A developmental atlas of male terminalia across twelve species of Drosophila.

How complex morphologies evolve is one of the central questions in evolutionary biology. Observing the morphogenetic events that occur during development provides a unique perspective on the origins and diversification of morphological novelty. One can trace the tissue of origin, emergence, and even regression of structures to resolve murky homology relationships between species. Here, we trace the developmental events that shape some of the most diverse organs in the animal kingdom-the male terminalia (genitalia and analia) of Drosophilids. Male genitalia are known for their rapid evolution with closely related species of the Drosophila genus demonstrating vast variation in their reproductive morphology. We used confocal microscopy to monitor terminalia development during metamorphosis in twelve related species of Drosophila. From this comprehensive dataset, we propose a new staging scheme for pupal terminalia development based on shared developmental landmarks, which allows one to align developmental time points between species. We were able to trace the origin of different substructures, find new morphologies and suggest possible homology of certain substructures. Additionally, we demonstrate that posterior lobe is likely originated prior to the split between the Drosophila melanogaster and the Drosophila yakuba clade. Our dataset opens up many new directions of research and provides an entry point for future studies of the Drosophila male terminalia evolution and development.

The Density of Regulatory Information Is a Major Determinant of Evolutionary Constraint on Noncoding DNA in Drosophila.

Evolutionary analyses have estimated that ∼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the ∼90 kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated.

Developmental phenomics suggests that H3K4 monomethylation confers multi-level phenotypic robustness.

How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. Here, we genetically reduce H3K4me1 deposition in Drosophila melanogaster and find that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness. Using a developmental phenomics approach, we find changes in morphology, metabolism, behavior, and offspring production. However, many phenotypic changes are only detected when hypomethylated flies develop outside of standard laboratory environments or with specific genetic backgrounds. Therefore, quantitative phenomics measurements can unravel how pleiotropic modulators of gene expression affect developmental robustness under conditions resembling the natural environments of a species.

Can changes in 3D genome architecture create new regulatory landscapes that contribute to phenotypic evolution?

Animal genomes are compartmentalized into insulated regulatory units named topology-associated domains (TADs). TADs insulate gene promoters from enhancers that occupy neighboring TADs. Chromosomal rearrangements that disrupt TAD structure can generate new regulatory interactions between enhancers and promoters that were once separated into different TADs, which might lead to new gene expression patterns. On the one hand, TAD rearrangements are known to cause deleterious phenotypes, but, on the other hand, rearrangements can also create novel expression patterns that may be selected during evolution because they generate advantageous phenotypes. Here, we review recent studies that explore the effects of chromosomal rearrangements and genetic perturbations on TAD structure and gene regulation in the context of development and evolution. We discuss the possible contribution of evolutionary breakpoints (EBRs) that affect TAD structure to the evolution of gene regulation and the phenotype.

Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors.

Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. In this work we describe a gene regulatory network consisting of three transcription factors-Prospero (Pros), D-Pax2, and Delilah (Dei)-that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. We show that D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. We further show that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.

A complex gene regulatory architecture underlies the development and evolution of cuticle morphology in Drosophila.

The cuticle of insects is decorated with non-sensory hairs called trichomes. A few Drosophila species independently lost most of the dorso-lateral trichomes on first instar larvae. Genetic experiments revealed that this naked cuticle phenotype was caused by the evolution of enhancer function at the ovo/shavenbaby (ovo/svb) locus. Here we explore how this discovery catalyzed major new insights into morphological evolution in different developmental contexts, enhancer pleiotropy in gene regulation and the functionality and evolution of the Svb gene regulatory network (GRN). Taken together this highlights the importance of understanding the architecture and evolution of gene regulatory networks in detail and the great potential for further study of the Svb GRN.

Actors with Multiple Roles: Pleiotropic Enhancers and the Paradigm of Enhancer Modularity.

The current paradigm in the field of gene regulation postulates that regulatory information for generating gene expression is organized into modules (enhancers), each containing the information for driving gene expression in a single spatiotemporal context. This modular organization is thought to facilitate the evolution of gene expression by minimizing pleiotropic effects. Here we review recent studies that provide evidence of quite the opposite: (i) enhancers can function in multiple developmental contexts, implying that enhancers can be pleiotropic, (ii) transcription factor binding sites within pleiotropic enhancers are reused in different contexts, and (iii) pleiotropy impacts the structure and evolution of enhancers. Altogether, this evidence suggests that enhancer pleiotropy is pervasive in animal genomes, challenging the commonly held view of modularity.

Comprehensive Analysis of a cis-Regulatory Region Reveals Pleiotropy in Enhancer Function.

Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. Here, we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. We explored how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases.

Evolved Repression Overcomes Enhancer Robustness.

Biological systems display extraordinary robustness. Robustness of transcriptional enhancers results mainly from clusters of binding sites for the same transcription factor, and it is not clear how robust enhancers can evolve loss of expression through point mutations. Here, we report the high-resolution functional dissection of a robust enhancer of the shavenbaby gene that has contributed to morphological evolution. We found that robustness is encoded by many binding sites for the transcriptional activator Arrowhead and that, during evolution, some of these activator sites were lost, weakening enhancer activity. Complete silencing of enhancer function, however, required evolution of a binding site for the spatially restricted potent repressor Abrupt. These findings illustrate that recruitment of repressor binding sites can overcome enhancer robustness and may minimize pleiotropic consequences of enhancer evolution. Recruitment of repression may be a general mode of evolution to break robust regulatory linkages.

Evolving Genital Structures: A Deep Look at Network Co-option.

Novel body structures are often generated by the redeployment of ancestral components of the genome. In this issue of Developmental Cell, Glassford et al. (2015) present a thorough analysis of the co-option of a gene regulatory network in the origin of an evolutionary novelty.

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos.

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2-5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.

Comparative spatiotemporal analysis of Hox gene expression in early stages of intermediate mesoderm formation.

BACKGROUND: Hox genes are key players in AP patterning of the vertebrate body plan and are necessary for organogenesis. Several studies provide evidence for the role Hox genes play during kidney development and especially regarding metanephros initiation and formation. However, the role Hox genes play during early stages of kidney development is largely unknown. A recent study in our lab revealed the role Hoxb4 plays in conferring the competence of intermediate mesodermal cells to respond to kidney inductive signals and express early kidney regulators. RESULTS: As a first step in understanding the role Hox genes play in setting the formation of the pronephros morphogenetic field and the expression of early regulators of kidney development, we studied in detail the expression pattern of 10 Hox genes in relation to the 6th somite axial level, the anterior sharp border of the kidney field. Despite the idea of spatial co-linearity as exemplified in the Hox gene expression pattern in late developmental stages, a very dynamic spatio-temporal expression of these genes was found in early stages. CONCLUSIONS: Since mesodermal patterning occurs at gastrula stages, the relevance of a "Hox code" at early stages is questioned in this study.

Interplay between activin and Hox genes determines the formation of the kidney morphogenetic field.

The kidney develops in a specific position along the anterior-posterior axis. All vertebrate kidney tissues are derived from the intermediate mesoderm (IM), and early kidney genes such as Lim1 and Pax2 are expressed in amniotes posterior to the sixth somite axial level. IM cells anterior to this level do not express kidney genes owing to changes in their competence to respond to kidney-inductive signals present along the entire axis. We aimed to understand the molecular mechanisms governing the loss of competence of anterior IM cells and the formation of the anterior border of the kidney morphogenetic field. We identified the dorsal neural tube as the potential kidney-inductive tissue and showed that activin, a secreted morphogen, is necessary but insufficient for Lim1 induction and establishment of the kidney field. Activin or activin-like and BMP signaling cascades are activated along the entire axis, including in anterior non-kidney IM, suggesting that competence to respond to these signals involves downstream or other components. Detailed expression pattern analysis of Hox genes during early chick development revealed that paralogous group four genes share the same anterior border as the kidney genes. Ectopic expression of Hoxb4 in anterior non-kidney IM, either by retinoic acid (RA) administration or plasmid-mediated overexpression, resulted in ectopic kidney gene expression. The anterior expansion of Lim1 expression was restrained when Hoxb4 was co-expressed with a truncated form of activin receptor. We suggest a model in which the competence of IM cells to respond to TGFbeta signaling and express kidney genes is driven by RA and mediated by Hoxb4.