Development Genes and Evolution

Analysis of the wnt1 regulatory chromosomal landscape Abstract One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain–hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.

GSK3β controls the timing and pattern of the fifth spiral cleavage at the 2–4 cell stage in Lymnaea stagnalis Abstract Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3β, β-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3β and β-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3β inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of β-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3β in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.

From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford Abstract The British Society for Developmental Biology Autumn Meeting, held in Oxford in September 2018, was the third in a series of international workshops which have been focussed on development at the extraembryonic-embryonic interface. This workshop, entitled "Embryonic-Extraembryonic Interactions: from Genetics to Environment" built on the two previous workshops held in 2011 (Leuven, Belgium) and 2015 (Göttingen, Germany). This workshop brought together researchers utilising a diverse range of organisms (including both vertebrate and invertebrate species) and a range of experimental approaches to answer core questions in developmental biology. This meeting report highlights some of the major themes emerging from the workshop including an evolutionary perspective as well as recent advances that have been made through the adoption of emerging techniques and technologies.

Transcriptome profiling reveals male- and female-specific gene expression pattern and novel gene candidates for the control of sex determination and gonad development in Xenopus laevis Abstract Xenopus laevis is an amphibian (frog) species widely used in developmental biology and genetics. To unravel the molecular machinery regulating sex differentiation of Xenopus gonads, we analyzed for the first time the transcriptome of developing amphibian gonads covering sex determination period. We applied microarray at four developmental stages: (i) NF50 (undifferentiated gonad during sex determination), (ii) NF53 (the onset of sexual differentiation of the gonads), (iii) NF56 (sexual differentiation of the gonads), and (iv) NF62 (developmental progression of differentiated gonads). Our analysis showed that during the NF50, the genetic female (ZW) gonads expressed more sex-specific genes than genetic male (ZZ) gonads, which suggests that a robust genetic program is realized during female sex determination in Xenopus. However, a contrasting expression pattern was observed at later stages (NF56 and NF62), when the ZW gonads expressed less sex-specific genes than ZZ gonads, i.e., more genes may be involved in further development of the male gonads (ZZ). We identified sexual dimorphism in the expression of several functional groups of genes, including signaling factors, proteases, protease inhibitors, transcription factors, extracellular matrix components, extracellular matrix enzymes, cell adhesion molecules, and epithelium-specific intermediate filaments. In addition, our analysis detected a sexually dimorphic expression of many uncharacterized genes of unknown function, which should be studied further to reveal their identity and if/how they regulate gonad development, sex determination, and sexual differentiation. Comparison between genes sex-specifically expressed in developing gonads of Xenopus and available transcriptome data from zebrafish, two reptile species, chicken, and mouse revealed significant differences in the genetic control of sex determination and gonad development. This shows that the genetic control of gonad development is evolutionarily malleable.

Divergent Axin and GSK-3 paralogs in the beta-catenin destruction complexes of tapeworms Abstract The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.

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

Independent evolution of complex development in animals and plants: deep homology and lateral gene transfer Abstract The evolution of multicellularity is a premier example of phenotypic convergence: simple multicellularity evolved independently many times, and complex multicellular phenotypes are found in several distant groups. Furthermore, both animal and plant lineages have independently reached extreme levels of morphological, functional, and developmental complexity. This study explores the genetic basis for the parallel evolution of complex multicellularity and development in the animal and green plant (i.e., green algae and land plants) lineages. Specifically, the study (i) identifies the SAND domain—a DNA-binding domain with important roles in the regulation of cell proliferation and differentiation, as unique to animals, green algae, and land plants; and (ii) suggests that the parallel deployment of this ancestral domain in similar regulatory roles could have contributed to the independent evolution of complex development in these distant groups. Given the deep animal-green plant divergence, the limited distribution of the SAND domain is best explained by invoking a lateral gene transfer (LGT) event from a green alga to an early metazoan. The presence of a sequence motif specifically shared by a family of SAND-containing transcription factors involved in the evolution of complex multicellularity in volvocine algae and two types of SAND proteins that emerged early in the evolution of animals is consistent with this scenario. Overall, these findings imply that (i) in addition to be involved in the evolution of similar phenotypes, deep homologous sequences can also contribute to shaping parallel evolutionary trajectories in distant lineages, and (ii) LGT could provide an additional source of latent homologous sequences that can be deployed in analogous roles and affect the evolutionary potentials of distantly related groups.

Early development of the feeding larva of the sea urchin Heliocidaris tuberculata : role of the small micromeres Abstract The two modes of development in sea urchins are direct development, in which the adult develops directly from the gastrula to the adult and does not feed, and indirect development, in which the adult develops indirectly through a feeding larva. In this account of the indirect, feeding larva of Heliocidaris tuberculata, the question raised is whether an evolutionary difference of unequal cell divisions contributes to the development of feeding structures in the indirect larva. In indirect development, the cell divisions at the fourth and fifth cell cycles of the zygote are unequal, with four small micromeres formed at the vegetal pole at the fifth cell division. In direct development, these cell divisions are not unequal. From their position at the head of the archenteron, the small micromeres are strategically located to contribute to the feeding tissues of the larva and the adult of H. tuberculata.

The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments Abstract During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins HFA, HLLAA and HWA. For in vivo analysis, UAS-lines HFA, HLLAA and HWA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the HLD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.

Silicatein expression in Haliclona indistincta (Phylum Porifera, Order Haplosclerida) at different developmental stages Abstract Silicatein is the main protein responsible for the formation of spicules, tiny structures that constitute the silica skeleton of marine demosponges (Phylum Porifera). A unique innovation in Porifera that evolved from the cathepsin L family of proteins, it has been reported that two amino acids (S and H) are necessary to form the catalytic triad (SHN) to enable silica condensation. However, a diversity of silicatein sequence variants has since been reported with a variable pattern of presence/absence across sponge groups. Variants containing CHN or C/SQN at the active site appear more common in sponges from the Haplosclerida. Here, we report the expression levels of five silicatein variants through different developmental stages in the haplosclerid Haliclona indistincta. All five silicatein variants were expressed at low levels in the free-swimming larvae, which lack spicules and expression significantly increased at the two developmental phases in which spicules were visible. At these two phases, silicateins of CHN and C/SQN types were much more highly expressed than the SHN type indicating a possible ability of active sites with these alternative amino acids to condense silica and a more complex evolutionary story for spicule formation in marine demosponges than previously understood.