The ftz upstream element drives late ftz stripes but is not required for regulation of Ftz target genes.

The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. Here, we asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS7S homozygotes, ftz stripes appear in the same order as wildtype, and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz stripe expression, with all 7 stripes decaying earlier than wildtype in ftzΔUPS7S homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. We propose that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, our results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development.

Segmental expression of two ecdysone pathway genes during embryogenesis of hemimetabolous insects.

Signaling networks are redeployed across different developmental times and places to generate phenotypic diversity from a limited genetic toolkit. Hormone signaling networks in particular have well-studied roles in multiple developmental processes. In insects, the ecdysone pathway controls critical events in late embryogenesis and throughout post-embryonic development. While this pathway has not been shown to function in the earliest stage of embryonic development in the model insect Drosophila melanogaster, one component of the network, the nuclear receptor E75A, is necessary for proper segment generation in the milkweed bug Oncopeltus fasciatus. Published expression data from several other species suggests possible conservation of this role across hundreds of millions of years of insect evolution. Previous work also demonstrates a second nuclear receptor in the ecdysone pathway, Ftz-F1, plays a role in segmentation in multiple insect species. Here we report tightly linked expression patterns of ftz-F1 and E75A in two hemimetabolous insect species, the German cockroach Blattella germanica and the two-spotted cricket Gryllus bimaculatus. In both species, the genes are expressed segmentally in adjacent cells, but they are never co-expressed. Using parental RNAi, we show the two genes have distinct roles in early embryogenesis. E75A appears necessary for abdominal segmentation in B. germanica, while ftz-F1 is essential for proper germband formation. Our results suggest that the ecdysone network is critical for early embryogenesis in hemimetabolous insects.

Genome editing of the vermilion locus generates a visible eye color marker for Oncopeltus fasciatus.

Insects display a vast array of eye and body colors. Genes encoding products involved in biosynthesis and deposition of pigments are ideal genetic markers, contributing, for example, to the power of Drosophila genetics. Oncopeltus fasciatus is an emerging model for hemimetabolous insects, a member of the piercing-sucking feeding order Hemiptera, that includes pests and disease vectors. To identify candidate visible markers for O. fasciatus, we used parental and nymphal RNAi to identify genes that altered eye or body color while having no deleterious effects on viability. We selected Of-vermilion for CRISPR/Cas9 genome editing, generating three independent loss-of-function mutant lines. These studies mapped Of-vermilion to the X-chromosome, the first assignment of a gene to a chromosome in this species. Of-vermilion homozygotes have bright red, rather than black, eyes and are fully viable and fertile. We used these mutants to verify a role for Of-xdh1, ortholog of Drosophila rosy, in contributing to red pigmentation using RNAi. Rather than wild-type-like red bodies, bugs lacking both vermilion and xdh1 have bright yellow bodies, suggesting that ommochromes and pteridines contribute to O. fasciatus body color. Our studies generated the first gene-based visible marker for O. fasciatus and expanded the genetic toolkit for this model system.

Anterior-posterior patterning of segments in Anopheles stephensi offers insights into the transition from sequential to simultaneous segmentation in holometabolous insects.

The gene regulatory network for segmentation in arthropods offers valuable insights into how networks evolve owing to the breadth of species examined and the extremely detailed knowledge gained in the model organism Drosophila melanogaster. These studies have shown that Drosophila's network represents a derived state that acquired changes to accelerate segment patterning, whereas most insects specify segments gradually as the embryo elongates. Such heterochronic shifts in segmentation have potentially emerged multiple times within holometabolous insects, resulting in many mechanistic variants and difficulties in isolating underlying commonalities that permit such shifts. Recent studies identified regulatory genes that work as timing factors, coordinating gene expression transitions during segmentation. These studies predict that changes in timing factor deployment explain shifts in segment patterning relative to other developmental events. Here, we test this hypothesis by characterizing the temporal and spatial expression of the pair-rule patterning genes in the malaria vector mosquito, Anopheles stephensi. This insect is a Dipteran (fly), like Drosophila, but represents an ancient divergence within this clade, offering a useful counterpart for evo-devo studies. In mosquito embryos, we observe anterior to posterior sequential addition of stripes for many pair-rule genes and a wave of broad timer gene expression across this axis. Segment polarity gene stripes are added sequentially in the wake of the timer gene wave and the full pattern is not complete until the embryo is fully elongated. This "progressive segmentation" mode in Anopheles displays commonalities with both Drosophila's rapid segmentation mechanism and sequential modes used by more distantly related insects.

The fushi tarazu zebra element is not required for Drosophila viability or fertility.

Expression of genes in precisely controlled spatiotemporal patterns is essential for embryonic development. Much of our understanding of mechanisms regulating gene expression comes from the study of cis-regulatory elements (CREs) that direct expression of reporter genes in transgenic organisms. This reporter-transgene approach identifies genomic regions sufficient to drive expression but fails to provide information about quantitative and qualitative contributions to endogenous expression, although such conclusions are often inferred. Here we evaluated the endogenous function of a classic Drosophila CRE, the fushi tarazu (ftz) zebra element. ftz is a pair-rule segmentation gene expressed in seven stripes during embryogenesis, necessary for formation of alternate body segments. Reporter transgenes identified the promoter-proximal zebra element as a major driver of the seven ftz stripes. We generated a precise genomic deletion of the zebra element (ftzΔZ) to assess its role in the context of native chromatin and neighboring CREs, expecting large decreases in ftz seven-stripe expression. However, significant reduction in expression was found for only one stripe, ftz stripe 4, expressed at ∼25% of wild type levels in ftzΔZ homozygotes. Defects in corresponding regions of ftzΔZ mutants suggest this level of expression borders the threshold required to promote morphological segmentation. Further, we established true-breeding lines of homozygous ftzΔZ flies, demonstrating that the body segments missing in the mutants are not required for viability or fertility. These results highlight the different types of conclusions drawn from different experimental designs and emphasize the importance of examining transcriptional regulatory mechanisms in the context of the native genomic environment.

Regulatory gene function handoff allows essential gene loss in mosquitoes.

Regulatory genes are often multifunctional and constrained, which results in evolutionary conservation. It is difficult to understand how a regulatory gene could be lost from one species' genome when it is essential for viability in closely related species. The gene paired is a classic Drosophila pair-rule gene, required for formation of alternate body segments in diverse insect species. Surprisingly, paired was lost in mosquitoes without disrupting body patterning. Here, we demonstrate that a paired family member, gooseberry, has acquired paired-like expression in the malaria mosquito Anopheles stephensi. Anopheles-gooseberry CRISPR-Cas9 knock-out mutants display pair-rule phenotypes and alteration of target gene expression similar to what is seen in Drosophila and beetle paired mutants. Thus, paired was functionally replaced by the related gene, gooseberry, in mosquitoes. Our findings document a rare example of a functional replacement of an essential regulatory gene and provide a mechanistic explanation of how such loss can occur.

High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus.

In this manuscript, we report that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is highly efficient in the hemipteran Oncopeltus fasciatus The white gene is well characterized in Drosophila where mutation causes loss of eye pigmentation; white is a reliable marker for transgenesis and other genetic manipulations. Accordingly, white has been targeted in a number of nonmodel insects to establish tools for genetic studies. Here, we generated mutations in the Of-white (Of-w) locus using CRISPR/Cas9. We found that Of-w is required for pigmentation throughout the body of Oncopeltus, not just the ommatidia. High rates of somatic mosaicism were observed in the injected generation, reflecting biallelic mutations, and a high rate of germline mutation was evidenced by the large proportion of heterozygous G1s. However, Of-w mutations are homozygous lethal; G2 homozygotes lacked pigment dispersion throughout the body and did not hatch, precluding the establishment of a stable mutant line. Embryonic and parental RNA interference (RNAi) were subsequently performed to rule out off-target mutations producing the observed phenotype and to evaluate the efficacy of RNAi in ablating gene function compared to a loss-of-function mutation. RNAi knockdowns phenocopied Of-w homozygotes, with an unusual accumulation of orange granules observed in unhatched embryos. This is, to our knowledge, the first CRISPR/Cas9-targeted mutation generated in Oncopeltus While we were unable to establish white as a useful visible marker for Oncopeltus, these findings are instructive for the selection of visible markers in nonmodel species and reveal an unusual role for an ortholog of a classic Drosophila gene.

Oncopeltus-like gene expression patterns in Murgantia histrionica, a new hemipteran model system, suggest ancient regulatory network divergence.

BACKGROUND: Much has been learned about basic biology from studies of insect model systems. The pre-eminent insect model system, Drosophila melanogaster, is a holometabolous insect with a derived mode of segment formation. While additional insect models have been pioneered in recent years, most of these fall within holometabolous lineages. In contrast, hemimetabolous insects have garnered less attention, although they include agricultural pests, vectors of human disease, and present numerous evolutionary novelties in form and function. The milkweed bug, Oncopeltus fasciatus (order: Hemiptera)-close outgroup to holometabolous insects-is an emerging model system. However, comparative studies within this order are limited as many phytophagous hemipterans are difficult to stably maintain in the lab due to their reliance on fresh plants, deposition of eggs within plant material, and long development time from embryo to adult. RESULTS: Here we present the harlequin bug, Murgantia histrionica, as a new hemipteran model species. Murgantia-a member of the stink bug family Pentatomidae which shares a common ancestor with Oncopeltus ~ 200 mya-is easy to rear in the lab, produces a large number of eggs, and is amenable to molecular genetic techniques. We use Murgantia to ask whether Pair-Rule Genes (PRGs) are deployed in ways similar to holometabolous insects or to Oncopeltus. Specifically, PRGs even-skipped, odd-skipped, paired and sloppy-paired are initially expressed in PR-stripes in Drosophila and a number of holometabolous insects but in segmental-stripes in Oncopeltus. We found that these genes are likewise expressed in segmental-stripes in Murgantia, while runt displays partial PR-character in both species. Also like Oncopeltus, E75A is expressed in a clear PR-pattern in blastoderm- and germband-stage Murgantia embryos, although it plays no role in segmentation in Drosophila. Thus, genes diagnostic of the split between holometabolous insects and Oncopeltus are expressed in an Oncopeltus-like fashion during Murgantia development. CONCLUSIONS: The similarity in gene expression between Murgantia and Oncopeltus suggests that Oncopeltus is not a sole outlier species in failing to utilize orthologs of Drosophila PRGs for PR-patterning. Rather, strategies deployed for PR-patterning, including the use of E75A in the PRG-network, are likely conserved within Hemiptera, and possibly more broadly among hemimetabolous insects.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Shifting roles of Drosophila pair-rule gene orthologs: segmental expression and function in the milkweed bug Oncopeltus fasciatus.

The discovery of pair-rule genes (PRGs) in Drosophila revealed the existence of an underlying two-segment-wide prepattern directing embryogenesis. The milkweed bug Oncopeltus fasciatus, a hemimetabolous insect, is a more representative arthropod: most of its segments form sequentially after gastrulation. Here, we report the expression and function of orthologs of the complete set of nine Drosophila PRGs in Oncopeltus Seven Of-PRG-orthologs are expressed in stripes in the primordia of every segment, rather than every other segment; Of-runt is PR-like and several orthologs are also expressed in the segment addition zone. RNAi-mediated knockdown of Of-odd-skipped, paired and sloppy-paired impacted all segments, with no indication of PR-like register. We confirm that Of-E75A is expressed in PR-like stripes, although it is not expressed in this way in Drosophila, demonstrating the existence of an underlying PR-like prepattern in Oncopeltus These findings reveal that a switch occurred in regulatory circuits, leading to segment formation: while several holometabolous insects are 'Drosophila-like', using PRG orthologs for PR patterning, most Of-PRGs are expressed segmentally in Oncopeltus, a more basally branching insect. Thus, an evolutionarily stable phenotype - segment formation - is directed by alternate regulatory pathways in diverse species.

Dynamic expression of Drosophila segmental cell surface-encoding genes and their pair-rule regulators.

Drosophila segmentation is regulated by a complex network of transcription factors that include products of the pair-rule genes (PRGs). PRGs are expressed in early embryos in the primorida of alternate segmental units, establishing the repeated, segmental body plan of the fly. Despite detailed analysis of the regulatory logic among segmentation genes, the relationship between these genes and the morphological formation of segments is still poorly understood, since regulation of transcription factor expression is not sufficient to explain how segments actually form and are maintained. Cell surface proteins containing Leucine rich repeats (LRR) play a variety of roles in development, and those expressed in segmental patterns likely impact segment morphogenesis. Here we explore the relationships between the PRG network and segmentally expressed LRR-encoding (sLRR) genes. We examined expression of Toll2, Toll6, Toll7, Toll8 and tartan (trn) in wild type or PRG mutant embryos. Expression of each sLRR-encoding gene is dynamic, but each has a unique register along the anterior-posterior axis. The registers for different sLRRs are off-set from one another resulting in a continually changing set of overlapping expression patterns among the sLRR-encoding genes themselves and between the sLRR-encoding genes and the PRGs. Accordingly, each sLRR-encoding gene is regulated by a unique combination of PRGs. These findings suggest that one role of the PRG network is to promote segmentation by establishing a cell surface code: each row of cells in the two-segment-wide primordia expresses a unique combination of sLRRs, thereby translating regulatory information from the PRGs to direct segment morphogenesis.

Effect of Genetic Diagnosis on Patients with Previously Undiagnosed Disease.

BACKGROUND: Many patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added. METHODS: We evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care. RESULTS: A total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes. CONCLUSIONS: The UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.).

Solution Nuclear Magnetic Resonance Studies of the Ligand-Binding Domain of an Orphan Nuclear Receptor Reveal a Dynamic Helix in the Ligand-Binding Pocket.

The ligand-binding domains (LBDs) of the NR5A subfamily of nuclear receptors activate transcription via ligand-dependent and ligand-independent mechanisms. The Drosophila Ftz-F1 receptor (NR5A3) belongs to the latter category, and its ligand independence is attributed to a short helical segment (α6) within the protein that resides in the canonical ligand-binding pocket (LBP) in the crystalline state. Here, we show that the α6 helix is dynamic in solution when Ftz-F1 is bound to the LxxLL motif of its cofactor Ftz, undergoing motions on the fast (picosecond to nanosecond) as well as slow (microsecond to millisecond) time scales. Motions on the slow time scale (∼10-3 s) appear to pervade throughout the domain, most prominently in the LBP and residues at or near the cofactor-binding site. We ascribe the fast time scale motions to a solvent-accessible conformation for the α6 helix akin to those described for its orthologs in higher organisms. We assign this conformation where the LBP is "open" to a lowly populated species, while the major conformer bears the properties of the crystal structure where the LBP is "closed". We propose that these conformational transitions could allow binding to small molecule ligands and/or play a role in dissociation of the cofactor from the binding site. Indeed, we show that the Ftz-F1 LBD can bind phospholipids, not unlike its orthologs. Our studies provide the first detailed insights into intrinsic motions occurring on a variety of time scales in a nuclear receptor LBD and reveal that potentially functionally significant motions pervade throughout the domain in solution, despite evidence to the contrary implied by the crystal structure.

Conservation and variation in pair-rule gene expression and function in the intermediate-germ beetle Dermestes maculatus.

A set of pair-rule (PR) segmentation genes (PRGs) promotes the formation of alternate body segments in Drosophila melanogaster Whereas Drosophila embryos are long-germ, with segments specified more or less simultaneously, most insects add segments sequentially as the germband elongates. The hide beetle Dermestes maculatus represents an intermediate between short- and long-germ development, ideal for comparative study of PRGs. We show that eight of nine Drosophila PRG orthologs are expressed in stripes in Dermestes Functional results parse these genes into three groups: Dmac-eve, -odd and -run play roles in both germband elongation and PR patterning; Dmac-slp and -prd function exclusively as complementary, classic PRGs, supporting functional decoupling of elongation and segment formation; and orthologs of ftz, ftz-f1, h and opa show more variable function in Dermestes and other species. While extensive cell death generally prefigured Dermestes PRG RNAi-mediated cuticle defects, an organized region with high mitotic activity near the margin of the segment addition zone is likely to have contributed to truncation of eveRNAi embryos. Our results suggest general conservation of clock-like regulation of PR stripe addition in sequentially segmenting species while highlighting regulatory rewiring involving a subset of PRG orthologs.

Establishment of molecular genetic approaches to study gene expression and function in an invasive hemipteran, Halyomorpha halys.

Hemiptera is a large clade of insects understudied in terms of developmental biology. Halyomorpha halys, the Brown Marmorated Stink Bug (BMSB, referred to throughout as H. halys), is an invasive hemipteran pest of the mid-Atlantic region of the USA that has rapidly spread to other regions in recent years, devastating a wide range of crops using a piercing and sucking mechanism. Its phylogenetic position, polyphagous habits, and rapid spread in the USA suggested that H. halys would be an ideal system to broaden our knowledge of developmental mechanisms in insects. We and others previously generated transcriptome sequences from different life stages of this insect. Here, we describe tools to examine gene expression patterns in whole-mount H. halys embryos and to test the response of H. halys to RNA interference (RNAi). We show that spatial and temporal patterns of gene expression in H. halys can be effectively monitored by both immunostaining and in situ hybridization. We also show that delivery of dsRNA to adult females knocks down gene function in offspring, using the homeotic gene Sex combs reduced (Scr). Knockdown of Hh-Scr resulted in dramatic malformations of the mouthparts, demonstrating for the first time that RNAi is effective in this species. Our results suggest that, despite difficulties with long-term laboratory culture of H. halys, this species shows promise as a developmental system.

The Function and Evolution of Nuclear Receptors in Insect Embryonic Development.

Nuclear receptors are a family of transcription factors that are often responsive to small ligands, allowing for efficient gene expression-level responses to a stimulus. The average insect has 21 genes encoding nuclear receptors, whose functions are especially well studied in developmental transitions during the insect life cycle, such as metamorphosis and molting. However, their utility as well-controlled transcriptional regulators also lends them to important roles in embryogenesis, neurogenesis, metabolism, and organogenesis. Such developmental functions have been explored in depth in the model organism Drosophila melanogaster. More recently, advances in genomic resources and functional genomic methodologies have allowed for comparison of nuclear receptor function among a wider range of insect species. As has been the trend throughout the field of Evo-Devo, these new data sets reveal that many genes are shared, but the ways in which they are utilized in different lineages are more variable. In this chapter, we describe the suite of nuclear receptor genes found in Drosophila and their developmental functions. We then compare and contrast these genes and their functions in diverse insects.

Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers.

The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern.

Evo-Devo: Discovery of Diverse Mechanisms Regulating Development.

Evo-devo is a relatively new field focusing on how mechanisms controlling development have changed during evolution. To date, studies of highly conserved members of an animal genetic toolkit have predominated in this field. These toolkit genes were originally identified in a small number of model organisms that do not represent the extant diversity of metazoans. We describe how this research paradigm came to be and what we have learned from it, and then argue that studies of other types of animals and of lineage-specific genes, in both model and nonmodel systems, are needed to expand our understanding of development and evolution.