Abstract: The ancestral arthropod is believed to have had a clustered arrangement of ten Hox genes. Within arthropods, Hox gene mutations result in transformation of segment identities. Despite the fact that variation in segment number/character was common in the diversification of arthropods, few examples of Hox gene gains/losses have been correlated with morphological evolution. Furthermore, a full appreciation of the variation in the genomic arrangement of Hox genes in extant arthropods has not been recognized, as genome sequences from each major arthropod clade have not been reported until recently. Initial genomic analysis of the chelicerate Tetranychus urticae suggested that loss of Hox genes and Hox gene clustering might be more common than previously assumed. To further characterize the genomic evolution of arthropod Hox genes, we compared the genomic arrangement and general characteristics of Hox genes from representative taxa from each arthropod subphylum. In agreement with others, we find arthropods generally contain ten Hox genes arranged in a common orientation in the genome, with an increasing number of sampled species missing either Hox3 or abdominal-A orthologs. The genomic clustering of Hox genes in species we surveyed varies significantly, ranging from 0.3 to 13.6 Mb. In all species sampled, arthropod Hox genes are dispersed in the genome relative to the vertebrate Mus musculus. Differences in Hox cluster size arise from variation in the number of intervening genes, intergenic spacing, and the size of introns and UTRs. In the arthropods surveyed, Hox gene duplications are rare and four microRNAs are, in general, conserved in similar genomic positions relative to the Hox genes. The tightly clustered Hox complexes found in the vertebrates are not evident within arthropods, and differential patterns of Hox gene dispersion are found throughout the arthropods. The comparative genomic data continue to support an ancestral arthropod Hox cluster of ten genes with a shared orientation, with four Hox gene-associated miRNAs, although the degree of dispersion between genes in an ancestral cluster remains uncertain. Hox3 and abdominal-A orthologs have been lost in multiple, independent lineages, and current data support a model in which inversions of the Abdominal-B locus that result in the loss of abdominal-A correlate with reduced trunk segmentation.

Abstract: Gonads are specialized gamete-producing structures that, despite their functional importance, are generated by diverse mechanisms across groups of animals and can be among the most plastic organs of the body. Annelids, the segmented worms, are a group in which gonads have been documented to be plastic and to be able to regenerate, but little is known about what factors influence gonad development or how these structures regenerate. In this study, we aimed to identify factors that influence the presence and size of gonads and to investigate gonad regeneration in the small asexually reproducing annelid, Pristina leidyi. We found that gonad presence and size in asexual adult P. leidyi are highly variable across individuals and identified several factors that influence these structures. An extrinsic factor, food availability, and two intrinsic factors, individual age and parental age, strongly influence the presence and size of gonads in P. leidyi. We also found that following head amputation in this species, gonads can develop by morphallactic regeneration in previously non-gonadal segments. We also identified a sexually mature individual from our laboratory culture that demonstrates that, although our laboratory strain reproduces only asexually, it retains the potential to become fully sexual. Our findings demonstrate that gonads in P. leidyi display high phenotypic plasticity and flexibility with respect to their presence, their size, and the segments in which they can form. Considering our findings along with relevant data from other species, we find that, as a group, clitellate annelids can form gonads in at least four different contexts: post-starvation refeeding, fission, morphallactic regeneration, and epimorphic regeneration. This group is thus particularly useful for investigating the mechanisms involved in gonad formation and the evolution of post-embryonic phenotypic plasticity.

Abstract: The Neotropical frog genera Ceratophrys, Chacophrys and Lepidobatrachus form the monophyletic family Ceratophryidae. Although in- and out-group relationships are not fully resolved, the monophyly of the three genera is well supported by both morphological and molecular data. Much is known about the morphology of the ceratophryids, but there is little comparative information on how modification of a common ancestral developmental pathway played a role in shaping their particular body plans. Herein, we review morphological variation during ceratophryid ontogeny in order to explore the role of development in their evolution. The ceratophryids are collectively characterized by rapid larval development with respect to other anurans, yet the three genera differ in their postmetamorphic growth rates to sexual maturity. Derived traits in the group can be divided into many homoplastic features that evolved in parallel with those of anurans with fossorial/burrowing behaviors in semiarid environments, and apomorphies. Morphological novelties have evolved in their feeding mechanism, which makes them capable of feeding on exceptional large prey. Lepidobatrachus is unusual in having reduced the ecomorphological differences between its larvae and adults. As a result, both the larvae and the frog are similarly able to capture large prey underwater. Some unique features in Lepidobatrachus are differentiated in the tadpole and then exaggerated in the adult (e.g., the posterior displaced jaw articulation) in a manner unobserved in any other anurans.

Abstract: Background Shifts in floral form across angiosperms, particularly from radially symmetrical to bilaterally symmetrical flowers, are often associated with shifts in speciation rates and changes in pollination syndrome. Growing evidence across both rosids and asterids indicates that CYCLOIDEA (CYC)-like transcription factors from the TCP gene family play a role in establishing the dorsoventral pattern of flower symmetry, which affects the development of both the corolla and androecium. Previous studies of CYC-like genes, especially of the CYC2 clade, indicate that these genes are dorsally restricted in bilaterally symmetrical flowers. Also, gene duplication of CYC-like genes often correlates with shifts in floral form in both individual flowers and head-like inflorescences (capitula).

Abstract: Genomic analysis has upended chordate phylogeny, placing the tunicates as the sister group to the vertebrates. This taxonomic rearrangement raises questions about the emergence of a tunicate/vertebrate ancestor. Characterization of developmental genes uniquely shared by tunicates and vertebrates is one promising approach for deciphering developmental shifts underlying acquisition of novel, ancestral traits. The matrix glycoprotein Fibronectin (FN) has long been considered a vertebrate-specific gene, playing a major instructive role in vertebrate embryonic development. However, the recent computational prediction of an orthologous “vertebrate-like” Fn gene in the genome of a tunicate, Ciona savignyi, challenges this viewpoint suggesting that Fn may have arisen in the shared tunicate/vertebrate ancestor. Here we verify the presence of a tunicate Fn ortholog. Transgenic reporter analysis was used to characterize a Ciona Fn enhancer driving expression in the notochord. Targeted knockdown in the notochord lineage indicates that FN is required for proper convergent extension. These findings suggest that acquisition of Fn was associated with altered notochord morphogenesis in the vertebrate/tunicate ancestor.

Abstract: The evolution of animal segmentation is a major research focus within the field of evolutionary–developmental biology. Most studied segmented animals generate their segments in a repetitive, anterior-to-posterior fashion coordinated with the extension of the body axis from a posterior growth zone. In the current study we ask which selection pressures and ordering of evolutionary events may have contributed to the evolution of this specific segmentation mode. To answer this question we extend a previous in silico simulation model of the evolution of segmentation by allowing the tissue growth pattern to freely evolve. We then determine the likelihood of evolving oscillatory sequential segmentation combined with posterior growth under various conditions, such as the presence or absence of a posterior morphogen gradient or selection for determinate growth. We find that posterior growth with sequential segmentation is the predominant outcome of our simulations only if a posterior morphogen gradient is assumed to have already evolved and selection for determinate growth occurs secondarily. Otherwise, an alternative segmentation mechanism dominates, in which divisions occur in large bursts through the entire tissue and all segments are created simultaneously. Our study suggests that the ancestry of a posterior signalling centre has played an important role in the evolution of sequential segmentation. In addition, it suggests that determinate growth evolved secondarily, after the evolution of posterior growth. More generally, we demonstrate the potential of evo-devo simulation models that allow us to vary conditions as well as the onset of selection pressures to infer a likely order of evolutionary innovations.

Abstract: Basic Helix-Loop-Helix (bHLH) genes encode a large family of eukaryotic transcription factors, categorized into six high-order groups: pan-eukaryotic group B involved in regulation of cell cycle, metabolism, and development; holozoan-specific groups C and F involved in development and maintenance of homeostasis; and metazoan-specific groups A, D and E including well-studied genes, such as Atonal, Twist and Hairy, with diverse developmental roles including control of morphogenesis and specification of neurons. Current scenarios of bHLH evolution in animals are mainly based on the bHLH gene set found in the genome of demosponge Amphimedon queenslandica. In this species, the majority of the 21 identified bHLH genes belong to group B, and the single group A gene is orthologous to several neurogenic bilaterian subfamilies, including atonal and neurogenin. Given recently discovered differences in developmental toolkit components between siliceous and calcareous sponges, we have carried out genome-wide analysis of bHLH genes in Sycon ciliatum, an emerging calcisponge model. We identified 30 bHLH genes in this species, representing 12 individual families, including four group A families not found in Amphimedon, and two larger family groupings. Notably, the families represented in Sycon are only partially overlapping with those represented in Amphimedon. Developmental expression analysis of a subset of the identified genes revealed patterns consistent with deeply conserved roles, such as specification of sensory cells by Atona-related and stem cells by Myc genes. Our results demonstrate independent gene loss events in demosponges and calcisponges, implying a complex bHLH toolkit in the last common metazoan ancestor.

Abstract: Annelida is a morphologically diverse animal group that exhibits a remarkable variety in nervous system architecture (e.g., number and location of longitudinal cords, architecture of the brain). Despite this heterogeneity of neural arrangements, the molecular profiles related to central nervous system patterning seem to be conserved even between distantly related annelids. In particular, comparative molecular studies on brain and anterior neural region patterning genes have focused so far mainly on indirect-developing macrofaunal taxa. Therefore, analyses on microscopic, direct-developing annelids are important to attain a general picture of the evolutionary events underlying the vast diversity of annelid neuroanatomy. We have analyzed the expression domains of 11 evolutionarily conserved genes involved in brain and anterior neural patterning in adult females of the direct-developing meiofaunal annelid Dinophilus gyrociliatus. The small, compact brain shows expression of dimmed, foxg, goosecoid, homeobrain, nk2.1, orthodenticle, orthopedia, pax6, six3/6 and synaptotagmin-1. Although most of the studied markers localize to specific brain areas, the genes six3/6 and synaptotagmin-1 are expressed in nearly all perikarya of the brain. All genes except for goosecoid, pax6 and nk2.2 overlap in the anterior brain region, while the respective expression domains are more separated in the posterior brain. Our findings reveal that the expression patterns of the genes foxg, orthodenticle, orthopedia and six3/6 correlate with those described in Platynereis dumerilii larvae, and homeobrain, nk2.1, orthodenticle and synaptotagmin-1 resemble the pattern of late larvae of Capitella teleta. Although data on other annelids are limited, molecular similarities between adult Dinophilus and larval Platynereis and Capitella suggest an overall conservation of molecular mechanisms patterning the anterior neural regions, independent from developmental and ecological strategies, or of the size and configuration of the nervous system.

Abstract: The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle “cellular conveyor belts” of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Abstract: Bocconia and Macleaya are the only genera of the poppy family (Papaveraceae) lacking petals; however, the developmental and genetic processes underlying such evolutionary shift have not yet been studied. We studied floral development in two species of petal-less poppies Bocconia frutescens and Macleaya cordata as well as in the closely related petal-bearing Stylophorum diphyllum. We generated a floral transcriptome of B. frutescens to identify MADS-box ABCE floral organ identity genes expressed during early floral development. We performed phylogenetic analyses of these genes across Ranunculales as well as RT-PCR and qRT-PCR to assess loci-specific expression patterns. We found that petal-to-stamen homeosis in petal-less poppies occurs through distinct developmental pathways. Transcriptomic analyses of B. frutescens floral buds showed that homologs of all MADS-box genes are expressed except for the APETALA3-3 ortholog. Species-specific duplications of other ABCE genes in B. frutescens have resulted in functional copies with expanded expression patterns than those predicted by the model. Petal loss in B. frutescens is likely associated with the lack of expression of AP3-3 and an expanded expression of AGAMOUS. The genetic basis of petal identity is conserved in Ranunculaceae and Papaveraceae although they have different number of AP3 paralogs and exhibit dissimilar floral groundplans.

Abstract: Organisms develop and evolve in a modular fashion, but how individual modules interact with the environment remains poorly understood. Phenotypically plastic traits are often under selection, and studies are needed to address how traits respond to the environment in a modular fashion. In this study, tissue-specific plasticity of melanic spots was examined in the large milkweed bug, Oncopeltus fasciatus. Although the size of the abdominal melanic bands varied according to rearing temperatures, wing melanic bands were more robust. To explore the regulation of abdominal pigmentation plasticity, candidate genes involved in abdominal melanic spot patterning and biosynthesis of melanin were analyzed. While the knockdown of dopa decarboxylase (Ddc) led to lighter pigmentation in both the wings and the abdomen, the shape of the melanic elements remained unaffected. Although the knockdown of Abdominal-B (Abd-B) partially phenocopied the low-temperature phenotype, the abdominal bands were still sensitive to temperature shifts. These observations suggest that regulators downstream of Abd-B but upstream of DDC are responsible for the temperature response of the abdomen. Ablation of wings led to the regeneration of a smaller wing with reduced melanic bands that were shifted proximally. In addition, the knockdown of the Wnt signaling nuclear effector genes, armadillo 1 and armadillo 2, altered both the melanic bands and the wing shape. Thus, the pleiotropic effects of Wnt signaling may constrain the amount of plasticity in wing melanic bands. We propose that when traits are regulated by distinct pre-patterning mechanisms, they can respond to the environment in a modular fashion, whereas when the environment impacts developmental regulators that are shared between different modules, phenotypic plasticity can manifest as a developmentally integrated system.

Abstract: Dbx1 is a homeodomain transcription factor involved in neuronal fate specification belonging to a widely conserved family among bilaterians. In mammals, Dbx1 was proposed to act as a transcriptional repressor by interacting with the Groucho corepressors to allow the specification of neurons involved in essential biological functions such as locomotion or breathing. Sequence alignments of Dbx1 proteins from different species allowed us to identify two conserved domains related to the Groucho-dependent Engrailed repressor domain (RD), as well as a newly described domain composed of clusterized acidic residues at the C-terminus (Cter) which is present in tetrapods but also several invertebrates. Using a heterologous luciferase assay, we showed that the two putative repressor domains behave as such in a Groucho-dependent manner, whereas the Cter does not bear any intrinsic transcriptional activity. Consistently with in vitro data, we found that both RDs are involved in cell fate specification using in vivo electroporation experiments in the chick spinal cord. Surprisingly, we show that the Cter domain is required for Dbx1 function in vivo, acting as a modulator of its repressive activity and/or imparting specificity. Our results strongly suggest that the presence of a Cter domain among tetrapods is essential for Dbx1 to regulate neuronal diversity and, in turn, nervous system complexity.

Abstract: Kinorhynchs are ecdysozoan animals with a phylogenetic position close to priapulids and loriciferans. To understand the nature of segmentation within Kinorhyncha and to infer a probable ancestry of segmentation within the last common ancestor of Ecdysozoa, the musculature and the nervous system of the allomalorhagid kinorhynch Pycnophyes kielensis were investigated by use of immunohistochemistry, confocal laser scanning microscopy, and 3D reconstruction software. The kinorhynch body plan comprises 11 trunk segments. Trunk musculature consists of paired ventral and dorsal longitudinal muscles in segments 1–10 as well as dorsoventral muscles in segments 1–11. Dorsal and ventral longitudinal muscles insert on apodemes of the cuticle inside the animal within each segment. Strands of longitudinal musculature extend over segment borders in segments 1–6. In segments 7–10, the trunk musculature is confined to the segments. Musculature of the digestive system comprises a strong pharyngeal bulb with attached mouth cone muscles as well as pharyngeal bulb protractors and retractors. The musculature of the digestive system shows no sign of segmentation. Judged by the size of the pharyngeal bulb protractors and retractors, the pharyngeal bulb, as well as the introvert, is moved passively by internal pressure caused by concerted action of the dorsoventral muscles. The nervous system comprises a neuropil ring anterior to the pharyngeal bulb. Associated with the neuropil ring are flask-shaped serotonergic somata extending anteriorly and posteriorly. A ventral nerve cord is connected to the neuropil ring and runs toward the anterior until an attachment point in segment 1, and from there toward the posterior with one ganglion in segment 6. Segmentation within Kinorhyncha likely evolved from an unsegmented ancestor. This conclusion is supported by continuous trunk musculature in the anterior segments 1–6, continuous pharyngeal bulb protractors and retractors throughout the anterior segments, no sign of segmentation within the digestive system, and the absence of ganglia in most segments. The musculature shows evidence of segmentation that fit the definition of an anteroposteriorly repeated body unit only in segments 7–10.

Abstract: Here we present a report on Ctenopalooza: A meeting of ctenophorologists held at the Whitney Laboratory for Marine Bioscience in St. Augustine, FL, USA, on March 14–15, 2016. In this report, we provide a summary of each of the sessions that occurred during this two-day meeting, which touched on most of the relevant areas of ctenophore biology. The report includes some major themes regarding the future of ctenophore research that emerged during Ctenopalooza. More information can be found at the meeting Web site: http://ctenopalooza.whitney.ufl.edu .

Abstract: An essential developmental pathway in sexually reproducing animals is the specification of germ cells and the differentiation of mature gametes, sperm and oocytes. The “germline” genes vasa, nanos and piwi are commonly identified in primordial germ cells, suggesting a molecular signature for the germline throughout animals. However, these genes are also expressed in a diverse set of somatic stem cells throughout the animal kingdom leaving open significant questions for whether they are required for germline specification. Similarly, members of the Dmrt gene family are essential components regulating sex determination and differentiation in bilaterian animals, but the functions of these transcription factors, including potential roles in sex determination, in early diverging animals remain unknown. The phylogenetic position of ctenophores and the genome sequence of the lobate Mnemiopsis leidyi motivated us to determine the compliment of these gene families in this species and determine expression patterns during development. Our phylogenetic analyses of the vasa, piwi and nanos gene families show that Mnemiopsis has multiple genes in each family with multiple lineage-specific paralogs. Expression domains of Mnemiopsis nanos, vasa and piwi, during embryogenesis from fertilization to the cydippid stage, were diverse, with little overlapping expression and no or little expression in what we think are the germ cells or gametogenic regions. piwi paralogs in Mnemiopsis had distinct expression domains in the ectoderm during development. We observed overlapping expression domains in the apical organ and tentacle apparatus of the cydippid for a subset of “germline genes,” which are areas of high cell proliferation, suggesting that these genes are involved with “stem cell” specification and maintenance. Similarly, the five Dmrt genes show diverse non-overlapping expression domains, with no clear evidence for expression in future gametogenic regions of the adult. We also report on splice variants for two Mnemiopsis Dmrt genes that impact the presence and composition of the DM DNA binding domain for these transcription factors. Our results are consistent with a potential role for vasa, piwi and nanos genes in the specification or maintenance of somatic stem cell populations during development in Mnemiopsis. These results are similar to previous results in the tentaculate ctenophore Pleurobrachia, with the exception that these genes were also expressed in gonads and developing gametes of adult Pleurobrachia. These differences suggest that the Mnemiopsis germline is either specified later in development than hypothesized, the germline undergoes extensive migration, or the germline does not express these classic molecular markers. Our results highlight the utility of comparing expression of orthologous genes across multiple species. We provide the first description of Dmrt expression in a ctenophore, which indicates that Dmrt genes are expressed in distinct structures and regions during development but not in future gametogenic regions, the only sex-specific structure for this hermaphroditic species.

Abstract: The evolutionary mechanisms involved in shaping complex gene regulatory networks (GRN) that encode for morphologically similar structures in distantly related animals remain elusive. In this context, echinoderm larval skeletons found in brittle stars and sea urchins provide an ideal system. Here, we characterize for the first time the development of the larval skeleton in the ophiuroid Amphiura filiformis and compare it systematically with its counterpart in sea urchin. We show that ophiuroids and euechinoids, that split at least 480 Million years ago (Mya), have remarkable similarities in tempo and mode of skeletal development. Despite morphological and ontological similarities, our high-resolution study of the dynamics of genetic regulatory states in A. filiformis highlights numerous differences in the architecture of their underlying GRNs. Importantly, the A.filiformis pplx, the closest gene to the sea urchin double negative gate (DNG) repressor pmar1, fails to drive the skeletogenic program in sea urchin, showing important evolutionary differences in protein function. hesC, the second repressor of the DNG, is co-expressed with most of the genes that are repressed in sea urchin, indicating the absence of direct repression of tbr, ets1/2, and delta in A. filiformis. Furthermore, the absence of expression in later stages of brittle star skeleton development of key regulatory genes, such as foxb and dri, shows significantly different regulatory states. Our data fill up an important gap in the picture of larval mesoderm in echinoderms and allows us to explore the evolutionary implications relative to the recently established phylogeny of echinoderm classes. In light of recent studies on other echinoderms, our data highlight a high evolutionary plasticity of the same nodes throughout evolution of echinoderm skeletogenesis. Finally, gene duplication, protein function diversification, and cis-regulatory element evolution all contributed to shape the regulatory program for larval skeletogenesis in different branches of echinoderms.

Abstract: Background: The microvillus is a versatile organelle that serves important functions in disparate animal cell types. However, from a molecular perspective, the microvillus has been well studied in only a few, predominantly vertebrate, contexts. Little is known about how differences in microvillar structure contribute to differences in function, and how these differences evolved. We sequenced the transcriptome of the freshwater sponge, Ephydatia muelleri, and examined the expression of vertebrate microvillar gene homologs in choanocytes—the only microvilli-bearing cell type present in sponges. Sponges offer a distant phylogenetic comparison with vertebrates, and choanocytes are central to discussions about early animal evolution due to their similarity with choanoflagellates, the single-celled sister line-age of modern animals. Results: We found that, from a genomic perspective, sponges have conserved homologs of most vertebrate microvillar genes, most of which are expressed in choanocytes, and many of which exhibit choanocyte-specific or choanocyte-enriched expression. Possible exceptions include the cadherins that form intermicrovillar links in the enterocyte brush border and hair cell stereocilia of vertebrates and cnidarians. No obvious orthologs of these proteins were detected in sponges, but at least four candidate cadherins were identified as choanocyte-enriched and might serve this function. In contrast to the evidence for conserved microvillar structure in sponges and vertebrates, we found that choanoflagellates and ctenophores lack homologs of many fundamental microvillar genes, suggesting that microvillar structure may diverge significantly in these lineages, warranting further study. Conclusions: The available evidence suggests that microvilli evolved early in the prehistory of modern animals and have been repurposed to serve myriad functions in different cellular contexts. Detailed understanding of the sequence by which different microvilli-bearing cell/tissue types diversified will require further study of microvillar composition and development in disparate cell types and lineages. Of particular interest are the microvilli of choano-flagellates, ctenophores, and sponges, which collectively bracket the earliest events in animal evolution.

Abstract: Background Obligate intracellular symbionts of insects are metabolically and developmentally integrated with their hosts. Typically, reproduction fails in many insect nutritional endosymbioses when host insects are cured of their bacterial symbionts, and yet remarkably little is known about the processes that developmentally integrate host and symbiont. Here in the best studied insect obligate intracellular symbiosis, that of the pea aphid, Acyrthosiphon pisum, with the gammaproteobacterium Buchnera aphidicola, we tracked the expression and localization of amino acid transporter ApGLNT1 gene products during asexual embryogenesis. Recently being characterized as a glutamine transporter, ApGLNT1 has been proposed to be a key regulator of amino acid biosynthesis in A. pisum bacteriocytes. To determine when this important mediator of the symbiosis becomes expressed in aphid embryonic bacteriocytes, we applied whole-mount in situ hybridization and fluorescent immunostaining with a specific anti-ApGLNT1 antibody to detect the temporal and spatial expression of ApGLNT1 gene products during asexual embryogenesis.

Abstract: Many insects host their obligate, maternally transmitted symbiotic bacteria in specialized cells called bacteriocytes. One of the best-studied insect nutritional endosymbioses is that of the aphid and its endosymbiont, Buchnera aphidicola. Aphids and Buchnera are metabolically and developmentally integrated, but the molecular mechanisms underlying Buchnera transmission and coordination with aphid development remain largely unknown. Previous work using electron microscopy to study aphid asexual embryogenesis has revealed that Buchnera transmission involves exocytosis from a maternal bacteriocyte followed by endocytotic uptake by a blastula. While the importance of exo- and endocytic cellular processes for symbiont transmission is clear, the molecular mechanisms that regulate these processes are not known. Here, we shed light on the molecular mechanisms that regulate Buchnera transmission and developmental integration. We present the developmental atlas of ACYPI000536 and ACYPI008904 mRNAs during asexual embryogenesis in the pea aphid, Acyrthosiphon pisum. Immediately before Buchnera invasion, transcripts of both genes were detected by whole-mount in situ hybridization in the posterior syncytial nuclei of late blastula embryos. Following Buchnera invasion, expression of both genes was identified in the region occupied by Buchnera throughout embryogenesis. Notably during Buchnera migration, expression of both genes was not concomitant with the entirety of the bacterial mass but rather expression colocalized with Buchnera in the anterior region of the bacterial mass. In addition, we found that ACYPI000536 was expressed in nuclei at the leading edge of the bacterial mass, joining the bacterial mass in subsequent developmental stages. Finally, quantitative reverse transcription real-time PCR suggested that early in development both transcripts were maternally provisioned to embryos. We venture that ACYPI000536 and ACYPI008904 function as nutrient sensors at the site of symbiont invasion to facilitate TOR-pathway-mediated endocytosis of Buchnera by the aphid blastula. Our data support earlier reports of bacteriocyte determination involving a two-step recruitment process but suggest that the second wave of recruitment occurs earlier than previously described. Finally, our work highlights that bacteriocyte-enriched amino acid transporter paralogs have additionally been retained to play novel developmental roles in both symbiont recruitment and bacteriome development.

Abstract: Platynereis and other polychaete annelids with homonomous segmentation are regarded to closely resemble ancestral forms of bilateria. The head region comprises the prostomium, the peristomium, a variable number of cephalized body segments and several appendages, like cirri, antennae and palps. The trunk of such polychaetes shows numerous, nearly identical segments. Each segment bears a parapodium with species-specific morphology on either side. The posterior end of the trunk features a segment proliferation zone and a terminal pygidium with the anus and anal cirri. The removal of a substantial part of the posterior trunk is by no means lethal. Cells at the site of injury dedifferentiate and proliferate forming a blastema to regenerate both the pygidium and the proliferation zone. The pygidium forms new anal cirri, and the proliferation zone generates new segments at a rapid pace. The formation of body appendages like the cirri and the segmental parapodia can thus be studied in the caudal regenerate of Platynereis within only a few days. The development of body appendages in Platynereis is regulated by a network of genes common to polychaetes but also shared by distant taxa. We isolated DNA sequences from P. dumerilii of five genes known to be involved in appendage formation within other groups: Meis/homothorax, Pbx1/extradenticle, Dlx/Distal-less, decapentaplegic and specific protein 1/buttonhead. Analyses of expression patterns during caudal regeneration by in situ hybridization reveal striking similarities related to expression in arthropods and vertebrates. All genes exhibit transient expression during differentiation and growth of segments. As was shown previously in other phyla Pdu-Meis/hth and Pdu-Pbx1/exd are co-expressed, although the expression is not limited to the proximal part of the parapodia. Pdu-Dll is prominent in parapodia but upregulated in the anal cirri. No direct dependence concerning Pdu-Dll and Pdu-sp/btd expression is observed in Platynereis. Pdu-dpp shows an expression pattern not comparable to its expression in other taxa. The expression patterns observed suggest conserved roles of these genes during appendage formation across different clades, but the underlying mechanisms utilizing this toolset might not be identical. Some genes show broad expression along the proximodistal axis indicating a possible role in proximodistal patterning of body appendages. Other genes exhibit expression patterns limited to specific parts and tissues of the growing parapodia, thus presumably being involved in formation of taxon-specific morphological differences.

Abstract: The aquiferous body plan of poriferans revolves around internal chambers comprised of choanocytes, a cell type structurally similar to choanoflagellates. These choanocyte chambers perform a range of physiological and developmental functions, including the capture of food and the generation of stem cells. Despite the increasing interest for choanocytes as sponge stem cells, there is limited knowledge on the development of choanocyte chambers. Using a combination of cell lineage tracing, antibody staining and EdU labeling, here we examine the development of choanocytes and the chambers they comprise during metamorphosis in the marine demosponge Amphimedon queenslandica. Lineage-tracing experiments show that larval epithelial cells transform into mesenchymal pluripotent stem cells, resembling archeocytes, within 24 h of initiating metamorphosis. By 36 h, some of these labeled archeocyte-like cells have differentiated into choanocytes that will form the first postlarval choanocyte chambers. Non-labeled cells also contribute to these primary choanocyte chambers, consistent with these chambers being a chimera of multiple transdifferentiated larval cell types and not the proliferation of a single choanocyte precursor. Moreover, cell proliferation assays demonstrate that, following the initial formation of choanocyte chambers, chambers grow at least partially by the proliferation of choanocytes within the chamber, although recruitment of individual cells into established chambers also appears to occur. EdU labeling of postlarvae and juveniles reveals that choanocyte chambers are the primary location of cell proliferation during metamorphosis. Our results show that multiple larval cell lineages typically contribute to formation of individual choanocyte chambers at metamorphosis, contrary to previous reports in other species that show sponge choanocyte chambers form clonally. Choanocytes in postlarval and juvenile A. queenslandica chambers can also divide, with choanocyte chambers being the primary location of cell proliferation. Interestingly, the level of cell proliferation varies greatly between chambers and appears to be contingent on the size, location and developmental state of the chamber. Small chambers on the periphery of the body tend to possess more dividing cells. As choanocytes can also dedifferentiate into archeocyte-like cells, cell proliferation in chambers may not only contribute to chamber growth and self-renewal but also increase the number of pluripotent archeocytes.

Abstract: New branches within the embryonic chicken lung form via apical constriction, in which epithelial cells in the primary bronchus become trapezoidal in shape. These branches form at precise locations along the primary bronchus that scale relative to the size of the organ. Here, we examined the extent to which this scaling relationship and branching mechanism are conserved within lungs of three species of birds. Analyzing the development of embryonic lungs from chicken, quail, and duck, as well as lungs explanted and cultured ex vivo, revealed that the patterns of branching are remarkably conserved. In particular, secondary bronchi form at identical positions in chicken and quail, the patterns of which are indistinguishable, consistent with the close evolutionary relationship of these two species. In contrast, secondary bronchi form at slightly different positions in duck, the lungs of which are significantly larger than those of chicken and quail at the same stage of development. Confocal analysis of fixed specimens revealed that each secondary bronchus forms by apical constriction of the dorsal epithelium of the primary bronchus, a morphogenetic mechanism distinct from that used to create branches in mammalian lungs. Our findings suggest that monopodial branching off the primary bronchus is driven by apical constriction in lungs of chicken, quail, and duck. The relative positions at which these branches form are also conserved relative to the evolutionary relationship of these species. It will be interesting to determine whether these mechanisms hold in more distant species of birds, and why they differ so significantly in mammals.

Abstract: Germ cells are specified during early development and are responsible for generating gametes in the adult. After germ cells are specified, they typically migrate to a particular niche in the organism where they reside for the remainder of its lifetime. For some model organisms, the specification and migration of germ cells have been extensively studied, but how these events occur in animals that reproduce both sexually and asexually is not well understood. We have identified a novel TGF-β family member in Botryllus schlosseri, tgfβ-f, and found that it is expressed by follicle cell progenitors and the differentiated follicle and support cells surrounding the maturing gametes. Using the expression of tgfβ-f and the germ cell marker vasa, we have found that nearly all germ cells in Botryllus are associated with tgfβ-f-expressing follicle progenitors in clusters consisting solely of those two cell types. These clusters were mostly small, consisting of ten or fewer cells, and generally contained between a 2:1 and 1:1 ratio of follicle progenitors to germ cells. Clusters of germ and follicle progenitor cells were primarily localized to niches in the primary and secondary buds, but could also be found in other locations including the vasculature. We analyzed the location of germ cell clusters throughout the asexual life cycle of Botryllus and found that at the stage when germ cells are first detected in the secondary bud niche, a dramatic change in the size and location of germ/follicle cell clusters also occurred. Our findings suggest that germ/follicle cell clusters have predictable migratory patterns during the weekly asexual developmental cycle in Botryllus. An increased number of small clusters and the presence of clusters in the vasculature coinciding with the appearance of clusters in the secondary bud suggest that fragmentation of clusters and the migration of smaller clusters through the vasculature may be an important aspect of Botryllus reproductive biology, ensuring the transmission of the germline to subsequent asexual generations.

Abstract: Cichlid fishes from the Rift Lakes of East Africa have undergone the most spectacular adaptive radiations in vertebrate history. Eco-morphological adaptations in lakes Victoria, Malawi and Tanganyika have resulted in a vast array of skull shapes and sizes, yet primary axes of morphological variation are conserved in all three radiations, prominently including the size of the preorbital region of the skull. This conserved pattern suggests that development may constrain the trajectories of cichlid head morphological evolution. Here, we (1) present a comparative analysis of adult head morphology in two sand-dweller cichlids from Lake Malawi with preorbital size differences representative of the main axis of variation among the three lakes and (2) analyze the ontogeny of shape and size differences by focusing on known developmental modules throughout the head. We find that (1) developmental differences between the two species correlate with known developmental modules; (2) differences in embryonic cartilage development result in phenotypically integrated changes among all bones derived from a single cartilage, while differences in dermal bone development tend to influence isolated regions within a bone; and lastly (3) species-specific morphologies appear in the embryo as subtle differences, which become progressively amplified throughout ontogeny. We propose that this amplification takes place at skeletal growth zones, the locations and shapes of which are patterned during embryogenesis. This study is the most anatomically comprehensive analysis of the developmental differences underlying cichlid skull evolution in the Rift Lakes of East Africa. The scale of our analysis reveals previously unnoticed correlations between developmental modules and patterns of phenotypic integration. We propose that the primary axes of morphological variation among East African cichlid adaptive radiations are constrained by the hierarchical modularity of the teleost head skeleton.

Abstract: The forelimb of the flightless emu is a vestigial structure, with greatly reduced wing elements and digit loss. To explore the molecular and cellular mechanisms associated with the evolution of vestigial wings and loss of flight in the emu, key limb patterning genes were examined in developing embryos. Limb development was compared in emu versus chicken embryos. Immunostaining for cell proliferation markers was used to analyze growth of the emu forelimb and hindlimb buds. Expression patterns of limb patterning genes were studied, using whole-mount in situ hybridization (for mRNA localization) and RNA-seq (for mRNA expression levels). The forelimb of the emu embryo showed heterochronic development compared to that in the chicken, with the forelimb bud being retarded in its development. Early outgrowth of the emu forelimb bud is characterized by a lower level of cell proliferation compared the hindlimb bud, as assessed by PH3 immunostaining. In contrast, there were no obvious differences in apoptosis in forelimb versus hindlimb buds (cleaved caspase 3 staining). Most key patterning genes were expressed in emu forelimb buds similarly to that observed in the chicken, but with smaller expression domains. However, expression of Sonic Hedgehog (Shh) mRNA, which is central to anterior–posterior axis development, was delayed in the emu forelimb bud relative to other patterning genes. Regulators of Shh expression, Gli3 and HoxD13, also showed altered expression levels in the emu forelimb bud. These data reveal heterochronic but otherwise normal expression of most patterning genes in the emu vestigial forelimb. Delayed Shh expression may be related to the small and vestigial structure of the emu forelimb bud. However, the genetic mechanism driving retarded emu wing development is likely to rest within the forelimb field of the lateral plate mesoderm, predating the expression of patterning genes.