Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms.

BACKGROUND: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. RESULTS: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository ( http://bitbucket.org/osiris_phylogenetics/pia/ ) and we demonstrate PIA on a publicly-accessible web server ( http://galaxy-dev.cnsi.ucsb.edu/pia/ ). CONCLUSIONS: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

Blue-light-receptive cryptochrome is expressed in a sponge eye lacking neurons and opsin.

Many larval sponges possess pigment ring eyes that apparently mediate phototactic swimming. Yet sponges are not known to possess nervous systems or opsin genes, so the unknown molecular components of sponge phototaxis must differ fundamentally from those in other animals, inspiring questions about how this sensory system functions. Here we present molecular and biochemical data on cryptochrome, a candidate gene for functional involvement in sponge pigment ring eyes. We report that Amphimedon queenslandica, a demosponge, possesses two cryptochrome/photolyase genes, Aq-Cry1 and Aq-Cry2. The mRNA of one gene (Aq-Cry2) is expressed in situ at the pigment ring eye. Additionally, we report that Aq-Cry2 lacks photolyase activity and contains a flavin-based co-factor that is responsive to wavelengths of light that also mediate larval photic behavior. These results suggest that Aq-Cry2 may act in the aneural, opsin-less phototaxic behavior of a sponge.

RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria.

BACKGROUND: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. RESULTS: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. CONCLUSION: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Gene duplication and the origins of morphological complexity in pancrustacean eyes, a genomic approach.

BACKGROUND: Duplication and divergence of genes and genetic networks is hypothesized to be a major driver of the evolution of complexity and novel features. Here, we examine the history of genes and genetic networks in the context of eye evolution by using new approaches to understand patterns of gene duplication during the evolution of metazoan genomes. We hypothesize that 1) genes involved in eye development and phototransduction have duplicated and are retained at higher rates in animal clades that possess more distinct types of optical design; and 2) genes with functional relationships were duplicated and lost together, thereby preserving genetic networks. To test these hypotheses, we examine the rates and patterns of gene duplication and loss evident in 19 metazoan genomes, including that of Daphnia pulex - the first completely sequenced crustacean genome. This is of particular interest because the pancrustaceans (hexapods+crustaceans) have more optical designs than any other major clade of animals, allowing us to test specifically whether the high amount of disparity in pancrustacean eyes is correlated with a higher rate of duplication and retention of vision genes. RESULTS: Using protein predictions from 19 metazoan whole-genome projects, we found all members of 23 gene families known to be involved in eye development or phototransduction and deduced their phylogenetic relationships. This allowed us to estimate the number and timing of gene duplication and loss events in these gene families during animal evolution. When comparing duplication/retention rates of these genes, we found that the rate was significantly higher in pancrustaceans than in either vertebrates or non-pancrustacean protostomes. Comparing patterns of co-duplication across Metazoa showed that while these eye-genes co-duplicate at a significantly higher rate than those within a randomly shuffled matrix, many genes with known functional relationships in model organisms did not co-duplicate more often than expected by chance. CONCLUSIONS: Overall, and when accounting for factors such as differential rates of whole-genome duplication in different groups, our results are broadly consistent with the hypothesis that genes involved in eye development and phototransduction duplicate at a higher rate in Pancrustacea, the group with the greatest variety of optical designs. The result that these genes have a significantly high number of co-duplications and co-losses could be influenced by shared functions or other unstudied factors such as synteny. Since we did not observe co-duplication/co-loss of genes for all known functional modules (e.g. specific regulatory networks), the interactions among suites of known co-functioning genes (modules) may be plastic at the temporal scale of analysis performed here. Other factors in addition to gene duplication - such as cis-regulation, heterotopy, and co-option - are also likely to be strong factors in the diversification of eye types.

Expansion, diversification, and expression of T-box family genes in Porifera.

Sponges are among the earliest diverging lineage within the metazoan phyla. Although their adult morphology is distinctive, at several stages of development, they possess characteristics found in more complex animals. The T-box family of transcription factors is an evolutionarily ancient gene family known to be involved in the development of structures derived from all germ layers in the bilaterian animals. There is an incomplete understanding of the role that T-box transcription factors play in normal sponge development or whether developmental pathways using the T-box family share similarities between parazoan and eumetazoan animals. To address these questions, we present data that identify several important T-box genes in marine and freshwater sponges, place these genes in a phylogenetic context, and reveal patterns in how these genes are expressed in developing sponges. Phylogenetic analyses demonstrate that sponges have members of at least two of the five T-box subfamilies (Brachyury and Tbx2/3/4/5) and that the T-box genes expanded and diverged in the poriferan lineage. Our analysis of signature residues in the sponge T-box genes calls into question whether "true" Brachyury genes are found in the Porifera. Expression for a subset of the T-box genes was elucidated in larvae from the marine demosponge, Halichondria bowerbanki. Our results show that sponges regulate the timing and specificity of gene expression for T-box orthologs across larval developmental stages. In situ hybridization reveals distinct, yet sometimes overlapping expression of particular T-box genes in free-swimming larvae. Our results provide a comparative framework from which we can gain insights into the evolution of developmentally important pathways.

Ontogeny of sexual dimorphism via tissue duplication in an ostracod (Crustacea).

The adaptive significance of specific sexual dimorphism is well studied. However, the evolutionary history and ontogenic origins of the dimorphism are often unknown. As dimorphism represents two phenotypes generated from relatively similar genotypes, it is of interest to understand both its evolutionary and developmental/genetic underpinnings. Here, we present the first ontogenetic examination of the eyes of philomedid ostracods (Crustacea), which exhibit extremely sexually dimorphic lateral eyes. Adult male philomedids have large compound lateral eyes, whereas females have rudimentary lateral eyes. First, we show that eye dimorphism is unlikely to be due to additional genes present on a male-specific chromosome because karyotype analysis suggests philomedids are XX/XO. We then examine the ontogeny of eye development and find that in at least two species of Euphilomedes, this dimorphism is not generated solely by differences in tissue growth rates, as has been commonly shown for sexually dimorphic characters of other species. Instead, the dimorphism appears to arise during development via tissue duplication, where a single tissue becomes two, perhaps with different developmental potentials. The second eye field is only observed in male Euphilomedes, producing most of the adult eye tissue. We point out that tissue duplication is a developmental process with evolutionary implications because novel characters could evolve via alternative modification of the duplicated fields, analogous to the origin of new genes by gene duplication and alternative modification. Depending on the evolutionary history of the duplicated field, it may have either facilitated or directly caused the observed sexual dimorphism of philomedid ostracods.

And Lophotrochozoa makes three: Notch/Hes signaling in annelid segmentation.

Segmentation is unquestionably a major factor in the evolution of complex body plans, but how this trait itself evolved is unknown. Approaching this problem requires comparing the molecular mechanisms of segmentation in diverse segmented and unsegmented taxa. Notch/Hes signaling is involved in segmentation in sequentially segmenting vertebrates and arthropods, as judged by patterns of expression of one or more genes in this network and by the disruption of segmental patterning when Notch/Hes signaling is disrupted. We have previously shown that Notch and Hes homologs are expressed in the posterior progress zone (PPZ), from which segments arise, in the leech Helobdella robusta, a sequentially segmenting lophotrochozoan (phylum Annelida). Here, we show that disrupting Notch/Hes signaling disrupts segmentation in this species as well. Thus, Notch/Hes functions in either the maintenance of the PPZ and/or the patterning processes of segmentation in representatives of all three superphyla of bilaterally symmetric animals. These results are consistent with two evolutionary scenarios. In one, segmentation was already present in the ancestor of all three superphyla. In the other, Notch/Hes signaling functioned in axial growth by terminal addition in an unsegmented bilaterian ancestor, and was subsequently exapted to function in segmentation as that process evolved independently in two or more taxa.

Secreted frizzled related protein is a target of PaxB and plays a role in aquiferous system development in the freshwater sponge, Ephydatia muelleri.

Canonical and non-canonical Wnt signaling, as well as the Pax/Six gene network, are involved in patterning the freshwater sponge aquiferous system. Using computational approaches to identify transcription factor binding motifs in a freshwater sponge genome, we located putative PaxB binding sites near a Secreted Frizzled Related Protein (SFRP) gene in Ephydatia muelleri. EmSFRP is expressed throughout development, but with highest levels in juvenile sponges. In situ hybridization and antibody staining show EmSFRP expression throughout the pinacoderm and choanoderm in a subpopulation of amoeboid cells that may be differentiating archeocytes. Knockdown of EmSFRP leads to ectopic oscula formation during development, suggesting that EmSFRP acts as an antagonist of Wnt signaling in E. muelleri. Our findings support a hypothesis that regulation of the Wnt pathway by the Pax/Six network as well as the role of Wnt signaling in body plan morphogenesis was established before sponges diverged from the rest of the metazoans.

Sexually Dimorphic Eye-Loss Driven by Ecological Selection in an Ostracod Crustacean: Support for the Reproductive Role Hypothesis.

Euphilomedes carcharodonta ostracods exhibit sex-specific eye-loss, with females lacking image-forming compound eyes compared with males and related species. The standard assumption is that sexual dimorphism is driven by sexual selection. However, previous work in E. carcharodonta suggests that male eyes are used to evade predators in the male-specific ecological niche, and that male-eyes lack the resolution to search for females. In this study, we examine whether sexual selection or ecological selection drives the retention of male eyes. Ecological niche differentiation was hypothesized by Darwin (1871) to be an alternative selective force for sexual dimorphism either through food competition or through dimorphic sex-role behavior, the reproductive role hypothesis. As of yet, there is little experimental evidence supporting this hypothesis. Here, we experimentally blindfold male E. carcharodonta to mimic the female phenotype and examine the effects on sex-specific niches and behavior. Blindfolding does not appear to grossly change male behavior, nor do females behave differently when exposed to blindfolded males. This lead us to conclude that the development of complex eyes in male E. arises from ecological selection rather than sexual selection.

Sexually dimorphic gene expression in the lateral eyes of Euphilomedes carcharodonta (Ostracoda, Pancrustacea).

BACKGROUND: The evolution and development of sexual dimorphism illuminates a central question in biology: How do similar genomes produce different phenotypes? In an XX/XO system especially the state of a sexually dimorphic trait is determined by differences in gene expression, as there are no additional genetic loci in either sex. Here, we examine the XX/XO ostracod crustacean species Euphilomedes carcharodonta. This species exhibits radical sexual dimorphism of their lateral eyes, females have only a tiny simple lateral eye while males have elaborate ommatidial eyes. RESULTS: We find that males express three of nine eye-development gene homologs at significantly higher levels during juvenile eye development, compared to females. We also find that most eye-development genes examined are pleiotropic, with high expression levels during embryonic development as well as during juvenile eye development. Later, in adults, we find that phototransduction genes are expressed at higher levels in males than in females, as we might expect when comparing ommatidial to simple eyes. CONCLUSIONS: We show here that expression changes of a handful of developmental genes may underlie the radical difference in a dimorphic character. This work gives an important point of comparison for studying eye evolution and development in the Pancrustacea.

Evasion of predators contributes to the maintenance of male eyes in sexually dimorphic Euphilomedes ostracods (Crustacea).

Sexual dimorphisms have long drawn the attention of evolutionary biologists. However, we still have much to learn about the evolutionary, genetic, and developmental drivers of sexual dimorphisms. Here, we introduce ostracods of the genus Euphilomedes (Myodocopida, Ostracoda, and Crustacea) as a promising new system in which to investigate why and how sexual dimorphisms evolve. First, we ask whether male-skewed selective pressure from pelagic predators may help explain a dramatic sexual dimorphism in which male Euphilomedes have compound eyes, but females do not. Manipulative experiments demonstrate that blindfolding reduces the survival rate of male Euphilomedes when they are exposed to predatory fish. Blindfolding of the female rudimentary eyes (rudiments) does not, however, similarly influence the survival rate of brooding females. Further, numerical estimates of sighting distances, based on reasonable extrapolations from Euphilomedes's eye morphology, suggest that the eyes of male Euphilomedes are useful for detecting objects roughly the size of certain pelagic predators, but not conspecifics. We conclude that eyes do not mediate direct interactions between male and female Euphilomedes, but that differences in predation pressure-perhaps associated with different reproductive behaviors-contribute to maintaining the sexually dimorphic eyes of these ostracods. Second, through transcriptome sequencing, we examined potential gene regulatory networks that could underlie sexual dimorphism in Euphilomedes' eyes. From the transcriptome of juvenile male Euphilomedes' eyes, we identified phototransduction genes and components of eye-related developmental networks that are well characterized in Drosophila and other species. The presence of suites of eye regulatory genes in our Euphilomedes juvenile male transcriptome will allow us, in future studies, to test how ostracods regulate the development of their sexually dimorphic eyes.

Genomics and the evolutionary origins of nervous system complexity.

Advances in genomics are leading to increased understanding of the evolution of complexity, especially by beginning to bridge genotype and phenotype. Here, using examples from nervous system evolution, we define general patterns of increased complexity seen across levels of biological organization. We also explore specific evolutionary mechanisms that increase complexity, namely those that increase the number of biological units (parts) in a system. We provide specific neurobiological examples of increased complexity in genes, gene networks, cell types, and tissues/organs. These examples illustrate that while a variety of different mechanisms increase biological complexity, they can be understood in a generalized comparative framework.

Furcation, field-splitting, and the evolutionary origins of novelty in arthropod photoreceptors.

Arthropod photoreceptor evolution is a prime example of how evolution has used existing components in the origin of new structures. Here, we outline a comparative approach to understanding the mutational origins of novel structures, describing multiple examples from arthropod photoreceptor evolution. We suggest that developmental mechanisms have often split photoreceptors during evolution (field-splitting) and we introduce "co-duplication" as a null model for the mutational origins of photoreceptor components. Under co-duplication, gene duplication events coincide with the origin of a higher level structure like an eye. If co-duplication is rejected for a component, that component probably came to be used in a new photoreceptor through regulatory mutations. If not rejected, a gene duplication mutation may have allowed the component to be used in a new structure. In multiple case studies in arthropod photoreceptor evolution, we consistently reject the null hypothesis of co-duplication of genetic components and photoreceptors. Nevertheless, gene duplication events have in some cases occurred later, allowing divergence of photoreceptors. These studies provide a new perspective on the evolution of arthropod photoreceptors and provide a comparative approach that generalizes to the study of any evolutionary novelty.

Characterization of Notch-class gene expression in segmentation stem cells and segment founder cells in Helobdella robusta (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae).

To understand the evolution of segmentation, we must compare segmentation in all three major groups of eusegmented animals: vertebrates, arthropods, and annelids. The leech Helobdella robusta is an experimentally tractable annelid representative, which makes segments in anteroposterior progression from a posterior growth zone consisting of 10 identified stem cells. In vertebrates and some arthropods, Notch signaling is required for normal segmentation and functions via regulation of hes-class genes. We have previously characterized the expression of an hes-class gene (Hro-hes) during segmentation in Helobdella, and here, we characterize the expression of an H. robusta notch homolog (Hro-notch) during this process. We find that Hro-notch is transcribed in the segmental founder cells (blast cells) and their stem-cell precursors (teloblasts), as well as in other nonsegmental tissues. The mesodermal and ectodermal lineages show clear differences in the levels of Hro-notch expression. Finally, Hro-notch is shown to be inherited by newly born segmental founder cells as well as transcribed by them before their first cell division.