One eye but no vision: cave fish with induced eyes do not respond to light

One of the most intriguing questions in evolutionary biology is the degree to which behavior is a necessary consequence of morphology. We explore this issue by examining phototactic behavior in epigean (eyed surface-dwelling) and troglomorphic (blind cave) forms of the teleost Astyanax fasciatus whose eyes were modified during embryogenesis by removing one or both lens vesicles from the epigean form or by transplanting the lens vesicle from an epigean fish into the optic cup of a blind cave form. Lens removal results in eye degeneration and blindness in adult epigean fish, whereas lens transplantation stimulates growth of the eye, inducing the development of optic tissues in the normally eyeless adult cave fish. Photoresponsiveness was examined by placing fish in an aquarium with one half illuminated and the other half dark and scoring their presence in the illuminated or dark half. Both the eyeless epigean fish and cave fish with induced eyes are indifferent to the illumination whereas the surface forms are scotophilic, suggesting that optic development and phototactic behavior are decoupled.     

Der Micos-Fisch,Höhlenfisch in statu nascendi oder Bastard ?

The Micos Population–cave fish in statu nascendi or hybrid? Observations on the evolution of cavernicoles The Micos-Cave in the Sierra de la Colmena in the State of San Luis Potosi, Mexico, contains a cavernicolous population of Astyanax mexicanus, whose members are for the most part blind, but, in contrast to other cave dwelling populations of the same species, appear almost normal in their pigmentation. Besides these, there are also large eyed and pigmented specimens to be found in this cave. Any transitional stages between the blind and the normal visioned fish are lacking. Offspring of the blind cavernicoles that are raised under light conditions develop a superficially lying eye which is markedly smaller than normal, attaining its size proportional to the light-intensity of the experimental conditions. The size and structure of the eye-remnants of the blind fish as well as the eyes of their offspring are considerably more variable than in the river specimens. Crossings of the blind Micos fish with the river fish Astyanax and also with a blind and unpigmented troglobiont of the same species - Sabinos fish - result in both cases in a more or less intermediate F¹-hybrid. A strict inbreeding within the Micos fish, selecting specimens with especially large eyes, produced animals whose eyes are comparable to those of the river fish after only three offspring generations. Electrophoresis studies on the allozyme variability at various loci prove that the Micos fish is genetically only slightly different compared to the river fish. On the other hand in some allele frequencies there is an alternative variation between the two. The Micos fish also differs from the typical troglobionts which are monomorphic at almost all loci examined and also possess alleles that are not found in the river form. Based on the genetic constitution, the Micos fish and the river fish found in the cave do not form a panmictic population. It is also doubtful that the Micos fish is the progeny of a hybrid swarm which previously resulted from a cross between a real troglobiont and the newly arrived river fish, because the Micos fish is in every characteristic genetically very similar to the river fish, whereas no clear traces of troglobiont relationship are found. Thus the Micos fish actually appears to be a cave form in statu nascendi against which the river fish that find their way in from time to time cannot compete.     

Phylogeography of surface and cave Astyanax (Teleostei) from Central and North America based on cytochrome b sequence data

Astyanax fasciatus has become a model organism for the study of regressive and adaptive evolution in cave animals. To fully understand these processes, it is important to have background information on the systematics and phylogeography of surface and cave populations of this species. Here we investigate the phylogeography of A. fasciatus in North and Central America and also the historical biogeography of this region. Phylogenetic analysis of part of the mtDNA cytochrome b gene from 26 surface and nine cave A. fasciatus populations revealed seven major clades, which, in principle, represent geographical patterns of distribution. However, the four strongly eye and pigment reduced cave populations, Piedras, Sabinos, Tinaja, and Curva, form a separate cluster, which is not sister group to the surface populations from the same locality. Similarly the Belizean populations do not cluster with their geographic neighbors from the Yucatan. The analyses indicate that there have been recurrent invasions of surface Astyanax from the south, that were most likely influenced by major climate changes during the Pleistocene. During this period, ancestors of the strongly eye and pigment reduced cave populations were able to survive underground as thermophilic relics when the surface populations became extinct. The high level of genetic divergence among the different clades shows that differing haplotype lineages must have reinvaded the surface waters from the south and/or back-colonized them from residual habitats and also penetrated into the caves. Nested clade analyses show that recurrent gene flow as well as historic processes like past fragmentation and range expansion have influenced current populations of A. fasciatus in Central and North America. Different haplotype clades of the phylogeny are not compatible with the present taxonomy of Astyanax and, therefore, we propose the application of a single systematic unit, called A. fasciatus.     

Regressive evolution: ontogeny and genetics of cavefish eye rudimentation

Two hypotheses exist to explain ontogenetic eye reduction in Astyanax cave fish: first, after lens induction by the primordial eye cup, the lens plays the role of a central regulator of eye and retina regression or, second, the retina itself is an independent unit of eye development. A comparative study of five blind cave fish populations and their surface sister form was performed to investigate the differences of ontogenetic eye regression between the cave populations during different stages of development. The study revealed that, in addition to the initial formation of smaller primordia, eye regression is also caused during later ontogeny by different relative growth and specific histological characteristics. Whereas the cave fish lens never properly differentiates, the regressive process of the retina is transitorily interrupted by ongoing differentiation. In the newly-discovered Molino cave population, even visual cells with well-organized outer segments develop, which are secondarily reduced at a later ontogenetic stage. This result shows that the retina and lens are independent developmental units within the eye ball. Presumably, the genetic systems responsible for both show independent inheritance, which is also corroborated by hybrids of F ₂-crosses between the cave and surface fish, in which lens and retina development do not correlate. During ontogeny, the eye size differs between the cave populations. In Pachón cave fish, the relatively large eye size correlates with an ancient introgression from a surface population, which may have delayed eye regression.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 287–296.     

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.     

Convergent evolution of the cavefish Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation

More than 20 populations of the cave-dwelling characid Astyanax occur within a restricted karst area in Mexico. The fish possess reduced eyes without lenses and visual cells. It is still an open question as to whether this condition evolved convergently after multiple entries of the surface ancestor into the different caves or whether a single cave ancestor, already characterized by reduced eyes, spread secondarily within them. In the crosses between specific populations, specimens appear that deviate considerably from those of the parents. They possess larger and better-developed eyes with histologically intact lenses and visual cells; they thus have the structural potential for vision. This indicates that in different cave populations, different mutations in the eye gene system have occurred. In cases where these non-functional rudimentary genes are recombined in hybrid specimens, gene expression may be restored. This is the result of separate evolution of several Astyanax cave populations.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 545−554.     

Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus)

The full length cDNA sequence of the myostatin gene was cloned from a teleostean fish, the Chilean flounder (Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5'- and 3'-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including the structural domains responsible for its biological activity. Amino acid sequence comparison revealed high sequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Gene expression analysis showed that cfMSTN mRNA is present in a wide variety of tissues in juvenile fish. In addition, we assessed the spatial expression pattern of the MSTN mRNA during embryos and larval stages through whole mount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days post fertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye, were positive for MSTN mRNA. Our results contribute to the knowledge of the MSTN system in larval and juvenile stages; in particular the strong expression observed in the notochord suggests that MSTN, in synchronization with positive growth signals, may play an important role in the control of the development of larvae somites.     

Development of pigment-cup eyes in the polychaete Platynereis dumerilii and evolutionary conservation of larval eyes in Bilateria

The role of Pax6 in eye development in insects and vertebrates supports the view that their eyes evolved from simple pigment-cup ocelli present in their last common ancestors (Urbilateria). The cerebral eyes in errant polychaetes represent prototype invertebrate pigment-cup ocelli and thus resemble the presumed ancestral eyes. We have analysed expression of conserved eye specification genes in the early development of larval and adult pigment-cup eyes in Platynereis dumerilii (Polychaeta, Annelida, Lophotrochozoa). Both larval and adult eyes form in close vicinity of the optic anlagen on both sides of the developing brain ganglia. While pax6 is expressed in the larval, but not in the developing, adult eyes, expression of six1/2 from trochophora stages onwards specifically outlines the optic anlagen and thus covers both the developing larval and adult eyes. Using Platynereis rhabdomeric opsin as differentiation marker, we show that the first pair of adult eye photoreceptor cells is detected within bilateral clusters that transitorily express ath, the Platynereis atonal orthologue, thus resembling proneural sensory clusters. Our data indicate that--similar to insects, but different from the vertebrates--polychaete six1/2 expression outlines the entire visual system from early developmental stages onwards and ath-positive clusters generate the first photoreceptor cells to appear. We propose that pax6-, six1/2- and ath-positive larval eyes, as found in today's trochophora, were present already in Urbilateria.     

Reaction to Alarm Substance in Cave Populations of Astyanax fasciatus (Characidae,Pisces)1

The effect of species-specific skin-extract containing alarm substance has been studied in two epigean and three hypogean populations of the Mexican-tetra Astyanax fasciatus. In all populations tested the feeding rate at the water surface decreased significantly after introducing the alarm substance and the fish avoided this surface area. Additionally epigean fish and some individuals from the Chica cave showed zigzag movements, rapid swimming and hiding. The fish did not respond to a skinextract of a species without alarm substance.     

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.     

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.     

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave-adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog-related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic 'hotspots' of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.     

Pax-6基因在发育中的表达与动物眼的进化

近年在脊椎动物和无脊椎动物中分离出Pax-6基因及其同源基因,这些基因都与动物的眼与神经系统的发育和形态发生有关.本文着重比较了无脊椎动物果蝇、文昌鱼、哺乳动物小鼠和人的Pax-6基因编码蛋白,Pax-6基因在发育过程中的表达,Pax-6与眼进化的关系等几个方面,并介绍Pax-6基因为靶基因的转基因果蝇的上游制作技术和原理.探讨了Pax-6基因作为眼发育的主导基因的作用和时空表达模式的保守性.     

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.     

twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development

The Drosophila Pax-6 gene eyeless (ey) plays a key role in eye development. Here we show tht Drosophila contains a second Pax-6 gene, twin of eyeless (toy), due to a duplication during insect evolution. Toy is more similar to vertebrate Pax-6 proteins than Ey with regard to overall sequence conservation, DNA-binding function, and early expression in the embryo, toy and ey share a similar expression pattern in the developing visual system, and targeted expression of Toy, like Ey, induces the formation of ectopic eyes. Genetic and biochemical evidence indicates, however, that Toy functions upstream of ey by directly regulating the eye-specific enhancer of ey. Toy is therefore required for initiation of ey expression in the embryo and acts through Ey to activate the eye developmental program.     

Fish can encode order in their spatial map

Animals must often orient through areas that are larger than their perceptual range. The blind Mexican cave fish, Astyanax fasciatus, depends on detecting self-induced near-field wave perturbations by objects via the use of its lateral line organ. Its perceptual range (less than or equal to 0.05 m) is greatly exceeded by its ecological ranging requirements (ca. 30 m). Although known to possess a spatial map of its environment, it is not known how this fish links places (or the area over which the perceptual range extends) together. Using the blind cave fish's propensity to accelerate when faced with objects or environments that are recognizably different, I used a behavioural assay to test whether fishes can learn and remember the order of a landmark sequence. I show, to my knowledge for the first time, that blind Mexican cave fish can encode order in their spatial map. The ability to represent the order in which a series of places are spatially linked is a powerful tool for animals that must orient beyond the limit of their perceptual range. The resulting spatial map would be analogous to a jigsaw puzzle, where each piece represents a place whose size is constrained by the animal's perceptual range.     

Pax-6, a murine paired box gene, is expressed in the developing CNS.

A multigene family of paired-box-containing genes (Pax genes) has been identified in the mouse. In this report, we describe the expression pattern of Pax-6 during embryogenesis and the isolation of cDNA clones spanning the entire coding region. The Pax-6 protein consists of 422 amino acids as deduced from the longest open reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with day 8 of gestation, Pax-6 is expressed in discrete regions of the forebrain and the hindbrain. In the neural tube, expression is mainly confined to mitotic active cells in the ventral ventricular zone along the entire anteroposterior axis starting at day 8.5 of development. Pax-6 is also expressed in the developing eye, the pituitary and the nasal epithelium.     

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.