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.     

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     

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.     

Restoring eye size in Astyanax mexicanus blind cavefish embryos through modulation of the Shh and Fgf8 forebrain organising centres

The cavefish morph of the Mexican tetra (Astyanax mexicanus) is blind at adult stage, although an eye that includes a retina and a lens develops during embryogenesis. There are, however, two major defects in cavefish eye development. One is lens apoptosis, a phenomenon that is indirectly linked to the expansion of ventral midline sonic hedgehog (Shh) expression during gastrulation and that induces eye degeneration. The other is the lack of the ventral quadrant of the retina. Here, we show that such ventralisation is not extended to the entire forebrain because fibroblast growth factor 8 (Fgf8), which is expressed in the forebrain rostral signalling centre, is activated 2 hours earlier in cavefish embryos than in their surface fish counterparts, in response to stronger Shh signalling in cavefish. We also show that neural plate patterning and morphogenesis are modified in cavefish, as assessed by Lhx2 and Lhx9 expression. Inhibition of Fgf receptor signalling in cavefish with SU5402 during gastrulation/early neurulation mimics the typical surface fish phenotype for both Shh and Lhx2/9 gene expression. Fate-mapping experiments show that posterior medial cells of the anterior neural plate, which lack Lhx2 expression in cavefish, contribute to the ventral quadrant of the retina in surface fish, whereas they contribute to the hypothalamus in cavefish. Furthermore, when Lhx2 expression is rescued in cavefish after SU5402 treatment, the ventral quadrant of the retina is also rescued. We propose that increased Shh signalling in cavefish causes earlier Fgf8 expression, a crucial heterochrony that is responsible for Lhx2 expression and retina morphogenesis defect.     

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.     

The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness

The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper (bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum^?/? zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4 cM interval on chromosome 7 with the closest markers located at 0.2 and 0 cM, respectively.     

Modularity and sense organs in the blind cavefish, Astyanax mexicanus

SUMMARY Mexican tetra (Astyanax mexicanus) exist as two morphs: a sighted (surface) form and a blind (cavefish) form. In the cavefish, some modules are lost, such as the eye and pigment modules, whereas others are expanded, such as the taste bud and cranial neuromast modules. We suggest that modularity can be viewed as being nested in a manner similar to Baupläne so that modules express unique sets of genes, cells, and processes. In terms of evolution, we conclude that natural selection can act on any of these hierarchical levels within modules or on all the sensory modules as a whole. We discuss interactions within and between modules with reference to the blind cavefish from both genetic and developmental perspectives. The cavefish represents an illuminating example of module interaction, uncoupling of modules, and module expansion.     

Embryonic development of Python sebae - I: Staging criteria and macroscopic skeletal morphogenesis of the head and limbs

This study explores the post-ovipositional craniofacial development of the African Rock Python (Python sebae). We first describe a staging system based on external characteristics and next use whole-mount skeletal staining supplemented with Computed tomography (CT) scanning to examine skeletal development. Our results show that python embryos are in early stages of organogenesis at the time of laying, with separate facial prominences and pharyngeal clefts still visible. Limb buds are also visible. By 11 days (stage 3), the chondrocranium is nearly fully formed; however, few intramembranous bones can be detected. One week later (stage 4), many of the intramembranous upper and lower jaw bones are visible but the calvaria are not present. Skeletal elements in the limbs also begin to form. Between stages 4 (day 18) and 7 (day 44), the complete set of intramembranous bones in the jaws and calvaria develops. Hindlimb development does not progress beyond stage 6 (33 days) and remains rudimentary throughout adult life. In contrast to other reptiles, there are two rows of teeth in the upper jaw. The outer tooth row is attached to the maxillary and premaxillary bones, whereas the inner row is attached to the pterygoid and palatine bones. Erupted teeth can be seen in whole-mount stage 10 specimens and are present in an unerupted, mineralized state at stage 7. Micro-CT analysis reveals that all the young membranous bones can be recognized even out of the context of the skull. These data demonstrate intrinsic patterning of the intramembranous bones, even though they form without a cartilaginous template. In addition, intramembranous bone morphology is established prior to muscle function, which can influence bone shape through differential force application. After careful staging, we conclude that python skeletal development occurs slowly enough to observe in good detail the early stages of craniofacial skeletogenesis. Thus, reptilian animal models will offer unique opportunities for understanding the early influences that contribute to perinatal bone shape.     

To see or not to see: evolution of eye degeneration in mexican blind cavefish

The evolutionary mechanisms responsible for the loss of eyesin cave animals are still unresolved. Hypotheses invoking naturalselection or neutral mutation have been advanced to explaineye regression. Here we describe comparative molecular and developmentalstudies in the teleost Astyanax mexicanus that shed new lighton this problem. A. mexicanus is a single species consistingof a sighted surface-dwelling form (surface fish) and many blindcave-dwelling forms (cavefish) from different caves. We firstreview the evolutionary relationships of Astyanax cavefish populationsand conclude that eye degeneration may have evolved multipletimes. We then compare the mechanisms of eye degeneration indifferent cavefish populations. We describe the results of experimentsshowing that programmed cell death of the lens plays a key rolein controlling eye degeneration in these cavefish populations.We also show that Pax6 gene expression and fate determinationin the optic primordia are modified similarly in different cavefishpopulations, probably due to hyperactive midline signaling.We discuss the contributions of the comparative developmentalapproach toward resolving the evolutionary mechanisms of eyedegeneration. A new hypothesis is presented in which both naturalselection and neutral mutation are proposed to have roles incavefish 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.     

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.     

Muscle‐induced loading as an important source of variation in craniofacial skeletal shape

The shape of the craniofacial skeleton is constantly changing through ontogeny and reflects a balance between developmental patterning and mechanical‐load‐induced remodeling. Muscles are a major contributor to producing the mechanical environment that is crucial for “normal” skull development. Here, we use an F₅ hybrid population of Lake Malawi cichlids to characterize the strength and types of associations between craniofacial bones and muscles. We focus on four bones/bone complexes, with different developmental origins, alongside four muscles with distinct functions. We used micro‐computed tomography to extract 3D information on bones and muscles. 3D geometric morphometrics and volumetric measurements were used to characterize bone and muscle shape, respectively. Linear regressions were performed to test for associations between bone shape and muscle volume. We identified three types of associations between muscles and bones: weak, strong direct (i.e., muscles insert directly onto bone), and strong indirect (i.e., bone is influenced by muscles without a direct connection). In addition, we show that although the shape of some bones is relatively robust to muscle‐induced mechanical stimulus, others appear to be highly sensitive to muscular input. Our results imply that the roles for muscular input on skeletal shape extend beyond specific points of origin or insertion and hold significant potential to influence broader patterns of craniofacial geometry. Thus, changes in the loading environment, either as a normal course of ontogeny or if an organism is exposed to a novel environment, may have pronounced effects on skeletal shape via near and far‐ranging effects of muscular loading.     

Gustatory apparatus of the oropharyngeal cavity in juvenile rainbow trout <Emphasis Type="Italic">Parassalmo mykiss</Emphasis>

Study of the structural organization of gustatory apparatus in rainbow trout Parasalmo mykiss performed using electron scanning microscopy demonstrated that external taste buds are absent in this species in skin covers of the head and in the circumoral region. In the oropharyngeal cavity (oral and gill cavities and pharynx) of the rainbow trout, a well-developed gustatory receptor apparatus was found. In correspondence with specific features of morphology and anatomy of the skull, taste buds form seven gustatory zones. Morphometric analysis demonstrated differences between gustatory zones in the pattern and density of distribution of taste buds, as well as in average sizes of their sensory field. Zones of similar innervation have many common features in morphology. Morphologically similar zones form three regions in the oropharyngeal cavity: rostral, central, and caudal. A tendency for a decrease in the concentration of taste buds in the rostrocaudal direction common for all sensory zones was revealed. The highest concentration of taste buds was recorded at papillae of rostral regions near big teeth. A typical feature of taste buds in rainbow trout is irregular shape of the taste pore. Analysis of ultrastructural specific features of apical processes of taste cells allows us to distinguish five cell shapes in the composition of taste buds. The numeric ratio of cell shapes varies in buds of different localization. The quantitative distribution of taste buds over sensory zones, specific features of morphology and sizes of their sensory field are discussed in relation to the feeding pattern of the species.     

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.     

A developmental staging series for the African house snake, Boaedon (Lamprophis) fuliginosus

Embryonic staging series are important tools in the study of morphological evolution as they establish a common standard for future studies. In this study, we describe the in ovo embryological development of the African house snake (Boaedon fuliginosus), a non-venomous, egg-laying species within the superfamily Elapoidea. We develop our staging series based on external morphology of the embryo including the head, eye, facial prominences, pharyngeal slits, heart, scales, and endolymphatic ducts. An analysis of embryonic growth in length and mass is presented, as well as preliminary data on craniofacial skeletal development. Our results indicate that B. fuliginosus embryos are well into organogenesis but lack well-defined facial prominences at the time of oviposition. Mandibular and maxillary processes extend rostrally within 8 days (stage 3), corresponding to the first appearance of Meckel's cartilages. Overall, the development of the craniofacial skeleton in B. fuliginosus appears similar to that of other snake species with intramembraneous bones (e.g., dentary and compound bones) ossifying before most of the endochondral bones, the first of which to ossify are the quadrate and the otic capsule. Our staging series is the first to describe the post-ovipositional development of a non-venomous elapoid based on external morphology. This species is an extremely tractable captive that can produce large clutches of eggs every 45 days throughout the year. As such, B. fuliginosus should be a good model for evolutionary developmental biologists focusing on the craniofacial skeleton, loss of limbs, generational teeth, and venom delivery systems.     

Structural Organization of the Taste Apparatus in Characins (Characidae,Teleostei)

The morphology and distribution of taste buds in the outer integument of the body and in the oral cavity of two forms (blind cave and sighted terrestrial ones) of the astyanax Astyanax fasciatus and in intact and blinded individuals of the Buenos Aires tetra Hyphessobrycon anisitsi have been studied using electronic scanning and light microscopy. In sighted individuals of both species, the morphometric parameters of the taste apparatus and the distribution of taste receptors are similar; the taste apparatus in the oral cavity is more developed than in the outer covers. Morphologically different taste zones were found in the oral cavity of characins. In blind fish, the taste apparatus of the maxillary zones is distinguished by smaller taste buds and a greater density of their distribution. The sensory field of taste buds in blind and sighted individuals of astyanax and tetra has a similar ultrastructure; it is formed by taste cells of three types. In blind astyanaxes and blinded individuals of tetra, numerous modified epidermal cells were found for the first time in the epithelium of the taste zones and in contact with taste buds, which are regarded as tactile receptors and a constituent element of polysensory taste-tactile complexes localized in blind fish in mainly ventral sensory zones.     

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.     

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.