Bayala, E. X. | bioRxiv Science

bioRxiv2022

Divergent expression of aristaless1 and aristaless2 is associated with embryonic appendage and pupal wing development in butterflies

Aristaless is a major regulator of developmental processes. It is well known for its role during appendage specification and extension across animals. Butterflies and moths have two copies of aristaless, aristaless1 (al1) and aristaless2 (al2), as a result of a gene duplication event. Previous work in Heliconius has shown that both copies appear to have novel functions related to wing color patterning. Here we expand our knowledge on the expression profiles associated with both ancestral and novel functions of Al1 across embryogenesis and wing pigmentation. Furthermore, we characterize Al2 expression, providing a comparative framework for understanding the role of gene duplicates in novel and ancestral roles. Our work shows that both Al1 and Al2 expression are associated with developing sensory appendages (leg, mouth, spines, and eyes) in embryos. Interestingly, Al1 appears to show higher expression earlier in embryogenesis while the highest levels of Al2 expression are shifted to later stages of embryonic development. Furthermore, Al1 localization appears extranuclear while Al2 co-localizes tightly with nuclei earlier, and then also expands outside the nucleus later in development. We observed similar cellular expression patterns for Al1 and Al2 in pupal wings when examining their roles in pigmentation. We also describe, for the first time, how Al1 localization appear to correlates with zones of Anterior/Posterior elongation of the body during embryonic growth, showcasing a possible new function related to Aristaless previously described role in appendage extension. Overall, these data suggest similar developmental roles associated with the extension/formation of specific appendages for both duplicates. However, we describe that such functions might be regulated by spatially and temporally complex patterns of expression for al1 and al2. This work expands our knowledge of Aristaless function and expression following gene duplication and the implications of the duplication on butterfly development. Finally, and more fundamentally, our study helps clarify principles behind sub-functionalization and gene expression evolution associated with developmental functions following gene duplication events.

developmental biology↗

bioRxiv2021

From the formation of embryonic appendages to the color of wings: Conserved and novel roles of aristaless1 in butterfly development

Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets and pharmacological manipulation of an upstream activator. We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this timepoint. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We also find that Al1 expression appears to downregulate the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3-Hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.

developmental biology↗