up to nine different individuals sequenced per morphotype (Warren, 2021). In the supplemental data, alignments of oca2 loci showed three specific deletion events (see section B) thought to be associated with loss of melanin pigmentation. Two of the smaller deletions in the Tinaja and Pachón morphotypes had specific deletion signatures showing distinct five and three prime (5´and 3´) boundaries. Another morphotype (Molino) had a larger oca2 deletion for which the specific boundaries have not yet been defined. These signatures may be the result of targeted transposon-mediated deletion activity associated with cave adaptations. Specific genome modifications that are regulated by transposons, including deletion events, are a well-documented phenomenon associated with a variety of developmental processes in animals (Bourque, 2018). At the ICR, genetic research on molecular mechanisms that may regulate adaptive traits in cavefish will include transposon activity. 2) Pleiotropy and genetic integration Another important component of trait development in cavefish comes from observations that multiple traits are influenced by the expression of single genes. The term ‘pleiotropy’ refers to the production of two or more unrelated effects produced by one gene. Our model of continuous environmental tracking (CET) would lead us (1) to question whether the effects are truly unrelated, and (2) to interpret the connectivity of multiple trait-specific effects as being coordinated, pre-programmed responses to changing environmental conditions. A prime example of inferred pleiotropy in cavefish describes the coupling of eye degeneration with enhancement of feeding anatomy (Yamamoto et al. 2004, 2009). Underlying the connectivity of these traits is a network of gene expression patterns. Upregulation or “hyperactivity” of the sonic hedgehog (shh) gene in the embryonic neural plate and dorsal anterior midline of cavefish meditates the expression levels of other genes. Specifically, increased levels of shh downregulates (represses) expression of pax6 and upregulates (induces) the expression of pax2 and vax1 (Yamamoto et al. 2009; Krishnan and Rohner, 2017). The relative expression of the pax6, pax2 and vax1 genes under the influence of hyperactive shh initiates the process of apoptosis (cell death) in the lens and retina, which causes degeneration and loss of eyes (Fig. 7). Importantly, correlated increase of shh expression in oral ectoderm and pharyngeal endoderm, also during embryonic cavefish development, induces the enhanced development of tastebuds and jaws (Yamamoto et al. 2009; Jeffery 2020). Following the onset eye degeneration, these cavefish embryos produce more tastebuds, at a faster rate (Varatharasan et al. 2009), and larger jaws than surface fish, which have functional eyes. And when shh is experimentally overexpressed in surface fish, there is a similar coupling of eye degeneration with enhancements of the gustatory system (taste buds and jaws) as observed in cavefish (Yamamoto et al. 2009). This complex pleiotropic effect is interpreted as a “developmental trade-off between these regressive and constructive traits” (Jeffery 2010). From our perspective of CET, coupled, coordinated, purposeful responses of vision and feeding within cave environments, both of which are clearly adaptive traits, would not imply some form of “trade-off” through an unguided natural mutation-selection process. Especially when such traits are also verified through reciprocal experiments with surface morphs of the same species. But there is more to pleiotropy and supposed ‘tradeoffs’. As mentioned above, reduction of melanophores in cavefish is controlled by oca2 (Protas et al. 2006; Klaassen et al. 2018), along with the melanocortin-1 receptor gene (mcr1), and others (Gross et al. 2009). In Astyanax cavefish, the melanin and catecholamine synthesis pathways diverge after conversion of L-DOPA. With a regulation and modification of the genome are most likely to be involved in cavefish eye degeneration. Thus, mechanisms for shutting down eye development in cavefish as an adaptive response to cave conditions must be due to built-in systems that regulate traits at the level of the genome. One of the more easily determined epigenetic modifications involves cytosine methylation where specified regulatory sections of the genome surrounding genes will have methyl groups attached to cytosine nucleotides along the genetic code. This type of site-directed methylation effectively downregulates or silences certain types of gene activity. Gore et al. (2018), determined that methylation-based epigenetic silencing was an adaptive mechanism for eye degeneration in Pachón cavefish. By performing parallel analyses in both blind cavefish and surface fish, and using zebrafish (Danio rerio) as a comparative sighted fish model, they discovered that DNA methylation of specific genomic sites confers eye-specific gene repression, and also regulates early eye development. Also, multiple cavefish genes with “promoter hypermethylation” were reported to be associated with eye disorders in humans and mice (Gore et al. 2018). These epigenetic data suggest that blindness in cavefish is inherent, pre-programmed and adaptive. Therefore, ‘mutations’ (random genetic errors) should be ruled out as having any selective value as credible explanations for eye loss in Astyanax cavefish. And in relation to our observations of rapid melanic pigmentation in commercial cavefish when exposed to light, it is probable that removal of methylation from euchromatin surrounding regulators of melanin production (e.g. oca2) may serve to upregulate the melanin synthesis pathway. It does not imply that the same epigenetic mechanism is active in cavefish where oca2 exon deletions have been confirmed (Klaassen et al. 2018). However it does mean that epigenetic methylation or acetylation patterns can inactivate or activate, respectively, specific gene loci (epialleles) or multiple phenotypic traits that are deployed in different environments (Cubas et al. 1999; Bertozzi and Ferguson-Smith 2020). In known cases of epigenetic inheritance, distinct regions of hypermethylated chromatin can be transferred between generations by epialleles. To assess generational transfer of genomic signatures of eye development and pigment regulation, we will need to characterize pan-epigenetic and specific epiallelic states in adult and larval cavefish, and in our Molino cavefish where melanic albinism is observed. Genetic deletions and insertions by transposable elements may indicate intentional regulatory events. With their known ability to rewire regulatory circuits (Feschotte, 2008), transposons could initiate or deactivate particular traits in cavefish during development. Transposable elements (e.g. Alu SINES), contain many binding sites for transcription factors that allow them to regulate developmental processes (Polak, 2006; Lynch, 2011). Indeed, research has shown that a very high proportion of cis-regulatory changes associated with development and adaptation are connected with transposition events (Chenais, 2012). The Zebrafish model genome has provided a wealth of information for not only animal genetics in general, but also Astyanax research as well (Chang, 2022). Recent research on the spatial and temporal expression of transposable elements during Zebrafish development provides a valuable reference resource for interpreting genomic regulation in cavefish. As developmental programs of zebrafish and Astyanax are very similar, we anticipate exploiting that resource for interpreting cavefish variation in developmental and adaptive morphotypes, especially as they relate to transposon-mediated genomic regulation. A number of specified changes to the genome may be occurring during the development of Astyanax cavefish. In the literature, we found one study that performed genome sequencing of a variety of different Astyanax cave morphotypes with BOYLE, ARLEDGE, THOMAS, TOMKINS, AND GULIUZZA Testing the cavefish model 2023 ICC 133
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