smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods.

Recently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualization and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

A postlarval instar of Phoxichilidium femoratum (Pycnogonida, Phoxichilidiidae) with an exceptional malformation.

Individuals of the marine chelicerate lineage Pycnogonida (sea spiders) show considerable regenerative capabilities after appendage injury or loss. In their natural habitats, especially the long legs of sea spiders are commonly lost and regenerated, as is evidenced by the frequent encounter of specimens with missing or miniature legs. In contrast to this, the collection of individuals with abnormally developed appendages or trunk regions is comparably rare. Here, we studied a remarkable malformation in a postlarval instar of the species Phoxichilidium femoratum (Rathke, 1799) and describe the external morphology and internal organization of the specimen using a combination of fluorescent histochemistry and scanning electron microscopy. The individual completely lacks the last trunk segment with leg pair 4 and the normally penultimate trunk segment bears only a single aberrant appendage resembling an extension of the anteroposterior body axis. Externally, the proximal units of the articulated appendage are unpaired, but further distally a bifurcation into two equally developed leg-like branches is found. Three-dimensional reconstruction of the musculature reveals components of two regular leg muscle sets in several of the proximal articles. This confirms interpretation of the entire appendage as a malformed leg and reveals an externally hidden paired organization along its entire proximodistal axis. To explain the origin of this unique malformation, early pioneering studies on the regenerative potential of pycnogonids are evaluated and (a) an injury-induced partial fusion of the developing limb buds of leg pair 3, as well as (b) irregular leg regeneration following near complete loss of trunk segments 3 and 4 are discussed. Which of the two hypotheses is more realistic remains to be tested by dedicated experimental approaches. These will have to rely on pycnogonid species with established laboratory husbandry in order to overcome the limitations of the few short-term regeneration studies performed to date.

Evolutionary dynamics of gene regulation.

The long controversy over the importance of changes in the regulatory genome has been resolved with the recognition that such changes are a fundamental component of evolutionary dynamics. Comparative studies have revealed four dominant modes of change as the regulatory genome evolved: (1) the origin of regulatory novelties such as distal enhancers and new types of promoters at the origin of Metazoa; (2) the expansion of regulatory capacity, most notably with diversification of transcription factors. Together these changes expanded the available combinatoric complexity of regulatory interactions and allow an increase in the variety of cell types. There are two more common modes of regulatory evolution: (3) Repatterning of gene regulatory networks. Such repatterning largely involves the introduction of transposons, promoter switching, co-option of regulatory genes or subcircuits, recombination, and the de novo generation of new regulatory sequences. Finally, (4) changes in enhancer and promoter specificity enable fine-scale adaptive changes. One of the outstanding issues at the intersection of evolutionary and developmental biology is how these various modes of regulatory evolution translate to morphological change, and particularly macro- and microevolutionary patterns and whether evolutionary novelties are associated with distinctive patterns of regulatory change.

Experimental duplication of bilaterian body axes in spider embryos: Holm's organizer and self-regulation of embryonic fields.

Bilaterally symmetric body plans of vertebrates and arthropods are defined by a single set of two orthogonal axes, the anterior-posterior (or head-tail) and dorsal-ventral axes. In vertebrates, and especially amphibians, complete or partial doubling of the bilaterian body axes can be induced by two different types of embryological manipulations: transplantation of an organizer region or bi-sectioning of an embryo. Such axis doubling relies on the ability of embryonic fields to flexibly respond to the situation and self-regulate toward forming a whole body. This phenomenon has facilitated experimental efforts to investigate the mechanisms of vertebrate body axes formation. However, few studies have addressed the self-regulatory capabilities of embryonic fields associated with body axes formation in non-vertebrate bilaterians. The pioneer spider embryologist Åke Holm reported twinning of spider embryos induced by both types of embryological manipulations in 1952; yet, his experiments have not been replicated by other investigators, and access to spider or non-vertebrate twins has been limited. In this review, we provide a historical background on twinning experiments in spiders, and an overview of current twinning approaches in familiar spider species and related molecular studies. Moreover, we discuss the benefits of the spider model system for a deeper understanding of the ancestral mechanisms of body axes formation in arthropods, as well as in bilaterians.

Functional analysis of engrailed in Tribolium segmentation.

The segment-polarity gene engrailed is required for segmentation in the early Drosophila embryo. Loss of Engrailed function results in segmentation defects that vary in severity from pair-rule phenotypes to a lawn phenotype lacking in obvious of segmentation. During segmentation, Engrailed is expressed in stripes with a single segmental periodicity in Drosophila, which is conserved in all arthropods examined so far. To define segments, the segmental stripes of Engrailed induce the segmental stripes of wingless at each parasegmental boundary. However, segmentation functions of orthologs of engrailed in non-Drosophila arthropods have yet to be reported. Here, we analyzed functions of the Tribolium ortholog of engrailed (Tc-engrailed) during embryonic segmentation. Larval cuticles with Tc-engrailed being knocked down had segmentation phenotypes including incomplete segment formation and loss of a group of segments. In agreement with the cuticle segmentation defects, segments developed incompletely and irregularly or did not form in Tribolium germbands where Tc-engrailed was knocked down. Furthermore, knock-down of Tc-engrailed did not properly express the segmental stripes of wingless in Tribolium germbands. Taken together with the conserved expression patterns of Engrailed in arthropod segmentation, our data suggest that Tc-engrailed is required for embryonic segmentation in Tribolium, and the genetic mechanism of Engrailed inducing wingless expression is conserved at least between Drosophila and Tribolium.

The hallmarks of GSK-3 in morphogenesis and embryonic development metabolism in arthropods.

Several research groups around the world have studied diverse aspects of energy metabolism in arthropod disease vectors, with the aim of discovering potential control targets. As in all oviparous organisms, arthropod embryonic development is characterized by the mobilization of maternally-derived metabolites for the formation of new tissues and organs. Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase described as an important regulator of metabolism and development in a wide range of organisms. GSK-3 was first identified based on its action upon glycogen synthase, a central enzyme in glycogen biosynthesis. Currently, it is recognized as a key component of multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, cell migration, and immune response. The present review will describe the current knowledge on GSK-3 activation and its role in morphogenesis and embryonic metabolism in arthropods. Altogether, the information discussed here can spark new approaches and strategies for further studies, enhancing our understanding of these important arthropod vectors and strengthening the resources in the search for novel control methods.

Arthropod segmentation.

There is now compelling evidence that many arthropods pattern their segments using a clock-and-wavefront mechanism, analogous to that operating during vertebrate somitogenesis. In this Review, we discuss how the arthropod segmentation clock generates a repeating sequence of pair-rule gene expression, and how this is converted into a segment-polarity pattern by 'timing factor' wavefronts associated with axial extension. We argue that the gene regulatory network that patterns segments may be relatively conserved, although the timing of segmentation varies widely, and double-segment periodicity appears to have evolved at least twice. Finally, we describe how the repeated evolution of a simultaneous (Drosophila-like) mode of segmentation within holometabolan insects can be explained by heterochronic shifts in timing factor expression plus extensive pre-patterning of the pair-rule genes.

Expression of NK genes that are not part of the NK cluster in the onychophoran Euperipatoides rowelli (Peripatopsidae).

BACKGROUND: NK genes are a group of homeobox transcription factors that are involved in various molecular pathways across bilaterians. They are typically divided into two subgroups, the NK cluster (NKC) and NK-linked genes (NKL). While the NKC genes have been studied in various bilaterians, corresponding data of many NKL genes are missing to date. To further investigate the ancestral roles of NK family genes, we analyzed the expression patterns of NKL genes in the onychophoran Euperipatoides rowelli. RESULTS: The NKL gene complement of E. rowelli comprises eight genes, including BarH, Bari, Emx, Hhex, Nedx, NK2.1, vax and NK2.2, of which only NK2.2 was studied previously. Our data for the remaining seven NKL genes revealed expression in different structures associated with the developing nervous system in embryos of E. rowelli. While NK2.1 and vax are expressed in distinct medial regions of the developing protocerebrum early in development, BarH, Bari, Emx, Hhex and Nedx are expressed in late developmental stages, after all major structures of the nervous system have been established. Furthermore, BarH and Nedx are expressed in distinct mesodermal domains in the developing limbs. CONCLUSIONS: Comparison of our expression data to those of other bilaterians revealed similar patterns of NK2.1, vax, BarH and Emx in various aspects of neural development, such as the formation of anterior neurosecretory cells mediated by a conserved molecular mechanism including NK2.1 and vax, and the development of the central and peripheral nervous system involving BarH and Emx. A conserved role in neural development has also been reported from NK2.2, suggesting that the NKL genes might have been primarily involved in neural development in the last common ancestor of bilaterians or at least nephrozoans (all bilaterians excluding xenacoelomorphs). The lack of comparative data for many of the remaining NKL genes, including Bari, Hhex and Nedx currently hampers further evolutionary conclusions. Hence, future studies should focus on the expression of these genes in other bilaterians, which would provide a basis for comparative studies and might help to better understand the role of NK genes in the diversification of bilaterians.

Chelicerates.

Compared to other arthropods, such as crustaceans or insects, the term 'chelicerate' often does not evoke a similar sense of recognition or familiarity. Yet the subphylum Chelicerata has been encountered by every living person today, frequently to the effect of fear, awe, or outright revulsion. Chelicerates include such familiar groups as spiders, scorpions, mites, and ticks, as well as an array of bizarre and unfamiliar forms, such as vinegaroons, camel spiders, and hooded tick spiders (Figure 1).

Embryonic expression patterns and phylogenetic analysis of panarthropod sox genes: insight into nervous system development, segmentation and gonadogenesis.

BACKGROUND: Sox (Sry-related high-mobility-group box) genes represent important factors in animal development. Relatively little, however, is known about the embryonic expression patterns and thus possible function(s) of Sox genes during ontogenesis in panarthropods (Arthropoda+Tardigrada+Onychophora). To date, studies have been restricted exclusively to higher insects, including the model system Drosophila melanogaster, with no comprehensive data available for any other arthropod group, or any tardigrade or onychophoran. RESULTS: This study provides a phylogenetic analysis of panarthropod Sox genes and presents the first comprehensive analysis of embryonic expression patterns in the flour beetle Tribolium castaneum (Hexapoda), the pill millipede Glomeris marginata (Myriapoda), and the velvet worm, Euperipatoides kanangrensis (Onychophora). 24 Sox genes were identified and investigated: 7 in Euperipatoides, 8 in Glomeris, and 9 in Tribolium. Each species possesses at least one ortholog of each of the five expected Sox gene families, B, C, D, E, and F, many of which are differentially expressed during ontogenesis. CONCLUSION: Sox gene expression (and potentially function) is highly conserved in arthropods and their closest relatives, the onychophorans. Sox B, C and D class genes appear to be crucial for nervous system development, while the Sox B genes Dichaete (D) and Sox21b likely play an additional conserved role in panarthropod segmentation. The Sox B gene Sox21a likely has a conserved function in foregut and Malpighian tubule development, at least in Hexapoda. The data further suggest that Sox D and E genes are involved in mesoderm differentiation, and that Sox E genes are involved in gonadal development. The new data expand our knowledge about the expression and implied function of Sox genes to Mandibulata (Myriapoda+Pancrustacea) and Panarthropoda (Arthropoda+Onychophora).

Embryonic expression of a Long Toll (Loto) gene in the onychophorans Euperipatoides kanangrensis and Cephalofovea clandestina.

Recent research has shown that Toll genes, and in particular a newly defined class of Toll genes, the so-called Long Toll Genes (Loto genes), are crucial factors in embryogenesis. In arthropods, they are involved in axis formation via a process called convergent extension (CE). A hallmark of Loto genes is their relatively (compared to other Toll genes) high number of leucine-rich repeat elements (LRRs) coupled with the fact that they are expressed in transverse stripes in all segments, or a subset of segments, patterns that are reminiscent of classical segmentation genes such as the pair-rule genes. Onychophorans represent a close outgroup to the arthropods; however, their embryonic development differs substantially. It is unclear if convergent extension contributes to onychophoran germ band formation and, if so, whether Loto genes are involved in governing this process. This study identifies a single onychophoran Toll gene from a sequenced embryonic transcriptome in two onychophoran species. The identified gene shows sequence and expression pattern characteristics of Loto genes. However, its expression pattern also comprises some general differences to arthropod Loto genes that are involved in CE.

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.

Metamorphosis Is Ancestral for Crown Euarthropods, and Evolved in the Cambrian or Earlier.

Macroevolutionary developmental biology employs fossilized ontogenetic data and phylogenetic comparative methods to probe the evolution of development at ancient nodes. Despite the prevalence of ecologically differentiated larval forms in marine invertebrates, it has been frequently presumed that the ancestors of arthropods were direct developers, and that metamorphosis may not have evolved until the Ordovician or later. Using fossils and new dated phylogenies, I infer that metamorphosis was likely ancestral for crown arthropods, contradicting this assumption. Based on a published morphological dataset encompassing 217 exceptionally preserved fossil and 96 extant taxa, fossils were directly incorporated into both the topology and age estimates, as in "tip dating" analyses. Using data from post-embryonic fossils representing 25 species throughout stem and crown arthropod lineages (as well as most of the 96 extant taxa), characters for metamorphosis were assigned based on inferred ecological changes in development (e.g., changes in habitat and adaptive landscape). Under all phylogenetic hypotheses, metamorphosis was supported as most likely ancestral to both ecdysozoans and euarthropods. Care must be taken to account for potential drastic post-embryonic morphological changes in evolutionary analyses. Many stem group euarthrpods may have had ecologically differentiated larval stages that did not preserve in the fossil record. Moreover, a complex life cycle and planktonic ecology may have evolved in the Ediacaran or earlier, and may have typified the pre-Cambrian explosion "wormworld" prior to the origin of crown group euarthropods.

Developmental and Evolutionary Perspectives on the Origin and Diversification of Arthropod Appendages.

Jointed, segmented appendages are a key innovation of arthropods. The subsequent diversification of these appendages, both along the body axis and across taxa, has contributed to the evolutionary success of arthropods. Both developmental and fossil data are informative for understanding how these transitions occurred. Comparative analyses help to pinpoint the developmental novelties that distinguish arthropod appendages from the lobopodous appendages of other panarthropods, and that distinguish different appendage types. The fossil record of stem group arthropods is diverse and preserves intermediate steps in these evolutionary transitions, including some that cannot be directly inferred based on extant taxa. These lead to hypotheses that can be tested with comparative developmental data, as well as to reinterpretations of developmental results. One developmental novelty of arthropods is the reiterated deployment of the joint formation network, which divides the appendages into segments. The fossil record raises questions about how this joint formation network was first deployed, given the contrasting morphologies of appendages in stem group versus extant arthropods. The fossil record supports a character tree for appendage diversification showing progressive individuation of appendages in an anterior-to-posterior sequence. However, to date, developmental evidence provides at best limited support for this character tree. Recent interpretations of the fossil record suggest that the labrum of extant arthropods is a greatly reduced protocerebral appendage pair; this hypothesis is consistent with the extensive shared developmental patterning of the labrum and jointed appendages. Reciprocal illumination from fossils and developmental patterning in a phylogenetic context both makes sense of some results and helps motivates questions for future research.

The Evolution of Arthropod Body Plans: Integrating Phylogeny, Fossils, and Development-An Introduction to the Symposium.

The last few years have seen a significant increase in the amount of data we have about the evolution of the arthropod body plan. This has come mainly from three separate sources: a new consensus and improved resolution of arthropod phylogeny, based largely on new phylogenomic analyses; a wealth of new early arthropod fossils from a number of Cambrian localities with excellent preservation, as well as a renewed analysis of some older fossils; and developmental data from a range of model and non-model pan-arthropod species that shed light on the developmental origins and homologies of key arthropod traits. However, there has been relatively little synthesis among these different data sources, and the three communities studying them have little overlap. The symposium "The Evolution of Arthropod Body Plans-Integrating Phylogeny, Fossils and Development" brought together leading researchers in these three disciplines and made a significant contribution to the emerging synthesis of arthropod evolution, which will help advance the field and will be useful for years to come.

Development, Trait Evolution, and the Evolution of Development in Trilobites.

Trilobites offer one of the best fossil records of any arthropod group. This is due to a number of factors, most notably the combination of (1) having inhabited areas where organisms are more likely to be buried and ultimately fossilized; and (2) having had a highly biomineralized exoskeleton more likely to survive the stresses of fossilization. This biomineralized exoskeleton was also morphologically complex, bearing traits that had ecological significance, and was present throughout postembryonic development, from larval to adult stages. Because the morphology of the exoskeleton changed gradually across molts during development, it is possible to reconstruct ontogenetic series for many species. Over the last decade, studies have documented both variation in modularity among closely related species and conserved developmental patterns among modules. In the latter case, trait evolution could still occur through modification of rates of morphological change along otherwise conserved ontogenetic trajectories. At the clade level, the pattern of expression and release of new exoskeletal segments during post-embryonic development was generally conserved across most species, but the relative timing of different segmentation events could vary, and developmental traits appear to have been relatively labile across the clade's evolutionary history. Most recently, comparative analyses indicate that the association between segmentation events and the timing of shifts in the rate of ontogenetic shape change varies across species. Despite these advances, we still know relatively little about how development constrained or contributed to trait evolution in trilobites, and almost nothing about the origin of novel traits in trilobites. A major (but removable) obstacle is the current lack of well-supported trilobite phylogenies that span higher taxonomic levels.

Conserved versus derived patterns of controlled cell death during the embryonic development of two species of Onychophora (velvet worms).

BACKGROUND: Apoptosis is involved in various developmental processes, including cell migration and tissue and organ formation. Some of these processes are conserved across metazoans, while others are specific to particular taxa. Although the patterns of apoptosis have been investigated in arthropods, no corresponding data are available from one of their closest relatives, the Onychophora (velvet worms). RESULTS: We analyzed the patterns of apoptosis in embryos of two onychophoran species: the lecithotrophic/matrotrophic viviparous peripatopsid Euperipatoides rowelli, and the placentotrophic viviparous peripatid Principapillatus hitoyensis. Our data show that apoptosis occurs early in development and might be responsible for the degeneration of extra-embryonic tissues. Moreover, apoptosis might be involved in the morphogenesis of the ventral and preventral organs in both species and occurs additionally in the placental stalk of P. hitoyensis. CONCLUSIONS: Despite the different developmental modes in these onychophoran species, our data suggest that patterns of apoptosis are conserved among onychophorans. While apoptosis in the dorsal extra-embryonic tissue might contribute to dorsal closure-a process also known from arthropods-the involvement of apoptosis in ventral closure might be unique to onychophorans. Apoptosis in the placental stalk of P. hitoyensis is most likely a derived feature of the placentotrophic onychophorans. Developmental Dynamics 246:403-416, 2017. © 2017 Wiley Periodicals, Inc.