Developmental evolution and developmental plasticity of the olfactory epithelium and olfactory skills in Mexican cavefish.

The fish Astyanax mexicanus comes in two forms: the normal surface-dwelling (SF) and the blind depigmented cave-adapted (CF) morphs. Among many phenotypic differences, cavefish show enhanced olfactory sensitivity to detect amino-acid odors and they possess large olfactory sensory organs. Here, we questioned the relationship between the size of the olfactory organ and olfactory capacities. Comparing olfactory detection abilities of CF, SF and F1 hybrids with various olfactory epithelium (OE) sizes in behavioral tests, we concluded that OE size is not the only factor involved. Other possibilities were envisaged. First, olfactory behavior was tested in SF raised in the dark or after embryonic lens ablation, which leads to eye degeneration and mimics the CF condition. Both absence of visual function and absence of visual organs improved the SF olfactory detection capacities, without affecting the size of their OE. This suggested that developmental plasticity occurs between the visual and the olfactory modalities, and can be recruited in SF after visual deprivation. Second, the development of the olfactory epithelium was compared in SF and CF in their first month of life. Proliferation, cell death, neuronal lifespan, and olfactory progenitor cell cycling properties were identical in the two morphs. By contrast, the proportions of the three main olfactory sensory neurons subtypes (ciliated, microvillous and crypt) in their OE differed. OMP-positive ciliated neurons were more represented in SF, TRPC2-positive microvillous neurons were proportionately more abundant in CF, and S100-positive crypt cells were found in equal densities in the two morphs. Thus, general proliferative properties of olfactory progenitors are identical but neurogenic properties differ and lead to variations in the neuronal composition of the OE in SF and CF. Together, these experiments suggest that there are at least two components in the evolution of cavefish olfactory skills: (1) one part of eye-dependent developmental phenotypic plasticity, which does not depend on the size of the olfactory organ, and (2) one part of developmental evolution of the OE, which may stem from embryonic specification of olfactory neurons progenitor pools.

Sensory evolution in blind cavefish is driven by early embryonic events during gastrulation and neurulation.

Natural variations in sensory systems constitute adaptive responses to the environment. Here, we compared sensory placode development in the blind cave-adapted morph and the eyed river-dwelling morph of Astyanax mexicanus Focusing on the lens and olfactory placodes, we found a trade-off between these two sensory components in the two morphs: from neural plate stage onwards, cavefish have larger olfactory placodes and smaller lens placodes. In a search for developmental mechanisms underlying cavefish sensory evolution, we analyzed the roles of Shh, Fgf8 and Bmp4 signaling, which are known to be fundamental in patterning the vertebrate head and are subtly modulated in space and time during cavefish embryogenesis. Modulating these signaling systems at the end of gastrulation shifted the balance toward a larger olfactory derivative. Olfactory tests to assess potential behavioral outcomes of such developmental evolution revealed that Astyanax cavefish are able to respond to a 105-fold lower concentration of amino acids than their surface-dwelling counterparts. We suggest that similar evolutionary developmental mechanisms may be used throughout vertebrates to drive adaptive sensory specializations according to lifestyle and habitat.

Enhanced prey capture skills in Astyanax cavefish larvae are independent from eye loss.

BACKGROUND: Enhanced food-finding efficiency is an obvious adaptive response to cave environments. Here, we have compared the food-finding abilities of Astyanax surface fish and blind cavefish young larvae in their first month of life, in the dark. RESULTS: Our results show that enhanced prey capture skills of cavefish are already in effect in fry soon after the yolk is depleted and the young larvae must find food for themselves. Moreover, using prey capture competition assays on surface fish fry with lensectomies, we showed that eye-dependent developmental processes are not the main determinant for enhanced prey capture skills. Finally, using F2 hybrid larvae resulting from crosses between surface fish and cavefish, we found that reduced eyes do not confer a selective advantage for prey capture by fry in the dark. CONCLUSION: We discuss these data with regards to our current developmental and genetic understanding of cavefish morphological and behavioral evolution.

Differences in chemosensory response between eyed and eyeless Astyanax mexicanus of the Rio Subterráneo cave.

BACKGROUND: In blind cave-dwelling populations of Astyanax mexicanus, several morphological and behavioral shifts occurred during evolution in caves characterized by total and permanent darkness. Previous studies have shown that sensory systems such as the lateral line (mechanosensory) and taste buds (chemosensory) are modified in cavefish. It has long been hypothesized that another chemosensory modality, the olfactory system, might have evolved as well to provide an additional mechanism for food-searching in troglomorphic Astyanax populations. FINDINGS: During a March 2013 cave expedition to the Sierra de El Abra region of San Luís Potosi, Mexico, we tested chemosensory capabilities of the Astyanax mexicanus of the Rio Subterráneo cave. This cave hosts a hybrid population presenting a wide range of troglomorphic and epigean mixed phenotypes. During a behavioral test performed in situ in the cave, a striking correlation was observed between the absence of eyes and an increased attraction to food extract. In addition, eyeless troglomorphic fish possessed significantly larger naris size than their eyed, nontroglomorphic counterparts. CONCLUSIONS: Our findings suggest that chemosensory capabilities might have evolved in cave-dwelling Astyanax mexicanus and that modulation of naris size might at least partially underlie this likely adaptive change.

Ectopic proliferation contributes to retinal dysplasia in the juvenile zebrafish patched2 mutant eye.

PURPOSE: Patched is a well-studied tumor suppressor and negative regulator of the Hedgehog (Hh) pathway. Earlier work in this laboratory has shown that embryonic zebrafish patched2 (ptc2) mutant retinas possess an expanded ciliary marginal zone (CMZ) and phenotypes similar to those in human patients with basal cell naevus syndrome (BCNS), a congenital disorder linked to mutations in the human PTCH gene. This study extends the analysis of retinal structure and homeostasis in ptc2-/- mutants to juvenile stages, to determine whether Patched 2 function is essential in the postembryonic eye. METHODS: Histologic, immunohistochemical, and molecular analyses were used to characterize retinal defects in the 6-week-old juvenile ptc2-/- retina. RESULTS: Juvenile ptc2-/- mutants exhibited peripheral retinal dysplasias that included the presence of ectopic neuronal clusters in the inner nuclear layer (INL) and regions of disrupted retinal lamination. Retinal dysplasias were locally associated with ectopic proliferation. BrdU/EdU labeling and immunohistochemistry assays demonstrated that a population of ectopically proliferating cells gave rise to the ectopic neuronal clusters in the INL of ptc2-/- mutants and that this contributed to retinal dysplasia in the mutant eye. CONCLUSIONS: These results demonstrate a direct link between overproliferation and retinal dysplasia in the ptc2-/- juvenile retina and establish ectopic proliferation as the likely cellular underpinning of retinal dysplasia in juvenile ptc2-/- mutants.

Toward a better understanding of human eye disease insights from the zebrafish, Danio rerio.

Visual impairment and blindness is widespread across the human population, and the development of therapies for ocular pathologies is of high priority. The zebrafish represents a valuable model organism for studying human ocular disease; it is utilized in eye research to understand underlying developmental processes, to identify potential causative genes for human disorders, and to develop therapies. Zebrafish eyes are similar in morphology, physiology, gene expression, and function to human eyes. Furthermore, zebrafish are highly amenable to laboratory research. This review outlines the use of zebrafish as a model for human ocular diseases such as colobomas, glaucoma, cataracts, photoreceptor degeneration, as well as dystrophies of the cornea and retinal pigmented epithelium.

Expanded progenitor populations, vitreo-retinal abnormalities, and Müller glial reactivity in the zebrafish leprechaun/patched2 retina.

BACKGROUND: The roles of the Hedgehog (Hh) pathway in controlling vertebrate retinal development have been studied extensively; however, species- and context-dependent findings have provided differing conclusions. Hh signaling has been shown to control both population size and cell cycle kinetics of proliferating retinal progenitors, and to modulate differentiation within the retina by regulating the timing of cell cycle exit. While cell cycle exit has in turn been shown to control cell fate decisions within the retina, a direct role for the Hh pathway in retinal cell fate decisions has yet to be established in vivo. RESULTS: To gain further insight into Hh pathway function in the retina, we have analyzed retinal development in leprechaun/patched2 mutant zebrafish. While lep/ptc2 mutants possessed more cells in their retinas, all cell types, except for Müller glia, were present at identical ratios as those observed in wild-type siblings. lep/ptc2 mutants possessed a localized upregulation of GFAP, a marker for 'reactive' glia, as well as morphological abnormalities at the vitreo-retinal interface, where Müller glial endfeet terminate. In addition, analysis of the over-proliferation phenotype at the ciliary marginal zone (CMZ) revealed that the number of proliferating progenitors, but not the rate of proliferation, was increased in lep/ptc2 mutants. CONCLUSION: Our results indicate that Patched2-dependent Hh signaling does not likely play an integral role in neuronal cell fate decisions in the zebrafish retina. ptc2 deficiency in zebrafish results in defects at the vitreo-retinal interface and Müller glial reactivity. These phenotypes are similar to the ocular abnormalities observed in human patients suffering from Basal Cell Naevus Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2), and point to the utility of the lep/ptc2 mutant line as a model for the study of BCNS-related ocular pathologies. Our findings regarding CMZ progenitor proliferation suggest that, in the zebrafish retina, Hh pathway activity may not affect cell cycle kinetics; rather, it likely regulates the size of the retinal progenitor pool in the CMZ.