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The evolution of sexual secondary characteristics necessitates regulatory factors that confer sexual identity to differentiating tissues and cells. InColias eurythemebutterflies, males exhibit two specialized wing scale types—ultraviolet-iridescent (UVI) and spatulate scales—which are absent in females and likely integral to male courtship behavior. This study investigates the regulatory mechanisms and single-nucleus transcriptomics underlying these two sexually dimorphic cell types during wing development. We show thatDoublesex(Dsx) expression is itself dimorphic and required to repress the UVI cell state in females, while unexpectedly, UVI activation in males is independent fromDsx. In the melanic marginal band,Dsxis required in each sex to enforce the presence of spatulate scales in males, and their absence in females. Single-nucleus RNAseq reveals that UVI and spatulate scale cell precursors each show distinctive gene expression profiles at 40% of pupal development, with marker genes that include regulators of transcription, cell signaling, cytoskeletal patterning, and chitin secretion. Both male-specific cell types share a low expression of theBric-a-brac(Bab) transcription factor, a key repressor of the UVI fate. Bab ChIP-seq profiling suggests that Bab binds thecis-regulatory regions of gene markers associated to UVI fate, including potential effector genes involved in the regulation of cytoskeletal processes and chitin secretion, and loci showing signatures of recent selective sweeps in a UVI-polymorphic population. These findings open new avenues for exploring wing patterning and scale development, shedding light on the mechanisms driving the specification of sex-specific cell states and the differentiation of specialized cell ultrastructures. 

ABSTRACT The success of butterflies and moths is tightly linked to the origin of scales within the group. A long-standing hypothesis postulates that scales are homologous to the well-described mechanosensory bristles found in the fruit fly Drosophila melanogaster, as both derive from an epithelial precursor. Previous histological and candidate gene approaches identified parallels in genes involved in scale and bristle development. Here, we provide developmental and transcriptomic evidence that the differentiation of lepidopteran scales derives from the sensory organ precursor (SOP). Live imaging in lepidopteran pupae shows that SOP cells undergo two asymmetric divisions that first abrogate the neurogenic lineage, and then lead to a differentiated scale precursor and its associated socket cell. Single-nucleus RNA sequencing using early pupal wings revealed differential gene expression patterns that mirror SOP development, suggesting a shared developmental program. Additionally, we recovered a newly associated gene, the transcription factor pdm3, involved in the proper differentiation of butterfly wing scales. Altogether, these data open up avenues for understanding scale type specification and development, and illustrate how single-cell transcriptomics provide a powerful platform for understanding evolution of cell types. 

Abstract Altered regulatory interactions during development likely underlie a large fraction of phenotypic diversity within and between species, yet identifying specific evolutionary changes remains challenging. Analysis of single-cell developmental transcriptomes from multiple species provides a powerful framework for unbiased identification of evolutionary changes in developmental mechanisms. Here, we leverage a “natural experiment” in developmental evolution in sea urchins, where a major life history switch recently evolved in the lineage leading to Heliocidaris erythrogramma, precipitating extensive changes in early development. Comparative analyses of single-cell transcriptome analysis (scRNA-seq) developmental time courses from H. erythrogramma and Lytechinus variegatus (representing the derived and ancestral states, respectively) reveal numerous evolutionary changes in embryonic patterning. The earliest cell fate specification events and the primary signaling center are co-localized in the ancestral developmental gene regulatory network; remarkably, in H. erythrogramma, they are spatially and temporally separate. Fate specification and differentiation are delayed in most embryonic cell lineages, although in some cases, these processes are conserved or even accelerated. Comparative analysis of regulator-target gene co-expression is consistent with many specific interactions being preserved but delayed in H. erythrogramma, while some otherwise widely conserved interactions have likely been lost. Finally, specific patterning events are directly correlated with evolutionary changes in larval morphology, suggesting that they are directly tied to the life history shift. Together, these findings demonstrate that comparative scRNA-seq developmental time courses can reveal a diverse set of evolutionary changes in embryonic patterning and provide an efficient way to identify likely candidate regulatory interactions for subsequent experimental validation. 

ABSTRACT Biphasic lifecycles are widespread among animals, but little is known about how the developmental transition between larvae and adults is regulated. Sea urchins are a unique system for studying this phenomenon because of the stark differences between their bilateral larval and pentaradial adult body plans. Here, we use single-cell RNA sequencing to analyze the development of Heliocidaris erythrogramma (He), a sea urchin species with an accelerated, non-feeding mode of larval development. The sequencing time course extends from embryogenesis to roughly a day before the onset of metamorphosis in He larvae, which is a period that has not been covered by previous datasets. We find that the non-feeding developmental strategy of He is associated with several changes in the specification of larval cell types compared to sea urchins with feeding larvae, such as the loss of a larva-specific skeletal cell population. Furthermore, the development of the larval and adult body plans in sea urchins may utilize largely different sets of regulatory genes. These findings lay the groundwork for extending existing developmental gene regulatory networks to cover additional stages of biphasic lifecycles. 

Abstract Chromatin accessibility plays an important role in shaping gene expression, yet little is known about the genetic and molecular mechanisms that influence the evolution of chromatin configuration. Both local (cis) and distant (trans) genetic influences can in principle influence chromatin accessibility and are based on distinct molecular mechanisms. We, therefore, sought to characterize the role that each of these plays in altering chromatin accessibility in 2 closely related sea urchin species. Using hybrids of Heliocidaris erythrogramma and Heliocidaris tuberculata, and adapting a statistical framework previously developed for the analysis of cis and trans influences on the transcriptome, we examined how these mechanisms shape the regulatory landscape at 3 important developmental stages, and compared our results to similar analyses of the transcriptome. We found extensive cis- and trans-based influences on evolutionary changes in chromatin, with cis effects generally larger in effect. Evolutionary changes in accessibility and gene expression are correlated, especially when expression has a local genetic basis. Maternal influences appear to have more of an effect on chromatin accessibility than on gene expression, persisting well past the maternal-to-zygotic transition. Chromatin accessibility near gene regulatory network genes appears to be distinctly regulated, with trans factors appearing to play an outsized role in the configuration of chromatin near these genes. Together, our results represent the first attempt to quantify cis and trans influences on evolutionary divergence in chromatin configuration in an outbred natural study system and suggest that chromatin regulation is more genetically complex than was previously appreciated. 

Abstract The developmental gene regulatory networks (dGRNs) of two sea urchin species,Lytechinus variegatus (Lv)andStrongylocentrotus purpuratus (Sp),have remained remarkably similar despite about 50 million years since a common ancestor. Hundreds of parallel experimental perturbations of transcription factors with similar outcomes support this conclusion. A recent scRNA-seq analysis suggested that the earliest expression of several genes within the dGRNs differs betweenLvandSp. Here, we present a careful reanalysis of the dGRNs in these two species, paying close attention to timing of first expression. We find that initial expression of genes critical for cell fate specification occurs during several compressed time periods in both species. Previously unrecognized feedback circuits are inferred from the temporally corrected dGRNs. Although many of these feedbacks differ in location within the respective GRNs, the overall number is similar between species. We identify several prominent differences in timing of first expression for key developmental regulatory genes; comparison with a third species indicates that these heterochronies likely originated in an unbiased manner with respect to embryonic cell lineage and evolutionary branch. Together, these results suggest that interactions can evolve even within highly conserved dGRNs and that feedback circuits may buffer the effects of heterochronies in the expression of key regulatory genes. 

Sea urchins have become significant mariculture species globally, and also serve as invertebrate model organisms in developmental biology. Cis-regulatory elements (enhancers) control development and physiology by regulating gene expression. Mutations that affect the function of these sequences may contribute to phenotypic diversity. Cis-regulatory targets offer new breeding potential for the future. Here, we use the CRISPR/Cas9 system to disrupt an enhancer of Endo16 in developing Lytechinus variegatus embryos, in consideration of the thorough research on Endo16’s regulatory region. We designed six gRNAs against Endo16 Module A (the most proximal region of regulatory sequences, which activates transcription in the vegetal plate and archenteron, specifically) and discovered that Endo16 Module A-disrupted embryos failed to undergo gastrulation at 20 h post fertilization. This result partly phenocopies morpholino knockdowns of Endo16. Moreover, we conducted qPCR and clone sequencing experiments to verify these results. Although mutations were not found regularly from sequencing affected individuals, we discuss some potential causes. In conclusion, our study provides a feasible and informative method for studying the function of cis-regulatory elements in sea urchins, and contributes to echinoderm precision breeding technology innovation and aquaculture industry development. 

Abstract Chromatin configuration is highly dynamic during embryonic development in animals, exerting an important point of control in transcriptional regulation. Yet there exists remarkably little information about the role of evolutionary changes in chromatin configuration to the evolution of gene expression and organismal traits. Genome-wide assays of chromatin configuration, coupled with whole-genome alignments, can help address this gap in knowledge in several ways. In this study we present a comparative analysis of regulatory element sequences and accessibility throughout embryogenesis in three sea urchin species with divergent life histories: a lecithotroph Heliocidaris erythrogramma, a closely related planktotroph H. tuberculata, and a distantly related planktotroph Lytechinus variegatus. We identified distinct epigenetic and mutational signatures of evolutionary modifications to the function of putative cis-regulatory elements in H. erythrogramma that have accumulated nonuniformly throughout the genome, suggesting selection, rather than drift, underlies many modifications associated with the derived life history. Specifically, regulatory elements composing the sea urchin developmental gene regulatory network are enriched for signatures of positive selection and accessibility changes which may function to alter binding affinity and access of developmental transcription factors to these sites. Furthermore, regulatory element changes often correlate with divergent expression patterns of genes involved in cell type specification, morphogenesis, and development of other derived traits, suggesting these evolutionary modifications have been consequential for phenotypic evolution in H. erythrogramma. Collectively, our results demonstrate that selective pressures imposed by changes in developmental life history rapidly reshape the cis-regulatory landscape of core developmental genes to generate novel traits and embryonic programs. 

As analyses of developmental mechanisms extend to ever more species, it becomes important to understand not just what is conserved or altered during evolution, but why. Closely related species that exhibit extreme phenotypic divergence can be uniquely informative in this regard. A case in point is the sea urchin genus Heliocidaris, which contains species that recently evolved a life history involving nonfeeding larvae following nearly half a billion years of prior evolution with feeding larvae. The resulting shift in selective regimes produced rapid and surprisingly extensive changes in developmental mechanisms that are otherwise highly conserved among echinoderm species. The magnitude and extent of these changes challenges the notion that conservation of early development in echinoderms is largely due to internal constraints that prohibit modification and instead suggests that natural selection actively maintains stability of inherently malleable trait developmental mechanisms over immense time periods. Knowing how and why natural selection changed during the evolution of nonfeeding larvae can also reveal why developmental mechanisms do and do not change in particular ways.