From genes to environment in shaping of an embryo: understanding embryonic-extraembryonic interactions at the BSDB autumn meeting in Oxford The authors of the article Ajduk & Duncan 2019 sincerely apologize for specifying the incorrect institutional affiliation for Professor Ali Brivanlou and also the incorrect spelling of Professor Brivanlou’s surname in the text of the article.

Transcriptome analysis provides insight into the role of the melanin pathway in two differently pigmented strains of the turtle Pelodiscus sinensis Abstract Pelodiscus sinensis is the most common turtle species raised in East and Southeast Asia. The Japanese strain and the Qingxi black strain are important aquatic breeds of P. sinensis in China with unique characteristics in terms of production performance and appearance, including skin color. In this study, melanin content measure and histological analysis on skin samples of these two strains were carried out to compare their color characteristics. The results showed that Qingxi black turtles clearly have a greater ability to deposit melanin than the Japanese strain especially in the abdomen. Then, de novo transcriptome assembly and differential expression profiling analyses on the ventral skin from the two strains were performed to identify the genes responsible for the differences in skin color using the Illumina RNA-Seq system with three biological replicates. A total of 19,331 annotated unigenes were found by aligning to the reference genome of P. sinensis using TopHat v2.0.12. Differential expression analysis revealed that 670 genes were expressed differently, including 185 upregulated genes and 485 downregulated genes in Qingxi black strain using the DESeq R package (|log2FoldChange| ≥ 1, padj < 0.05). Sixteen differentially expressed genes (DEGs), which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). GO and KEGG analyses revealed four DEGs (agouti signaling protein, frizzled family receptor 1, phospholipase C, and protein kinase C) were related to melanogenesis pathway. Gene expression levels of the four DEGs as well as three genes from the tyrosinase gene family were measured by qRT-PCR. The results indicated that agouti signaling protein, tyrosinase-related protein, and dopachrome tautomerase could be the main genes responsible for the difference in abdominal skin color between the two turtle strains. This study provided valuable information for further analysis of the melanogenesis mechanisms in different varieties of P. sinensis.

RNA-seq analysis provides insight into molecular adaptations of Andrias davidianus Abstract The Chinese giant salamander Andrias davidianus is regarded as an ideal model for studying local adaptations, such as longevity, tolerance to starvation, and cutaneous respiration. Transcriptome analysis is useful for studying the large and complex genomes of amphibians. Based on the coding gene set of adult A. davidianus, dozens of A. davidianus–specific genes were identified and three signaling pathway (JAK-STAT, HIF-1, and FoxO) genes were expanded as compared with other amphibians. The results of the pathway analysis of A. davidianus–specific genes indicated that the molecular adaptation of A. davidianus may have required a more rapid evolution of the immune system. Additionally, for the first time, the gene expressions in different parts of the skin tissue were compared. The results of the comparison analysis demonstrated that lateral skin could be more focused on mucus secretion, dorsal skin on immunity and melanogenesis, and abdominal skin on water and salt metabolism. This study provides the first insight into studying longevity and starvation tolerance in A. davidianus, and offers a basis for further investigation of the molecular mechanisms of adaptations in amphibians.

Expression and potential functions of KIF3A/3B to promote nuclear reshaping and tail formation during Larimichthys polyactis spermiogenesis Abstract KIF3A and KIF3B are homologous motor subunits of the Kinesin II protein family. KIF3A, KIF3B, and KAP3 form a heterotrimeric complex and play a significant role in spermatogenesis. Here, we first cloned full-length kif3a/3b cDNAs from Larimichthys polyactis. Lp-kif3a/3b are highly related to their homologs in other animals. The proteins are composed of three domains, an N-terminal head domain, a central stalk domain, and a C-terminus tail domain. Lp-kif3a/3b mRNAs were found to be ubiquitously expressed in the examined tissues, with high expression in the testis. Fluorescence in situ hybridization (FISH) was used to analyze the expression of Lp-kif3a/3b mRNAs during spermiogenesis. The results showed that Lp-kif3a/3b mRNAs had similar expression pattern and were continuously expressed during spermiogenesis. From middle spermatid to mature sperm, Lp-kif3a/3b mRNAs gradually localized to the side of the spermatid where the midpiece and tail form. In addition, we used immunofluorescence (IF) to observe that Lp-KIF3A protein co-localizes with tubulin during spermiogenesis. In early spermatid, Lp-KIF3A protein and microtubule signals were randomly distributed in the cytoplasm. In middle spermatid, however, the protein was detected primarily around the nucleus. In late spermatid, the protein migrated primarily to one side of the nucleus where the tail forms. In mature sperm, Lp-KIF3A and microtubules accumulated in the midpiece. Moreover, Lp-KIF3A co-localized with the mitochondria. In mature sperm, Lp-KIF3A and mitochondria were present in the midpiece. Therefore, Lp-KIF3A/KIF3B may be involved in spermiogenesis in L. polyactis, particularly during nuclear reshaping and tail formation.

Primary myogenesis in the sand lizard ( Lacerta agilis ) limb bud Abstract Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks.

BioCell2XML : a novel tool for converting cell lineage data from SIMI BioCell to MaMuT (Fiji) Abstract Computer-assisted 4D manual cell tracking has been a valuable method for understanding spatial-temporal dynamics of embryogenesis (e.g., Stach & Anselmi BMC Biol, 13(113), 1–11 2015; Vellutini et al. BMC Biol, 15(33), 1–28 2017; Wolff et al. eLife, 7, e34410 2018) since the method was introduced in the late 1990s. Since two decades SIMI® BioCell (Schnabel et al. Dev Biol, 184, 234–265 1997), a software which initially was developed for analyzing data coming from the, at that time new technique of 4D microscopy, is in use. Many laboratories around the world use SIMI BioCell for the manual tracing of cells in embryonic development of various species to reconstruct cell genealogies with high precision. However, the software has several disadvantages: limits in handling very large data sets, the virtually no maintenance over the last 10 years (bound to older Windows versions), the difficulty to access the created cell lineage data for analyses outside SIMI BioCell, and the high cost of the program. Recently, bioinformatics, in close collaboration with biologists, developed new lineaging tools that are freely available through the open source image processing platform Fiji. Here we introduce a software tool that allows conversion of SIMI BioCell lineage data to a format that is compatible with the Fiji plugin MaMuT (Wolff et al. eLife, 7, e34410 2018). Hereby we intend to maintain the usability of SIMI BioCell created cell lineage data for the future and, for investigators who wish to do so, facilitate the transition from this software to a more convenient program.

Embryonic expression of priapulid Wnt genes Abstract Posterior elongation of the developing embryo is a common feature of animal development. One group of genes that is involved in posterior elongation is represented by the Wnt genes, secreted glycoprotein ligands that signal to specific receptors on neighbouring cells and thereby establish cell-to-cell communication. In segmented animals such as annelids and arthropods, Wnt signalling is also likely involved in segment border formation and regionalisation of the segments. Priapulids represent unsegmented worms that are distantly related to arthropods. Despite their interesting phylogenetic position and their importance for the understanding of ecdysozoan evolution, priapulids still represent a highly underinvestigated group of animals. Here, we study the embryonic expression patterns of the complete sets of Wnt genes in the priapulids Priapulus caudatus and Halicryptus spinulosus. We find that both priapulids possess a complete set of 12 Wnt genes. At least in Priapulus, most of these genes are expressed in and around the posterior-located blastopore and thus likely play a role in posterior elongation. Together with previous work on the expression of other genetic factors such as caudal and even-skipped, this suggests that posterior elongation in priapulids is under control of the same (or very similar) conserved gene regulatory network as in arthropods.

The cleavage pattern of calanoid copepods—a case study Abstract Many crustacean groups show stereotyped cleavage patterns during early ontogeny. However, these patterns differ between the various major crustacean taxa, and a general mode is difficult to extract. Previous studies suggested that also copepods undergo an early cleavage with a more or less stereotyped pattern of blastomere divisions and fates. Yet, copepod embryology has been largely neglected. The last investigation of this kind dates back more than a century and the results are somewhat contradictory when compared with those of other researchers. To overcome these problems, we studied the early development of a so far undescribed calanoid copepod species, Skistodiaptomus sp., applying histochemical staining, confocal laser scanning microscopy, and bifocal 4D microscopy. The blastomere arrangement of the four-cell stage of this species varies to a large degree. It can either form a typical radial pattern with the four blastomeres lying in one plane or a tilted orientation of the axes connecting the sister cells of the previous division. In both cases, a stereotyped division pattern is maintained inside each quadrant during subsequent cleavages. In addition, we found two types of blastomere arrangements with a mirror symmetry. Most divisions within the quadrants follow the perpendicularity rule until the eighth cleavage. Deviations from this rule occur only in the narrow regions where the different quadrants touch and near the site of gastrulation. Gastrulation is initiated around the descendants of one individually identifiable blastomere of the 16-cell stage. This cell divides in a specific manner forming a characteristic cell arrangement, the gastrulation triangle. This gastrulation triangle initiates the internalization process of the gastrulation and it is encircled by another characteristic cell type, the crown cells. Our observations reveal several similarities to the early development of Calanus finmarchicus, another calanoid species. These relate to blastomere arrangements and divisions and the pattern of gastrulation. As Calanoida represent a basal or near basal branch of the copepod tree, this description will provide the ground for reconstruction of the cleavage pattern of the last common ancestor of Copepoda.

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.

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 wnt1 regulation. 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.