Amanda K. Powers

Natural bone fragmentation in the blind cave‐dwelling fish, Astyanax mexicanus: candidate gene identification through integrative comparative genomics

Animals that colonize dark and nutrient‐poor subterranean environments evolve numerous extreme phenotypes. These include dramatic changes to the craniofacial complex, many of which are under genetic control. These phenotypes can demonstrate asymmetric genetic signals wherein a QTL is detected on one side of the face but not the other. The causative gene(s) underlying QTL are difficult to identify with limited genomic resources. We approached this task by searching for candidate genes mediating fragmentation of the third suborbital bone (SO3) directly inferior to the orbit of the eye. We integrated positional genomic information using emerging Astyanax resources, and linked these intervals to homologous (syntenic) regions of the Danio rerio genome. We identified a discrete, approximately 6 Mb, conserved region wherein the gene causing SO3 fragmentation likely resides. We interrogated this interval for genes demonstrating significant differential expression using mRNA‐seq analysis of cave and surface morphs across life history. We then assessed genes with known roles in craniofacial evolution and development based on GO term annotation. Finally, we screened coding sequence alterations in this region, identifying two key genes: transforming growth factor β3 (tgfb3) and bone morphogenetic protein 4 (bmp4). Of these candidates, tgfb3 is most promising as it demonstrates significant differential expression across multiple stages of development, maps close (<1 Mb) to the fragmentation critical locus, and is implicated in a variety of other animal systems (including humans) in non‐syndromic clefting and malformations of the cranial sutures. Both abnormalities are analogous to the failure‐to‐fuse phenotype that we observe in SO3 fragmentation. This integrative approach will enable discovery of the causative genetic lesions leading to complex craniofacial features analogous to human craniofacial disorders. This work underscores the value of cave‐dwelling fish as a powerful evolutionary model of craniofacial disease, and demonstrates the power of integrative system‐level studies for informing the genetic basis of craniofacial aberrations in nature.

A pleiotropic interaction between vision loss and hypermelanism in Astyanax mexicanus cave x surface hybrids.

BackgroundCave-dwelling animals evolve various traits as a consequence of life in darkness. Constructive traits (e.g., enhanced non-visual sensory systems) presumably arise under strong selective pressures. The mechanism(s) driving regression of features, however, are not well understood. Quantitative trait locus (QTL) analyses in Astyanax mexicanus Pachón cave x surface hybrids revealed phenotypic effects associated with vision and pigmentation loss. Vision QTL were uniformly associated with reductions in the homozygous cave condition, however pigmentation QTL demonstrated mixed phenotypic effects. This implied pigmentation might be lost through both selective and neutral forces. Alternatively, in this report, we examined if a pleiotropic interaction may exist between vision and pigmentation since vision loss has been shown to result in darker skin in other fish and amphibian model systems.ResultsWe discovered that certain members of Pachón x surface pedigrees are significantly darker than surface-dwelling fish. All of these “hypermelanic” individuals demonstrated severe visual system malformations suggesting they may be blind. A vision-mediated behavioral assay revealed that these fish, in stark contrast to surface fish, behaved the same as blind cavefish. Further, hypermelanic melanophores were larger and more dendritic in morphology compared to surface fish melanophores. However, hypermelanic melanophores responded normally to melanin-concentrating hormone suggesting darkening stemmed from vision loss, rather than a defect in pigment cell function. Finally, a number of genomic regions were coordinately associated with both reduced vision and increased pigmentation.ConclusionsThis work suggests hypermelanism in hybrid Astyanax results from blindness. This finding provides an alternative explanation for phenotypic effect studies of pigmentation QTL as stemming (at least in part) from environmental, rather than exclusively genetic, interactions between two regressive phenotypes. Further, this analysis reveals persistence of background adaptation in Astyanax. As the eye was lost in cave-dwelling forms, enhanced pigmentation resulted. Given the extreme cave environment, which is often devoid of nutrition, enhanced pigmentation may impose an energetic cost. Such an energetic cost would be selected against, as a means of energy conservation. Thus, the pleiotropic interaction between vision loss and pigmentation may reveal an additional selective pressure favoring the loss of pigmentation in cave-dwelling animals.

Asymmetric Facial Bone Fragmentation Mirrors Asymmetric Distribution of Cranial Neuromasts in Blind Mexican Cavefish

Craniofacial asymmetry is a convergent trait widely distributed across animals that colonize the extreme cave environment. Although craniofacial asymmetry can be discerned easily, other complex phenotypes (such as sensory organ position and numerical variation) are challenging to score and compare. Certain bones of the craniofacial complex demonstrate substantial asymmetry, and co-localize to regions harboring dramatically expanded numbers of mechanosensory neuromasts. To determine if a relationship exists between this expansion and bone fragmentation in cavefish, we developed a quantitative measure of positional symmetry across the left-right axis. We found that three different cave-dwelling populations were significantly more asymmetric compared to surface-dwelling fish. Moreover, cave populations did not differ in the degree of neuromast asymmetry. This work establishes a method for quantifying symmetry of a complex phenotype, and demonstrates that facial bone fragmentation mirrors the asymmetric distribution of neuromasts in different cavefish populations. Further developmental studies will provide a clearer picture of the developmental and cellular changes that accompany this extreme phenotype, and help illuminate the genetic basis for facial asymmetry in vertebrates.

The Evolution of the Cavefish Craniofacial Complex

The vertebrate skull is a highly complex, integrated morphological structure derived from distinct embryonic tissues. Owing to its origin among vertebrates, this structure lies at the intersection of several important developmental, evolutionary, and fundamental biological questions. Although craniofacial phenotypes were some of the first features investigated in Mexican cavefish, our understanding of the genetic and developmental underpinnings of these traits are only just emerging. Interestingly, multiple cavefish populations of Astyanax mexicanus have converged on similar craniofacial aberrations. These alterations differ from the related surface-dwelling forms with respect to bone sizes, shape, and positioning within the craniofacial complex. Several of these characters have long been appreciated in the literature, and played a role in the early taxonomic classification of cavefish in the 1940s. Historically, craniofacial changes were considered to be solely a consequence of eye regression in cavefish. Developmental studies support the notion that circumorbital bone shape and positioning are a consequence of eye loss; however, other traits, such as bony fusion and fragmentation, do not appear to be associated with eye regression. Further, these traits are often asymmetric across the left-right axis. Many craniofacial features harbor a genetic basis; however, the precise mechanism that underlies their origin remains unknown. In this chapter, we discuss the historical relevance of the craniofacial complex in Astyanax and document the convergent appearance of similar craniofacial features in distinct cave populations. In addition, we review current knowledge of genetic, developmental, and evolutionary mechanisms influencing craniofacial evolution in cavefish, and provide suggestions for future research.

Facial bone fragmentation in blind cavefish arises through two unusual ossification processes

The precise mechanisms underlying cranial bone development, evolution and patterning remain incompletely characterised. This poses a challenge to understanding the etiologies of craniofacial malformations evolving in nature. Capitalising on natural variation, “evolutionary model systems” provide unique opportunities to identify underlying causes of aberrant phenotypes as a complement to studies in traditional systems. Mexican blind cavefish are a prime evolutionary model for cranial disorders since they frequently exhibit extreme alterations to the skull and lateral asymmetries. These aberrations occur in stark contrast to the normal cranial architectures of closely related surface-dwelling fish, providing a powerful comparative paradigm for understanding cranial bone formation. Using a longitudinal and in vivo analytical approach, we discovered two unusual ossification processes in cavefish that underlie the development of ‘fragmented’ and asymmetric cranial bones. The first mechanism involves the sporadic appearance of independent bony elements that fail to fuse together later in development. The second mechanism involves the “carving” of channels in the mature bone, a novel form of post-ossification remodeling. In the extreme cave environment, these novel mechanisms may have evolved to augment sensory input, and may indirectly result in a trade-off between sensory expansion and cranial bone development.

Canal neuromast position prefigures developmental patterning of the suborbital bone series in Astyanax cave- and surface-dwelling fish

Developmental patterning is a complex biological phenomenon, involving integrated cellular and molecular signaling across diverse tissues. In Astyanax cavefish, the lateral line sensory system is dramatically expanded in a region of the cranium marked by significant bone abnormalities. This system provides the opportunity to understand how facial bone patterning can become altered through sensory system changes. Here we investigate a classic postulation that mechanosensory receptor neuromasts seed intramembranous facial bones in aquatic vertebrates. Using an in vivo staining procedure across individual life history, we observed infraorbital canal neuromasts serving as sites of ossification for suborbital bones. The manner in which cavefish departed from the stereotypical and symmetrical canal neuromast patterns of closely-related surface-dwelling fish were associated with specific changes to the suborbital bone complex. For instance, bony fusion, rarely observed in surface fish, was associated with shorter distances between canal neuromasts in cavefish, suggesting that closer canal neuromasts result in bony fusions. Additionally, cavefish lacking the sixth suborbital bone (SO6) uniformly lacked the associated (sixth) canal neuromast. This study suggests that patterning of canal neuromasts may impact spatial position of suborbital bones across development. The absence of an eye and subsequent orbital collapse in cavefish appears to influence positional information normally inherent to the infraorbital canal. These alterations result in coordinated changes to adult neuromast and bone structures. This work highlights complex interactions between visual, sensory and bony tissues during development that explain certain abnormal craniofacial features in cavefish.

A natural animal model system of craniofacial anomalies: The blind Mexican cavefish: Cavefish as an extreme model system

Natural model systems evolving under extreme environmental pressures provide the opportunity to advance our knowledge of how the craniofacial complex evolves in nature. Unlike traditional models, natural systems are less inbred, and, therefore, better model the complex variation of the human population. Owing to the nature of certain craniofacial aberrations in blind Mexican cavefish, we suggest that this organism can provide new insights to a variety of craniofacial changes. Diverse cranial features have evolved in natural cave‐dwelling Astyanax fish, which have thrived in the unforgiving darkness and nutrient‐poor environment of the cave for countless generations. While the genetic and environmental underpinnings of various cranial anomalies have been investigated for decades, a comprehensive characterization of their molecular and developmental origins remains incomplete. Cavefish provide numerous advantages given the availability of genomic resources, developmental and molecular tools, and the presence of a normative surface‐dwelling “ancestral” surrogate for comparative studies. By leveraging the frequency of abnormal and asymmetric cranial features in cavefish, we anticipate advances in our knowledge of the etiologies of irregular cranial features. Extreme adaptations in cavefish are expected to offer new insights into the complex and multifactorial nature of craniofacial disorders and facial asymmetry. Anat Rec, 2018.

Digest: Old world climates give rise to “young” lizard skulls*

Shared environmental pressures often give rise to the convergence of morphological characters in unrelated and geographically distinct species. Darwin (1859) wrote that “analogous variation” of traits in different organisms could be explained by similar influences or challenges in their environment. Convergence of traits can be driven by adaptive radiation when animals invade similar ecological feeding niches and consequently converge on similar morphologies, such as broader beak shape in seed-crushing species of Darwin’s finches (Grant, 1999). Climate can also be a factor, as demonstrated by morphological convergence of rodents from distinct arid habitats in the Sonoran (SW United States) and Monte (NW Argentina; Mares, 1976) Deserts. Observed patterns of ecomorphology suggest that overall body size decreases with warmer climates (Bergmann’s Rule), while limb length increases (Allen’s Rule; Schreider, 1951). Despite clear evidence of a relationship between morphology and environment, the mechanisms underlying trait convergence are not always clear. Until recently, it has been difficult to determine if convergent evolution occurs as a result of adaptations to a particular habitat, or if trait evolution is constrained by developmental mechanisms. In this issue, Hipsley and Müller (2017) address this fundamental biological question by investigating ecomorphological convergence in skull shape within a broad family of lacertid lizards that span across Eurasia and Africa, inhabiting vastly different climates over their distribution. Using landmark-based geometric morphometrics, Hipsley and Müller (2017) compared derived lizards adapted for dry climates to their basal ancestors that inhabited moderately moist (“mesic”) habitats. Desert-dwelling species’ skull shape was sig-