize melanin from exogenous l-dopa, they are unable to convert exogenous l-tyrosine to l-dopa and melanin. We conclude that pigment cell regression in cavefish is mediated by an evolutionary change late in melanogenesis that may involve an impediment in the ability to convert l-tyrosine to l-dopa and melanin.

The ascidian tadpole larva has two brain sensory organs containing melanocytes: the otolith, a gravity receptor, and the ocellus, part of a photoreceptor. One or both of these sensory organs are absent in molgulid ascidians. We show here that developmental changes leading to the loss of sensory pigment cells occur by different mechanisms in closely related molgulid species. Sensory pigment cells are formed through a bilateral determination pathway in which two or more precursor cells are specified as an equivalence group on each side of the embryo. The precursor cells subsequently converge at the midline after neurulation and undergo cell interactions that decide the fates of the otolith and ocellus. Molgula occidentalis and M. oculata, which exhibit a tadpole larva with an otolith but lacking an ocellus, have conserved the bilateral pigment cell determination pathway. Programmed cell death (PCD) is superimposed on this pathway late in development to eliminate the ocellus precursor and supernumerary pigment cells, which do not differentiate into either an otolith or ocellus. In contrast to molgulids with tadpole larvae, no pigment cell precursors are specified on either side of the M. occulta embryo, which forms a tailless (anural) larva lacking both sensory organs, suggesting that the bilateral pigment cell determination pathway has been lost. The bilateral pigment cell determination pathway and superimposed PCD can be restored in hybrids obtained by fertilizing M. occulta eggs with M. oculata sperm, indicating control by a zygotic process. We conclude that PCD plays an important role in the evolution and development of brain sensory organs in molgulid ascidians.

We studied the development and evolution of craniofacial features in the teleost fish, Astyanax mexicanus. This species has an eyed surface dwelling form (surface fish) and many different cave dwelling forms (cavefish) with various degrees of reduced eyes and pigmentation. The craniofacial features we examined are the tooth-bearing maxillary bones, the nasal and antorbital bones, the circumorbital bones, and the opercular bones, all of which show evolutionary modifications in different cavefish populations. Manipulations of eye formation by transplantation of the embryonic lens, by lentectomy, or by removing the optic vesicle showed that eye-dependent and -independent processes change both the surface fish and cavefish craniofacial skeletons. The size of the olfactory pits, which the nasal and antorbital bones define, and the size and positioning of the circumorbital bones were found to correlate with eye development. For the six suborbital bones (SO1-6), the relationship with the developing eye appears to be due to ossification initiated from foci in the suborbital canal of cranial neuromasts, whose patterning is also highly correlated with the presence or absence of an eye. By contrast, we found that the number of maxillary teeth, the number of SO3 bone elements, the positioning of SO4-6 with respect to the opercular bone, and the shape of the opercular bone are not dependent on eye formation and vary among different cavefish populations. The results suggest that evolution of the cavefish craniofacial skeleton is controlled by multiple developmental events, some a direct consequence of eye degeneration and others unrelated to loss of the eye.

The Mexican tetra Astyanax mexicanus has many of the favorable attributes that have made the zebrafish a model system in developmental biology. The existence of eyed surface (surface fish) and blind cave (cavefish) dwelling forms in Astyanax also provides an attractive system for studying the evolution of developmental mechanisms. The polarity of evolutionary changes and the environmental conditions leading to the cavefish phenotype are known with certainty, and several different cavefish populations have evolved constructive and regressive changes independently. The constructive changes include enhancement of the feeding apparatus (jaws, taste buds, and teeth) and the mechanosensory system of cranial neuromasts. The homeobox gene Prox 1, which is expressed in the expanded taste buds and cranial neuromasts, is one of the genes involved in the constructive changes in sensory organ development. The regressive changes include loss of pigmentation and eye degeneration. Although adult cavefish lack functional eyes, small eye primordia are formed during embryogenesis, which later arrest in development, degenerate, and sink into the orbit. Apoptosis and lens signaling to other eye parts, such as the cornea, iris, and retina, result in the arrest of eye development and ultimate optic degeneration. Accordingly, an eye with restored cornea, iris, and retinal photoreceptor cells is formed when a surface fish lens is transplanted into a cavefish optic cup, indicating that cavefish optic tissues have conserved the ability to respond to lens signaling. Genetic analysis indicates that multiple genes regulate eye degeneration, and molecular studies suggest that Pax6 may be one of the genes controlling cavefish eye degeneration. Further studies of the Astyanax system will contribute to our understanding of the evolution of developmental mechanisms in vertebrates.

We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidians Molgula oculata and Molgula occulta are closely related species with different modes of development. M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin gene MocuMA1 was isolated from an M. oculata genomic library. MocuMA1 is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5' upstream region of MocuMA1 is sufficient to drive expression of a lacZ fusion construct in the larval muscle cells, implying that it is a functional gene. MocuMA1 mRNA first appears in the prospective muscle cells of M. oculata embryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA1a and MoccMA1b were isolated from a M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5' upstream regions of the M. occulta muscle actin genes also contain numerous changes, expression of MoccMA1a/lacZ and MoccMA1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMAlb. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.

The ascidian tadpole larva is regarded as a prototype of the ancestral chordate. Here we consider recent studies on the development of the tadpole larva that provide new insights into chordate origins and evolution. The notochord of ascidian larvae and vertebrates appear to be homologous structures based on their induction by endoderm and expression of the Brachyury (T) gene. The muscle cells of ascidian larvae also appear homologous to those of vertebrates based on their expression of bHLH myogenic and muscle-type actin genes, although they are specified by cytoplasmic determinants localized in the egg as well as embryonic induction. Studies of the tailless larvae of anural ascidians have resulted in the identification of Manx, a gene that may control tail development and evolution. These and other results support the ascidian tadpole prototype for the ancestral chordate.

We have used a subtractive procedure to isolate cDNA clones encoding genes expressed differentially in ascidian species with alternate modes of development. The ascidians used in this study were Molgula occulta, which develops a tailed (urodele) larva, and Molgula occulta, which develops a tailless (anural) larva. Two of the identified clones, Uro-2 and Uro-11, are described. Southern blots show that the Uro-2 and Uro-11 genes are present in both species, but the corresponding mRNAs are expressed preferentially in the urodele species. In situ hybridization showed that Uro-2 and Uro-11 transcripts accumulate in small oocytes during oogenesis. The maternal Uro-2 and Uro-11 transcripts were distributed throughout the oocyte cytoplasm. Transcript concentrations declined during vitellogenesis, but mature eggs still contain detectable levels of Uro-2 and Uro-11 mRNA. After fertilization, the maternal Uro-2 and Uro-11 transcripts were localized in the ectoplasm of uncleaved zygotes and mostly entered the ectoderm cells during cleavage. The Uro-2 gene appears to produce only maternal transcripts. In contrast, the Uro-11 gene may also produce zygotic transcripts, which accumulate between gastrulation and neurulation in posterior epidermis, neural and tail muscle cells. Zygotic expression of the Uro-11 gene was not detected in embryos of the anural species. The deduced amino acid sequences of the Uro-2 and Uro-11 cDNAs suggest that they encode novel basic proteins with distinctive structural features. The predicted Uro-2 protein contain, a leucine zipper motif, suggesting that it may dimerize with another protein. The predicted Uro-11 protein contains a nuclear localization signal, a region with similarity to part of the DNA-binding motif in the bacterial histone-like HU and IHF proteins, 12 repeats of the proposed DNA-binding motif S(T)PXX, and a potential zinc finger of the C6 or C6H2 class, suggesting that it may be a DNA-binding protein. The Uro-2 and Uro-11 proteins are candidates for regulatory factors involved in the evolutionary transition from urodele to anural development.

Ascidians have evolved alternate modes of development in which the conventional tadpole larva is remodeled or eliminated. Adultation, the precocious development of adult features in the larval head, is caused by superimposing the larval and adult differentiation programs. Caudalization, the addition of muscle cells to the larval tail, is caused by enhancing muscle induction or increasing the number of muscle cell divisions before terminal differentiation. Adultation and caudalization are correlated with increased egg size, suggesting dependence on maternal processes. Anural development, the elimination of the larval stage, is caused by maternal and zygotic events resulting in abbreviation and deletion of larval developmental programs. An example of a maternal change in anural species is the modification of the egg cytoskeleton during oogenesis, whereas a zygotic change may involve altered cell interactions during embryogenesis. Interspecific hybridization experiments suggest that some aspects of anural development may be caused by loss-of-function mutations. The dissociation of developmental programs is a key process in changing the mode of development in ascidians.

Anural ascidians do not develop into a conventional tailed larva with differentiated muscle cells, however, embryos of some anural ascidian species retain the ability to express acetylcholinesterase (AChE) in a vestigial muscle cell lineage. This study examines the number of AChE-positive cells that develop in the anural ascidian Molgula occulta relative to that in the closely related urodele (tailed) species, Molgula oculata. Histochemical assays showed that M. oculata embryos develop 36 to 38 AChE-positive cells, consistent with the number of tail muscle cells expressed in other urodele ascidians. In contrast, M. occulta embryos develop a mean of only 20 AChE-positive cells in their vestigial muscle lineage. Cleavage-arrested embryos of the anural species express AChE only in B-line blastomeres, showing that the vestigial muscle lineage cells are derived from the primary muscle lineage. Less than the expected number of AChE-positive B-line cells develop in cleavage-arrested anural embryos, however, implying that the allocation of primary muscle lineage cells is decreased. Eggs of the anural species can be fertilized with sperm of the urodele species resulting in the development of some larvae that contain a short tail and/or a brain melanocyte, specific features of urodele larvae. The typical urodele number of AChE-positive cells is restored in some of these hybrid embryos. Both primary and secondary muscle lineages are restored because cleavage-arrested hybrid embryos develop more AChE-positive cells in the B-line blastomeres and supernumerary AChE-positive cells in the A-line blastomeres. Hybrid embryos that develop the urodele complement of AChE-positive cells also form a tail and/or a brain melanocyte showing that restoration of muscle lineage cells is coupled to the development of other urodele features. AChE expression occurred in anural embryos with disorganized or dissociated blastomeres, indicating that AChE expression is determined autonomously. It is concluded that an evolutionary change in the allocation of larval muscle lineage cells occurs during development of the anural ascidian M. occulta which can be restored by interspecific hybridization with the urodele ascidian M. oculata.