Evolution of Eye Regression in the Cavefish Astyanax: Apoptosis and the Pax-6 Gene

SYNOPSIS. The eye is an extraordinary organ in terms of itsdevelopment and evolution. In cave animals, the eye is sometimesreduced or eliminated as a consequence of adaptation to lifein perpetual darkness. We have used the characid teleost Astyanaxmexicanus as a model system to investigate the mechanisms ofeye degeneration during the evolution of a cave vertebrate.Eyed surface populations of Astyanax entered caves during thePleistocene, and their descendants lost their eyes and pigmentation.Astyanax populations exhibiting various degrees of eye regressionhave been reported in 29 Mexican caves. Surface populationswith characteristics of the ancestral stock still exist in thevicinity of these caves. Thus, Astyanax represents one of thefew instances in which the ancestral (surface fish) and thederived (cavefish) developmental modes are extant and availablefor comparative studies. The cavefish embryo develops an opticprimordium consisting of a lens vesicle and optic cup but therudimentary eye arrests in development and degenerates. Herewe report that eye degeneration is accompanied by extensiveapoptosis and downregulation of the Pax-6 gene in the developinglens. The results suggest that alterations in lens developmentare important factors in eye regression during cavefish evolution.     

Adaptive evolution of eye degeneration in the Mexican blind cavefish

The evolutionary mechanisms responsible for eye degeneration in cave-adapted animals have not been resolved. Opposing hypotheses invoking neural mutation or natural selection, each with certain genetic and developmental expectations, have been advanced to explain eye regression, although little or no experimental evidence has been presented to support or reject either theory. Here we review recent developmental and molecular studies in the teleost Astyanax mexicanus, a single species consisting of a sighted surface-dwelling form (surface fish) and many blind cave-dwelling forms (cavefish), which shed new light on this problem. The manner of eye development and degeneration, the ability to experimentally restore eyes, gene expression patterns, and comparisons between different cavefish populations all provide important clues for understanding the evolutionary forces responsible for eye degeneration. A key discovery is that Hedgehog midline signaling is expanded and inhibits eye formation by inducing lens apoptosis in cavefish embryos. Accordingly, eyes could have been lost by default as a consequence of natural selection for constructive traits, such as feeding structures, which are positively regulated by Hh signaling. We conclude from these studies that eye degeneration in cavefish may be caused by adaptive evolution and pleiotropy.     

Prox 1 in eye degeneration and sensory organ compensation during development and evolution of the cavefish Astyanax

We have investigated expression of the homeobox gene Prox 1 during eye degeneration and sensory organ compensation in cavefish embryos. The teleost Astyanax mexicanus consists of sighted surface-dwelling forms (surface fish) and several populations of blind cave-dwelling forms (cavefish), which have evolved independently. Eye formation is initiated during cavefish development, but the lens vesicle undergoes apoptosis, and the eye subsequently arrests and degenerates. The requirement of Prox 1 for lens fiber differentiation and γ-crystallin expression in the mouse suggests that changes in the expression of this gene could be involved in cavefish eye degeneration. Surface fish and cavefish embryos stained with a Prox 1 antibody showed Prox 1 expression in the lens, neuroretina, myotomes, heart, hindbrain, and gut, as reported in other vertebrates. We found that Prox 1 expression is not altered during cavefish lens development. Prox 1 protein was detected in the lens vesicle as soon as it formed and persisted until the time of lens degeneration in each cavefish population. The cavefish lens vesicle was also shown to express a γ-crystallin gene, suggesting that Prox 1 is functional in cavefish lens development. In addition to the tissues described above, Prox 1 is expressed in developing taste buds and neuromasts in cavefish, which are enhanced to compensate for blindness. It is concluded that the Prox 1 gene is not involved in lens degeneration, but that expansion of the Prox 1 expression domain occurs during taste bud and neuromast development in cavefish. Received: 31 July 1999 / Accepted: 8 November 1999     

Complex Evolutionary and Genetic Patterns Characterize the Loss of Scleral Ossification in the Blind Cavefish Astyanax mexicanus

The sclera is the tough outer covering of the eye that provides structural support and helps maintain intraocular pressure. In some fishes, reptiles, and birds, the sclera is reinforced with an additional ring of hyaline cartilage or bone that forms from scleral ossicles. Currently, the evolutionary and genetic basis of scleral ossification is poorly understood, especially in teleost fishes. We assessed scleral ossification among several groups of the Mexican tetra (Astyanax mexicanus), which exhibit both an eyed and eyeless morph. Although eyed Astyanax surface fish have bony sclera similar to other teleosts, the ossicles of blind Astyanax cavefish generally do not form. We first sampled cavefish from multiple independent populations and used ancestral character state reconstructions to determine how many times scleral ossification has been lost. We then confirmed these results by assessing complementation of scleral ossification among the F₁ hybrid progeny of two cavefish populations. Finally, we quantified the number of scleral ossicles present among the F₂ hybrid progeny of a cross between surface fish and cavefish, and used this information to identify quantitative trait loci (QTL) responsible for this trait. Our results indicate that the loss of scleral ossification is common–but not ubiquitous–among Astyanax cavefish, and that this trait has been convergently lost at least three times. The presence of wild-type, ossified sclera among the F₁ hybrid progeny of a cross between different cavefish populations confirms the convergent evolution of this trait. However, a strongly skewed distribution of scleral ossicles found among surface fish x cavefish F₂ hybrids suggests that scleral ossification is a threshold trait with a complex genetic basis. Quantitative genetic mapping identified a single QTL for scleral ossification on Astyanax linkage group 1. We estimate that the threshold for this trait is likely determined by at least three genetic factors which may control the severity and onset of lens degeneration in cavefishes. We conclude that complex evolutionary and genetic patterns underlie the loss of scleral ossification in Astyanax cavefish.     

The lens controls cell survival in the retina: Evidence from the blind cavefish Astyanax

The lens influences retinal growth and differentiation during vertebrate eye development but the mechanisms are not understood. The role of the lens in retinal growth and development was studied in the teleost Astyanax mexicanus, which has eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms. A lens and laminated retina initially develop in cavefish embryos, but the lens dies by apoptosis. The cavefish retina is subsequently disorganized, apoptotic cells appear, the photoreceptor layer degenerates, and retinal growth is arrested. We show here by PCNA, BrdU, and TUNEL labeling that cell proliferation continues in the adult cavefish retina but the newly born cells are removed by apoptosis. Surface fish to cavefish lens transplantation, which restores retinal growth and rod cell differentiation, abolished apoptosis in the retina but not in the RPE. Surface fish lens deletion did not cause apoptosis in the surface fish retina or affect RPE differentiation. Neither lens transplantation in cavefish nor lens deletion in surface fish affected retinal cell proliferation. We conclude that the lens acts in concert with another optic component, possibly the RPE, to promote retinal cell survival. Accordingly, deficiency in both optic structures may lead to eye degeneration in cavefish.     

Quantitative Genetic Analysis of Retinal Degeneration in the Blind Cavefish Astyanax mexicanus

The retina is the light-sensitive tissue of the eye that facilitates vision. Mutations within genes affecting eye development and retinal function cause a host of degenerative visual diseases, including retinitis pigmentosa and anophthalmia/microphthalmia. The characin fish Astyanax mexicanus includes both eyed (surface fish) and eyeless (cavefish) morphs that initially develop eyes with normal retina; however, early in development, the eyes of cavefish degenerate. Since both surface and cave morphs are members of the same species, they serve as excellent evolutionary mutant models with which to identify genes causing retinal degeneration. In this study, we crossed the eyed and eyeless forms of A. mexicanus and quantified the thickness of individual retinal layers among 115 F₂ hybrid progeny. We used next generation sequencing (RAD-seq) and microsatellite mapping to construct a dense genetic map of the Astyanax genome, scan for quantitative trait loci (QTL) affecting retinal thickness, and identify candidate genes within these QTL regions. The map we constructed for Astyanax includes nearly 700 markers assembled into 25 linkage groups. Based on our scans with this map, we identified four QTL, one each associated with the thickness of the ganglion, inner nuclear, outer plexiform, and outer nuclear layers of the retina. For all but one QTL, cavefish alleles resulted in a clear reduction in the thickness of the affected layer. Comparative mapping of genetic markers within each QTL revealed that each QTL corresponds to an approximately 35 Mb region of the zebrafish genome. Within each region, we identified several candidate genes associated with the function of each affected retinal layer. Our study is the first to examine Astyanax retinal degeneration in the context of QTL mapping. The regions we identify serve as a starting point for future studies on the genetics of retinal degeneration and eye disease using the evolutionary mutant model Astyanax.     

Cavefish as a model system in evolutionary developmental biology

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.     

A Potential Benefit of Albinism in Astyanax Cavefish: Downregulation of the oca2 Gene Increases Tyrosine and Catecholamine Levels as an Alternative to Melanin Synthesis

Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.     

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.

     

Evolution of pigment cell regression in the cavefish Astyanax: a late step in melanogenesis

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.     

Albinism in phylogenetically and geographically distinct populations of Astyanax cavefish arises through the same loss-of-function Oca2 allele

The Mexican tetra, Astyanax mexicanus, comprises 29 populations of cave-adapted fish distributed across a vast karst region in northeastern Mexico. These populations have a complex evolutionary history, having descended from ‘old'' and ‘young'' ancestral surface-dwelling stocks that invaded the region ∼6.7 and ∼2.8 MYa, respectively. This study investigates a set of captive, pigmented Astyanax cavefish collected from the Micos cave locality in 1970, in which albinism appeared over the past two decades. We combined novel coloration analyses, coding sequence comparisons and mRNA expression level studies to investigate the origin of albinism in captive-bred Micos cavefish. We discovered that albino Micos cavefish harbor two copies of a loss-of-function ocular and cutaneous albinism type II (Oca2) allele previously identified in the geographically distant Pachón cave population. This result suggests that phylogenetically young Micos cavefish and phylogenetically old Pachón cave fish inherited this Oca2 allele from the ancestral surface-dwelling taxon. This likely resulted from the presence of the loss-of-function Oca2 haplotype in the ‘young'' ancestral surface-dwelling stock that colonized the Micos cave and also introgressed into the ancient Pachón cave population. The appearance of albinism in captive Micos cavefish, caused by the same loss-of-function allele present in Pachón cavefish, implies that geographically and phylogenetically distinct cave populations can evolve the same troglomorphic phenotype from standing genetic variation present in the ancestral taxon.     

Early and late changes in Pax6 expression accompany eye degeneration during cavefish development

We have compared Pax6 expression during embryonic development in the eyed surface form (surface fish) and several different eyeless cave forms (cavefish) of the teleost Astyanax mexicanus. Despite lacking functional eyes as adults, cavefish embryos form small optic primordia, which later arrest in development and show various degrees of eye degeneration. The pattern of Pax6 mRNA expression was modified early and late during cavefish development. In early surface fish embryos, two bilateral Pax6 expression domains are present in the anterior neural plate, which extend across the midline and fuse to form the forebrain and optic primordia. In cavefish embryos, these Pax6 domains are diminished in size and remain separated, resulting in an anterior gap in Pax6 expression and presumably the formation of smaller optic primordia. The anterior gap in Pax6 expression was confirmed by double staining for Pax6 and distalless-3 mRNA, which marks the anterior margin of the neural plate and is unaltered in cavefish. Similar anterior gaps in Pax6 expression occurred in independently derived cavefish populations, suggesting that they are important in eye degeneration. Later during surface fish development, Pax6 protein is expressed in the cornea, lens, and ganglion and amacrine cells of the neural retina. Pax6 expression was gradually reduced during cavefish lens development, concomitant with lens arrest and degeneration, and was absent in the corneal epithelium, which does not differentiate in cavefish. In contrast, Pax6 expression in the retinal ganglion and amarcine cells is unmodified in cavefish, despite retarded retinal development. The results suggest that changes in Pax6 expression are involved in the evolution of cavefish eye degeneration.     

Conservation of retinal circadian rhythms during cavefish eye degeneration

Regressive evolution of morphological features is a common evolutionary event. However, the relationship between structural degeneration and loss of physiological function is often unclear because the ancestral and derived states of a character are usually not available for comparison. Here, we report studies on retinomotor rhythms during development of the blind cavefish Astyanax mexicanus, a single teleost species consisting of a sighted surface-dwelling form (surface fish) and several blind cave-dwelling (cavefish) forms. The eyed and blind forms of Astyanax diverged from a common sighted ancestor within the past million years. Despite the absence of functional eyes in cavefish adults, optic primordia are formed in embryos, but then gradually arrest in development, degenerate, and sink into the orbits. Although a layered retina is formed in cavefish embryos, it is deficient in photoreceptor cells, and in some cases the retinal pigment epithelium has lost its pigmentation. We show that the capacity to exhibit light-entrained retinomotor rhythms has been conserved in the degenerating embryonic eyes of two different Astyanax cavefish populations. The results indicate that loss of circadian retinal function does not precede and is therefore not required for eye degeneration in the blind cavefish.     

Development and evolution of craniofacial patterning is mediated by eye-dependent and -independent processes in the cavefish Astyanax

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.     

A Novel Role for Mc1r in the Parallel Evolution of Depigmentation in Independent Populations of the Cavefish Astyanax mexicanus

The evolution of degenerate characteristics remains a poorly understood phenomenon. Only recently has the identification of mutations underlying regressive phenotypes become accessible through the use of genetic analyses. Focusing on the Mexican cave tetra Astyanax mexicanus, we describe, here, an analysis of the brown mutation, which was first described in the literature nearly 40 years ago. This phenotype causes reduced melanin content, decreased melanophore number, and brownish eyes in convergent cave forms of A. mexicanus. Crosses demonstrate non-complementation of the brown phenotype in F₂ individuals derived from two independent cave populations: Pachón and the linked Yerbaniz and Japonés caves, indicating the same locus is responsible for reduced pigmentation in these fish. While the brown mutant phenotype arose prior to the fixation of albinism in Pachón cave individuals, it is unclear whether the brown mutation arose before or after the fixation of albinism in the linked Yerbaniz/Japonés caves. Using a QTL approach combined with sequence and functional analyses, we have discovered that two distinct genetic alterations in the coding sequence of the gene Mc1r cause reduced pigmentation associated with the brown mutant phenotype in these caves. Our analysis identifies a novel role for Mc1r in the evolution of degenerative phenotypes in blind Mexican cavefish. Further, the brown phenotype has arisen independently in geographically separate caves, mediated through different mutations of the same gene. This example of parallelism indicates that certain genes are frequent targets of mutation in the repeated evolution of regressive phenotypes in cave-adapted species.     

Retinal homeobox genes and the role of cell proliferation in cavefish eye degeneration

The teleost Astyanax mexicanus exhibits eyed surface dwelling (surface fish) and blind cave dwelling (cavefish) forms. Despite lacking functional eyes as adults, cavefish embryos form eye primordia, which later arrest in development, degenerate and sink into the orbit. We are comparing the expression patterns of various eye regulatory genes during surfacefish and cavefish development to determine the cause of eye degeneration. Here we examine Rx and Chx/Vsx family homeobox genes, which have a major role in cell proliferation in the vertebrate retina. We isolated and sequenced a full-length RxcDNA clone (As-Rx1) and part of a Chx/Vsx(As-Vsx2) gene, which appear to be most closely related to the zebrafish Rx1 and Alx/Vsx2 genes respectively. In situ hybridization shows that these genes have similar but non-identical expression patterns during Astyanax eye development. Expression is first detected in the optic vesicle, then throughout the presumptive retina of the optic cup, and finally in the ciliary marginal zone (CMZ), the region of the growing retina where most new retinoblasts are formed. In addition, As-Rx1 is expressed in the outer nuclear layer (ONL) of the retina, which contains the photoreceptor cells, and As-Vsx2 is expressed in the inner nuclear layer, probably in the bipolar cells. With the exception of reduced As-Rx-1 expression in the ONL, the As-Rx1 and As-Vsx2 expression patterns were unchanged in the developing retina of two different cavefish populations, suggesting that cell proliferation is not inhibited. These results were confirmed by using PCNA and BrdU markers for retinal cell division. We conclude that the CMZ is active in cell proliferation long after eye growth is diminished and is therefore not the major cause of eye degeneration.     

Blind cavefish and heat shock protein chaperones: a novel role for hsp90alpha in lens apoptosis

Lens apoptosis plays a central role in cavefish eye degeneration. Heat shock proteins (hsps) can regulate apoptosis; therefore, we examined the relationship between constitutive hsp70 and hsp90 expression and lens apoptosis. The model system is Astyanax mexicanus, a teleost species consisting of an eyed surface-dwelling (surface fish) form and numerous blind cave-dwelling (cavefish) forms. Optic primordia are formed in the cavefish embryo but they subsequently undergo lens apoptosis, arrest in development and degenerate. Astyanax hsp90 and hsp70 DNAs were isolated to use as probes to compare gene expression during surface fish and cavefish development. Hsp90beta, which encodes one of two hsp90 isoforms, was not expressed in the surface fish or cavefish lens, whereas hsp70 was expressed in the lens of both forms, suggesting that neither is directly involved in lens apoptosis. In contrast, hsp90alpha, the other hsp90 isoform, was expressed in the cavefish but not the surface fish lens. Hsp90alpha expression peaked shortly before the beginning of lens apoptosis in three convergent cavefish populations, suggesting a close relationship with lens apoptosis. The absence of hsp90beta in the lens allowed us to use geldanamycin and radicicol, specific inhibitors of hsp90 chaperone function, to determine whether lens cell death requires hsp90alpha expression. Both inhibitors blocked TUNEL labeling in the cavefish lens, suggesting that hsp90alpha is required for apoptosis. In contrast to their effects on the lens, these inhibitors induced TUNEL labeling in the surface epidermis, presumably due to effects on hsp90beta function, implying that the two-hsp90 isoforms may have contrasting roles in cell survival. We conclude that hsp90alpha plays a novel role in lens apoptosis and cavefish eye degeneration.     

Early Lens Ablation Causes Dramatic Long-Term Effects on the Shape of Bones in the Craniofacial Skeleton of Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, exists as two morphs of a single species, a sighted surface morph and a blind cavefish. In addition to eye regression, cavefish have an increased number of taste buds, maxillary teeth and have an altered craniofacial skeleton compared to the sighted morph. We investigated the effect the lens has on the development of the surrounding skeleton, by ablating the lens at different time points during ontogeny. This unique long-term study sheds light on how early embryonic manipulations on the eye can affect the shape of the adult skull more than a year later, and the developmental window during which time these effects occur. The effects of lens ablation were analyzed by whole-mount bone staining, immunohistochemisty and landmark based morphometric analyzes. Our results indicate that lens ablation has the greatest impact on the skeleton when it is ablated at one day post fertilisation (dpf) compared to at four dpf. Morphometric analyzes indicate that there is a statistically significant difference in the shape of the supraorbital bone and suborbital bones four through six. These bones expand into the eye orbit exhibiting plasticity in their shape. Interestingly, the number of caudal teeth on the lower jaw is also affected by lens ablation. In contrast, the shape of the calvariae, the length of the mandible, and the number of mandibular taste buds are unaltered by lens removal. We demonstrate the plasticity of some craniofacial elements and the stability of others in the skull. Furthermore, this study highlights interactions present between sensory systems during early development and sheds light on the cavefish phenotype.     

Evolution of insect eye development: first insights from fruit fly, grasshopper and flour beetle

The molecular genetic dissection of Drosophila eye developmentled to the exciting discovery of a surprisingly large panelof genes and gene activities, which are functionally conservedacross phyla. Little effort has yet been made towards pinpointingnon-conserved gene functions in the developing Drosophila eye.This neglects the fact that Drosophila visual system developmentis a highly derived process. The comparative analysis of Drosophilaeye development within insects can be expected to enhance resolutionand accuracy of between phyla comparisons of eye development,and to reveal molecular developmental changes that facilitatedthe evolutionary transition from hemimetabolous to holometabolousinsect development. Here we review aspects of early Drosophilaeye development, which are likely to have diverged from thesituation in more primitive insects, as indicated by resultsfrom work in the flour beetle Tribolium castaneum and the grasshopperSchistocerca americana.