Drosophila Segmentation Research Articles

Domain-specific phenotypes in LINS1-related disorder-A Chinese family with the Q92X variant and literature review.

LINS1 is the human homolog of the Drosophila segment polarity gene that encodes an essential regulator of the wingless/Wnt signaling. By 2011, only seven pedigrees (16 patients) with eight causative variants in LINS1 gene have been reported. These cases mainly presented with infancy-/child-onset neurodevelopmental disorders, facial dysmorphia, and other clinical features, and a wide spectrum of clinically distinct phenotypes were also manifested. In our study, two brothers in a family were admitted and diagnosed with child-onset movement disorders, slight intellectual disability, psychological symptoms, eye problems, urinary and bowel dysfunction, mitral value prolapse, and Q-T prolongation. By exome sequencing, we identified a nonsense homozygous pathogenic variant (LINS1: c.274C > T (p.Q92X)), which had been reported in a case diagnosed with intellectual disability and psychiatric disorders (such as schizophrenia and anxiety). Compared with this case, the clinical features of our cases were distinct. In particular, our cases displayed unusual features of heart and blood system. Furthermore, the genotype-phenotype relationship analysis suggested that distinct phenotypes presented in cases carrying variants in different domains of the LINS1 gene. In conclusions, our findings suggest the high clinical variations in the LINS1 variants-related disorders. Moreover, the Q92X might be a recurrent variant in Hans of Southern China.

Founding the Wnt gene family: How wingless was found to be a positional signal and oncogene homolog.

The Wnt family of developmental regulators were named after the Drosophila segmentation gene wingless and the murine proto-oncogene int-1. Homology between these two genes connected oncogenesis to cell-cell signals in development. I review how wingless was initially characterized, and cloned, as part of the quest to identify developmental cell-to-cell signals, based on predictions of the Positional Information Model, and on the properties of homeotic and segmentation gene mutants. The requirements and cell-nonautonomy of wingless in patterning multiple embryonic and adult structures solidified its status as a candidate signaling molecule. The physical location of wingless mutations and transcription unit defined the gene and its developmental transcription pattern. When the Drosophila homolog of int-1 was then isolated, and predicted to encode a secreted proto-oncogene homolog, it's identity to the wingless gene confirmed that a developmental cell-cell signal had been identified and connected cancer to development.

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 striped expression, with all 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.

Automatic mechanistic inference from large families of Boolean models generated by Monte Carlo tree search.

Many important processes in biology, such as signaling and gene regulation, can be described using logic models. These logic models are typically built to behaviorally emulate experimentally observed phenotypes, which are assumed to be steady states of a biological system. Most models are built by hand and therefore researchers are only able to consider one or perhaps a few potential mechanisms. We present a method to automatically synthesize Boolean logic models with a specified set of steady states. Our method, called MC-Boomer, is based on Monte Carlo Tree Search an efficient, parallel search method using reinforcement learning. Our approach enables users to constrain the model search space using prior knowledge or biochemical interaction databases, thus leading to generation of biologically plausible mechanistic hypotheses. Our approach can generate very large numbers of data-consistent models. To help develop mechanistic insight from these models, we developed analytical tools for multi-model inference and model selection. These tools reveal the key sets of interactions that govern the behavior of the models. We demonstrate that MC-Boomer works well at reconstructing randomly generated models. Then, using single time point measurements and reasonable biological constraints, our method generates hundreds of thousands of candidate models that match experimentally validated in-vivo behaviors of the Drosophila segment polarity network. Finally we outline how our multi-model analysis procedures elucidate potentially novel biological mechanisms and provide opportunities for model-driven experimental validation.

Shell field morphogenesis in the polyplacophoran mollusk Acanthochitona rubrolineata

BackgroundThe polyplacophoran mollusks (chitons) possess serially arranged shell plates. This feature is unique among mollusks and believed to be essential to explore the evolution of mollusks as well as their shells. Previous studies revealed several cell populations in the dorsal epithelium (shell field) of polyplacophoran larvae and their roles in the formation of shell plates. Nevertheless, they provide limited molecular information, and shell field morphogenesis remains largely uninvestigated.ResultsIn the present study, we investigated shell field development in the chiton Acanthochitona rubrolineata based on morphological characteristics and molecular patterns. A total of four types of tissue could be recognized from the shell field of A. rubrolineata. The shell field comprised not only the centrally located, alternatively arranged plate fields and ridges, but also the tissues surrounding them, which were the precursors of the girdle and we termed as the girdle field. The girdle field exhibited a concentric organization composed of two circularly arranged tissues, and spicules were only developed in the outer circle. Dynamic engrailed expression and F-actin (filamentous actin) distributions revealed relatively complicated morphogenesis of the shell field. The repeated units (plate fields and ridges) were gradually established in the shell field, seemingly different from the manners used in the segmentation of Drosophila or vertebrates. The seven repeated ridges also experienced different modes of ontogenesis from each other. In the girdle field, the presumptive spicule-formation cells exhibited different patterns of F-actin aggregations as they differentiate.ConclusionsThese results reveal the details concerning the structure of polyplacophoran shell field as well as its morphogenesis. They would contribute to exploring the mechanisms of polyplacophoran shell development and molluscan shell evolution.

Patterning, From Conifers to Consciousness: Turing's Theory and Order From Fluctuations.

This is a brief account of Turing’s ideas on biological pattern and the events that led to their wider acceptance by biologists as a valid way to investigate developmental pattern, and of the value of theory more generally in biology. Periodic patterns have played a key role in this process, especially 2D arrays of oriented stripes, which proved a disappointment in theoretical terms in the case of Drosophila segmentation, but a boost to theory as applied to skin patterns in fish and model chemical reactions. The concept of “order from fluctuations” is a key component of Turing’s theory, wherein pattern arises by selective amplification of spatial components concealed in the random disorder of molecular and/or cellular processes. For biological examples, a crucial point from an analytical standpoint is knowing the nature of the fluctuations, where the amplifier resides, and the timescale over which selective amplification occurs. The answer clarifies the difference between “inelegant” examples such as Drosophila segmentation, which is perhaps better understood as a programmatic assembly process, and “elegant” ones expressible in equations like Turing’s: that the fluctuations and selection process occur predominantly in evolutionary time for the former, but in real time for the latter, and likewise for error suppression, which for Drosophila is historical, in being lodged firmly in past evolutionary events. The prospects for a further extension of Turing’s ideas to the complexities of brain development and consciousness is discussed, where a case can be made that it could well be in neuroscience that his ideas find their most important application.

The Nasonia pair-rule gene regulatory network retains its function over 300 million years of evolution.

ABSTRACTInsect segmentation is a well-studied and tractable system with which to investigate the genetic regulation of development. Though insects segment their germband using a variety of methods, modelling work implies that a single gene regulatory network can underpin the two main types of insect segmentation. This means limited genetic changes are required to explain significant differences in segmentation mode between different insects. This idea needs to be tested in a wider variety of species, and the nature of the gene regulatory network (GRN) underlying this model has not been tested. Some insects, e.g. Nasonia vitripennis and Apis mellifera segment progressively, a pattern not examined in previous studies of this segmentation model, producing stripes at different times progressively through the embryo, but not from a segment addition zone. Here, we aim to understand the GRNs patterning Nasonia using a simulation-based approach. We found that an existing model of Drosophila segmentation ( Clark, 2017) can be used to recapitulate the progressive segmentation of Nasonia, if provided with altered inputs in the form of expression of the timer genes Nv-caudal and Nv-odd paired. We predict limited topological changes to the pair-rule network and show, by RNAi knockdown, that Nv-odd paired is required for morphological segmentation. Together this implies that very limited changes to the Drosophila network are required to simulate Nasonia segmentation, despite significant differences in segmentation modes, implying that Nasonia use a very similar version of an ancestral GRN used by Drosophila, which must therefore have been conserved for at least 300 million years.

Practicing logical reasoning through Drosophila segmentation gene mutants.

Laboratory practical sessions are critical to scientific training in biology but usually fail to promote logical and hypothesis-driven reasoning and rely heavily on the teacher's instructions. This paper describes a 2-day laboratory practicum in which students prepare and analyze larval cuticle preparations of Drosophila segmentation gene mutant strains. Embryonic segmentation involves three major classes of genes according to their loss-of-function phenotypes: the establishment of broad regions by gap genes, the specification of the segments by the pair-rule genes, and the compartments within segments by the segment polarity genes. Students are asked to sort undefined segmentation mutants into gap, pair-rule, or segment polarity categories based on their knowledge of the Drosophila segmentation process and the microscopic anatomical traits they are capable of finding in the sample preparations. This technically simple practicum prompts students to pay attention to detailed observation to detect anatomic markers of intrasegmental compartments and thorax versus abdomen cuticle, and promote their logical reasoning in hypothesizing to which segmentation type a given mutant fits best.

Dynamic Modeling of Transcriptional Gene Regulatory Networks.

Diverse cellular phenotypes are determined by groups of transcription factors (TFs) and other regulators that influence each others' gene expression, forming transcriptional gene regulatory networks (GRNs). In many biological contexts, especially in development and associated diseases, the expression of the genes in GRNs is not static but evolves in time. Modeling the dynamics of GRN state is an important approach for understanding diverse cellular phenomena such as cell-fate specification, pluripotency and cell-fate reprogramming, oncogenesis, and tissue regeneration. In this protocol, we describe how to model GRNs using a data-driven dynamic modeling methodology, gene circuits. Gene circuits do not require knowledge of the GRN topology and connectivity but instead learn them from training data, making them very general and applicable to diverse biological contexts. We utilize the MATLAB-based gene circuit modeling software Fast Inference of Gene Regulation (FIGR) for training the model on quantitative gene expression data and simulating the GRN. We describe all the steps in the modeling life cycle, from formulating the model, training the model using FIGR, simulating the GRN, to analyzing and interpreting the model output. This protocol highlights these steps with the example of a dynamical model of the gap gene GRN involved in Drosophila segmentation and includes example MATLAB statements for each step.

Transcription Factor Binding Affinities and DNA Shape Readout.

SummaryAn essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. Here, we use a binding assay we recently established to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, we find that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates our approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.

Zygotic pioneer factor activity of Odd-paired/Zic is necessary for late function of the Drosophila segmentation network.

Because chromatin determines whether information encoded in DNA is accessible to transcription factors, dynamic chromatin states in development may constrain how gene regulatory networks impart embryonic pattern. To determine the interplay between chromatin states and regulatory network function, we performed ATAC-seq on Drosophila embryos during the establishment of the segmentation network, comparing wild-type and mutant embryos in which all graded maternal patterning inputs are eliminated. While during the period between zygotic genome activation and gastrulation many regions maintain stable accessibility, cis-regulatory modules (CRMs) within the network undergo extensive patterning-dependent changes in accessibility. A component of the network, Odd-paired (opa), is necessary for pioneering accessibility of late segmentation network CRMs. opa-driven changes in accessibility are accompanied by equivalent changes in gene expression. Interfering with the timing of opa activity impacts the proper patterning of expression. These results indicate that dynamic systems for chromatin regulation directly impact the reading of embryonic patterning information.

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

BackgroundMuch 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.ResultsHere 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.ConclusionsThe 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.

Post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced.

Homeotic selector (HOX) transcription factors (TFs) regulate gene expression that determines the identity of Drosophila segments along the anterior-posterior (A-P) axis. The current challenge with HOX proteins is understanding how they achieve their functional specificity while sharing a highly conserved homeodomain (HD) that recognize the same DNA binding sites. One mechanism proposed to regulate HOX activity is differential post-translational modification (PTM). As a first step in investigating this hypothesis, the sites of PTM on a Sex combs reduced protein fused to a triple tag (SCRTT) extracted from developing embryos were identified by Tandem Mass Spectrometry (MS/MS). The PTMs identified include phosphorylation at S185, S201, T315, S316, T317 and T324, acetylation at K218, S223, S227, K309, K434 and K439, formylation at K218, K309, K325, K341, K369, K434 and K439, methylation at S19, S166, K168 and T364, carboxylation at D108, K298, W307, K309, E323, K325 and K369, and hydroxylation at P22, Y87, P107, D108, D111, P269, P306, R310, N321, K325, Y334, R366, P392 and Y398. Of the 44 modifications, 18 map to functionally important regions of SCR. Besides a highly conserved DNA-binding HD, HOX proteins also have functionally important, evolutionarily conserved small motifs, which may be Short Linear Motifs (SLiMs). SLiMs are proposed to be preferential sites of phosphorylation. Although 6 of 7 phosphosites map to regions of predicted SLiMs, we find no support for the hypothesis that the individual S, T and Y residues of predicted SLiMs are phosphorylated more frequently than S, T and Y residues outside of predicted SLiMs.

Two pair-rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo.

While many developmentally relevant enhancers act in a modular fashion, there is growing evidence for nonadditive interactions between distinct cis-regulatory enhancers. We investigated if nonautonomous enhancer interactions underlie transcription regulation of the Drosophila segment polarity gene, wingless. We identified two wg enhancers active at the blastoderm stage: wg 3613u, located from -3.6 to -1.3 kb upstream of the wg transcription start site (TSS) and 3046d, located in intron two of the wg gene, from 3.0 to 4.6 kb downstream of the TSS. Genetic experiments confirm that Even Skipped (Eve), Fushi-tarazu (Ftz), Runt, Odd-paired (Opa), Odd-skipped (Odd), and Paired (Prd) contribute to spatially regulated wg expression. Interestingly, there are enhancer specific differences in response to the gain or loss of function of pair-rule gene activity. Although each element recapitulates aspects of wg expression, a composite reporter containing both enhancers more faithfully recapitulates wg regulation than would be predicted from the sum of their individual responses. These results suggest that the regulation of wg by pair-rule genes involves nonadditive interactions between distinct cis-regulatory enhancers.

Quantitative analysis reveals genotype- and domain- specific differences between mRNA and protein expression of segmentation genes in Drosophila

In many biological systems gene expression at mRNA and protein levels is not identical. Rigorous comparison of such differences on a spatio-temporal scale is still not feasible by high-throughput transcriptomic and proteomic analyses of early embryo development. Here, we characterize differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. We obtained quantitative imaging data on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression we use several criteria including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains we show examples where protein expression does not repeat mRNA expression even after a temporal delay. We calculated time delays between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+/Kr- heterozygotes. We detect that in wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. We further show that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, we show that the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, we analyze differences in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo.

Growth zone segmentation in the milkweed bug Oncopeltus fasciatus sheds light on the evolution of insect segmentation

BackgroundOne of the best studied developmental processes is the Drosophila segmentation cascade. However, this cascade is generally considered to be highly derived and unusual, with segments being patterned simultaneously, rather than the ancestral sequential segmentation mode. We present a detailed analysis of the segmentation cascade of the milkweed bug Oncopletus fasciatus, an insect with a more primitive segmentation mode, as a comparison to Drosophila, with the aim of reconstructing the evolution of insect segmentation modes.ResultsWe document the expression of 12 genes, representing different phases in the segmentation process. Using double staining we reconstruct the spatio-temporal relationships among these genes. We then show knock-down phenotypes of representative genes in order to uncover their roles and position in the cascade.ConclusionsWe conclude that sequential segmentation in the Oncopeltus germband includes three slightly overlapping phases: Primary pair-rule genes generate the first segmental gene expression in the anterior growth zone. This pattern is carried anteriorly by a series of secondary pair-rule genes, expressed in the transition between the growth zone and the segmented germband. Segment polarity genes are expressed in the segmented germband with conserved relationships. Unlike most holometabolous insects, this process generates a single-segment periodicity, and does not have a double-segment pattern at any stage. We suggest that the evolutionary transition to double-segment patterning lies in mutually exclusive expression patterns of secondary pair-rule genes. The fact that many aspects of the putative Oncopeltus segmentation network are similar to those of Drosophila, is consistent with a simple transition between sequential and simultaneous segmentation.

A quantitative study of the diversity of stripe-forming processes in an arthropod cell-based field undergoing axis formation and growth

One of the conserved traits of arthropod embryonic development is striped expression of homologs of Drosophila segment polarity genes, including hedgehog (hh). Although a diversity of stripe-forming processes is recognized among arthropod embryos, such varied stripe-forming processes have not been well characterized from cellular and quantitative perspectives. The spider Parasteatoda tepidariorum embryo, which has a hh-dependent mechanism of axis formation, offers a cell-based field where the stripes of Pt-hh (a hh homolog) expression dynamically develop in accordance with axis formation and growth, with the patterning processes varying among the regions of the field. In this study, using cell labeling, we mapped the future body subdivisions to the germ disc in the spider embryo and provided substantial evidence for the occurrence of kinetic waves of Pt-hh expression in the presumptive head and opisthosomal (or abdominal) regions of the embryonic field. Notably, combined with cell tracking, we showed that surface cells at and near the center of the germ disc persist in the posterior portion of the field from where Pt-hh stripes sequentially arise, suggesting the operation of ordered oscillations of Pt-hh expression. We then conducted a quantitative analysis of forming/formed Pt-hh stripes using serially timed fixation of sibling embryos. By utilizing length measurements that reflect the axis growth of the embryonic field, we reconstructed the pattern dynamics, which captured repeated splitting of Pt-hh stripes and oscillations of Pt-hh expression in the presumptive head and opisthosomal regions, respectively. In the intermediate thoracic region, three stripes of Pt-hh expression showed a late appearance, with the segmental units specified much earlier by another mechanism. Analyses provided quantitative estimates related to axis growth and stripe-splitting and oscillation events, including the periods of the patterning cycles. This work characterizes the diversity of stripe-forming processes in a cell-based field in a common spatiotemporal framework and highlights the contrasting dynamics of splitting versus oscillation. The cellular and quantitative data presented here provide the foundation for experimental, theoretical and evolutionary studies of cell-based pattern formation, especially body axis segmentation in arthropods.

A damped oscillator imposes temporal order on posterior gap gene expression in Drosophila

Insects determine their body segments in two different ways. Short-germband insects, such as the flour beetle Tribolium castaneum, use a molecular clock to establish segments sequentially. In contrast, long-germband insects, such as the vinegar fly Drosophila melanogaster, determine all segments simultaneously through a hierarchical cascade of gene regulation. Gap genes constitute the first layer of the Drosophila segmentation gene hierarchy, downstream of maternal gradients such as that of Caudal (Cad). We use data-driven mathematical modelling and phase space analysis to show that shifting gap domains in the posterior half of the Drosophila embryo are an emergent property of a robust damped oscillator mechanism, suggesting that the regulatory dynamics underlying long- and short-germband segmentation are much more similar than previously thought. In Tribolium, Cad has been proposed to modulate the frequency of the segmentation oscillator. Surprisingly, our simulations and experiments show that the shift rate of posterior gap domains is independent of maternal Cad levels in Drosophila. Our results suggest a novel evolutionary scenario for the short- to long-germband transition and help explain why this transition occurred convergently multiple times during the radiation of the holometabolan insects.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

Asymmetrically deployed actomyosin-based contractility generates a boundary between developing leg segments in Drosophila

The formation of complex tissues from simple epithelial sheets requires the regional subdivision of the developing tissue. This is initially accomplished by a sequence of gene regulatory hierarchies that set up distinct fates within adjacent territories, and rely on cross-regulatory interactions to do so. However, once adjacent territories are established, cells that confront one another across territorial boundaries must actively participate in maintaining separation from each other. Classically, it was assumed that adhesive differences would be a primary means of sorting cells to their respective territories. Yet it is becoming clear that no single, simple mechanism is at play. In the few instances studied, an emergent theme along developmental boundaries is the generation of asymmetry in cell mechanical properties. The repertoire of ways in which cells might establish and then put mechanical asymmetry to work is not fully appreciated since only a few boundaries have been molecularly studied. Here, we characterize once such boundary in the develop leg epithelium of Drosophila. The region of the pretarsus / tarsus is a known gene expression boundary that also exhibits a lineage restriction (Sakurai et al., 2007). We now show that the interface comprising this boundary is strikingly aligned compared to other cell interfaces across the disk. The boundary also exhibits an asymmetry for both Myosin II accumulation as well as one of its activators, Rho Kinase. Furthermore, the enrichment correlates with increased mechanical tension across that interface, and that tension is Rho Kinase-dependent. Lastly, interfering with actomyosin contractility, either by depletion of myosin heavy chain or expression of a phosphomimetic variant of regulatory light chain causes defects in alignment of the interfaces. These data suggest strongly that mechanical asymmetries are key in establishing and maintaining this developmental boundary.