Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi.

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

A major role for noncoding regulatory mutations in the evolution of enzyme activity.

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila.

Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. Here, we sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. We compared the activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster and found strong functional conservation among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, we found three novel wg enhancer activities in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. Our results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. We suggest that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers.

Evolution of yellow gene regulation and pigmentation in Drosophila.

BACKGROUND: Changes in developmental gene expression are central to phenotypic evolution, but the genetic mechanisms underlying these changes are not well understood. Interspecific differences in gene expression can arise from evolutionary changes in cis-regulatory DNA and/or in the expression of trans-acting regulatory proteins, but few case studies have distinguished between these mechanisms. Here, we compare the regulation of the yellow gene, which is required for melanization, among distantly related Drosophila species with different pigment patterns and determine the phenotypic effects of divergent Yellow expression. RESULTS: Yellow expression has diverged among D. melanogaster, D. subobscura, and D. virilis and, in all cases, correlates with the distribution of black melanin. Species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression. Evolutionary changes in the activity of orthologous cis-regulatory elements are responsible for differences in abdominal Yellow expression; however, cis-regulatory element evolution is not the sole cause of divergent Yellow expression patterns. Transformation of the D. melanogaster yellow gene into D. virilis altered its expression pattern, indicating that trans-acting factors that regulate the D. melanogaster yellow gene have also diverged between these two species. Finally, we found that the phenotypic effects of evolutionary changes in Yellow expression depend on epistatic interactions with other genes. CONCLUSIONS: Evolutionary changes in Yellow expression correlate with divergent melanin patterns and are a result of evolution in both cis- and trans-regulation. These changes were likely necessary for the divergence of pigmentation, but evolutionary changes in other genes were also required.