Journal of experimental zoology. Part B, Molecular and developmental evolution最新文献

A defining feature of sea urchins is their extreme fecundity. Urchins produce millions of transparent, synchronously developing embryos, ideal for spatial and temporal analysis of development. This biological feature has been effectively utilized for ensemble measurement of biochemical changes. However, it has been underutilized in imaging studies, where single embryo measurements are used. Here we present an example of how stable genetics and high content imaging, along with machine learning-based image analysis, can be used to exploit the fecundity and synchrony of sea urchins in imaging-based drug screens. Building upon our recently created sea urchin ABCB1 knockout line, we developed a high-throughput assay to probe the role of this drug transporter in embryos. We used high content imaging to compare accumulation and toxicity of canonical substrates and inhibitors of the transporter, including fluorescent molecules and antimitotic cancer drugs, in homozygous knockout and wildtype embryos. To measure responses from the resulting image data, we used a nested convolutional neural network, which rapidly classified embryos according to fluorescence or cell division. This approach identified sea urchin embryos with 99.8% accuracy and determined two-cell and aberrant embryos with 96.3% and 89.1% accuracy, respectively. The results revealed that ABCB1 knockout embryos accumulated the transporter substrate calcein 3.09 times faster than wildtypes. Similarly, knockouts were 4.71 and 3.07 times more sensitive to the mitotic poisons vinblastine and taxol. This study paves the way for large scale pharmacological screens in the sea urchin embryo.

Germ cells (reproductive cells and their progenitors) give rise to the next generation in sexually reproducing organisms. The loss or removal of germ cells often leads to sterility in established research organisms such as the fruit fly, nematodes, frog, and mouse. The failure to regenerate germ cells in these organisms reinforced the dogma of germline–soma barrier in which germ cells are set-aside during embryogenesis and cannot be replaced by somatic cells. However, in stark contrast, many animals including segmented worms (annelids), hydrozoans, planaria, sea stars, sea urchins, and tunicates can regenerate germ cells. Here I review germ cell and gonad regeneration in annelids, a rich history of research that dates back to the early 20th century in this highly regenerative group. Examples include annelids from across the annelid phylogeny, across developmental stages, and reproductive strategies. Adult annelids regenerate germ cells as a part of regeneration, grafting, and asexual reproduction. Annelids can also recover germ cells after ablation of germ cell progenitors in the embryos. I present a framework to investigate cellular sources of germ cell regeneration in annelids, and discuss the literature that supports different possibilities within this framework, where germ–soma separation may or may not be preserved. With contemporary genetic-lineage tracing and bioinformatics tools, and several genetically enabled annelid models, we are at the brink of answering the big questions that puzzled many for over more than a century.

Among 36 known chromosomal hybrid zones of the common shrew Sorex araneus, the Moscow–Seliger hybrid zone is of special interest because inter-racial complex heterozygotes (F₁ hybrids) produce the longest meiotic configuration, consisting of 11 chromosomes with monobrachial homology (undecavalent or chain-of-eleven: CXI). Different studies suggest that such a multivalent may negatively affect meiotic progression and in general should significantly reduce fertility of hybrids. In this work, by immunocytochemical and electron microscopy methods, we investigated for the first time chromosome synapsis, recombination and meiotic silencing in pachytene spermatocytes of natural inter-racial heterozygous shrew males carrying CXI configurations. Despite some abnormalities detected in spermatocytes, such as associations of chromosomes, stretched centromeres, and the absence of recombination nodules in some arms of the multivalent, a large number of morphologically normal spermatozoa were observed. Possible low stringency of pachytene checkpoints may mean that even very long meiotic configurations do not cause complete sterility of such complex inter-racial heterozygotes.

Sperm cryopreservation is a critical tool for safeguarding and managing valuable genetic resources. Protocols for cryopreservation of Xenopus laevis sperm were available but lacking sperm quality evaluation and scalability and the outcomes were inconsistent. The goal of this study was to begin developing a center-level cryopreservation pathway for this species by integrating French straws as containers that would facilitate germplasm repository development. The objectives were to analyze the effect of: (1) three sperm concentrations (33, 50, and 100 × 10⁶ sperm/mL) on post-thaw fertilization, (2) three final concentrations (2.5%, 5%, and 10%) of dimethyl sulfoxide, methanol, and dimethylformamide (DMFA) on sperm membrane integrity of fresh and frozen samples, (3) two concentrations (5% and 10%) of DMFA with and without 5% sucrose at four cooling rates (5, 10, 20, and 40°C/min) on sperm membrane integrity and motility, and (4) egg exposure to different concentrations of DMFA on fertilization. Few differences in sperm viability were found among fresh samples incubated in cryoprotectants, but thawed samples frozen in methanol or DMFA presented higher membrane integrity. Samples frozen in 10% DMFA at 20°C/min showed higher membrane integrity (60 ± 7%) than other DMFA concentrations and cooling rates, and the same total motility (30 ± 7%) as at 10°C/min. Higher DMFA concentrations (10%–13%) were detrimental for embryo development compared to lower concentrations (<6%). This study provided a reliable protocol for sperm cryopreservation in Xenopus laevis to yield an application pathway with potential for high throughput that can be used as a roadmap for work with other species.

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

During early development, sea lamprey embryos undergo programmatic elimination of DNA from somatic progenitor cells in a process termed programmed genome rearrangement (PGR). Eliminated DNA eventually becomes condensed into micronuclei, which are then physically degraded and permanently lost from the cell. Previous studies indicated that many of the genes eliminated during PGR have mammalian homologs that are bound by polycomb repressive complex (PRC) in embryonic stem cells. To test whether PRC components play a role in the faithful elimination of germline-specific sequences, we used a combination of CRISPR/Cas9 and lightsheet microscopy to investigate the impact of gene knockouts on early development and the progression through stages of DNA elimination. Analysis of knockout embryos for the core PRC2 subunits EZH, SUZ12, and EED show that disruption of all three genes results in an increase in micronucleus number, altered distribution of micronuclei within embryos, and an increase in micronucleus volume in mutant embryos. While the upstream events of DNA elimination are not strongly impacted by loss of PRC2 components, this study suggests that PRC2 plays a role in the later stages of elimination related to micronucleus condensation and degradation. These findings also suggest that other genes/epigenetic pathways may work in parallel during DNA elimination to mediate chromatin structure, accessibility, and the ultimate loss of germline-specific DNA.

The Xiphophorus melanoma receptor kinase gene, xmrk, is a bona fide oncogene driving melanocyte tumorigenesis of Xiphophorus fish. When ectopically expressed in medaka, it not only induces development of several pigment cell tumor types in different strains of medaka but also induces different tumor types within the same animal, suggesting its oncogenic activity has a transcriptomic background effect. Although the central pathways that xmrk utilizes to lead to melanomagenesis are well documented, genes and genetic pathways that modulate the oncogenic effect and alter the course of disease have not been studied so far. To understand how the genetic networks between different histocytes of xmrk-driven tumors are composed, we isolated two types of tumors, melanoma and xanthoerythrophoroma, from the same xmrk transgenic medaka individuals, established the transcriptional profiles of both xmrk-driven tumors, and compared (1) genes that are co-expressed with xmrk in both tumor types, and (2) differentially expressed genes and their associated molecular functions, between the two tumor types. Transcriptomic comparisons between the two tumor types show melanoma and xanthoerythrophoroma are characterized by transcriptional features representing varied functions, indicating distinct molecular interactions between the driving oncogene and the cell-type-specific transcriptomes. Melanoma tumors exhibit gene signatures that are relevant to proliferation and invasion, while xanthoerythrophoroma tumors are characterized by expression profiles related to metabolism and DNA repair. We conclude the transcriptomic backgrounds, exemplified by cell-type-specific genes that are downstream of xmrk effected signaling pathways, contribute the potential to change the course of tumor development and may affect overall tumor outcomes.

During the early stages of limb and fin regeneration in aquatic vertebrates (i.e., fishes and amphibians), blastema undergo transcriptional rewiring of innate immune signaling pathways to promote immune cell recruitment. In mammals, a fundamental component of innate immune signaling is the cytosolic DNA sensing pathway, cGAS-STING. However, to what extent the cGAS-STING pathway influences regeneration in aquatic anamniotes is unknown. In jawed vertebrates, negative regulation of cGAS-STING activity is accomplished by suppressors of cytosolic DNA such as Trex1, Pml, and PML-like exon 9 (Plex9) exonucleases. Here, we examine the expression of these suppressors of cGAS-STING, as well as inflammatory genes and cGAS activity during caudal fin and limb regeneration using the spotted gar (Lepisosteus oculatus) and axolotl (Ambystoma mexicanum) model species, and during age-related senescence in zebrafish (Danio rerio). In the regenerative blastema of wounded gar and axolotl, we observe increased inflammatory gene expression, including interferon genes and interleukins 6 and 8. We also observed a decrease in axolotl Trex1 and gar pml expression during the early phases of wound healing which correlates with a dramatic increase in cGAS activity. In contrast, the plex9.1 gene does not change in expression during wound healing in gar. However, we observed decreased expression of plex9.1 in the senescing cardiac tissue of aged zebrafish, where 2′3′-cGAMP levels are elevated. Finally, we demonstrate a similar pattern of Trex1, pml, and plex9.1 gene regulation across species in response to exogenous 2′3′-cGAMP. Thus, during the early stages of limb-fin regeneration, Pml, Trex1, and Plex9.1 exonucleases are downregulated, presumably to allow an evolutionarily ancient cGAS-STING activity to promote inflammation and the recruitment of immune cells.

Hearing in infants is essential for brain development, acquisition of verbal language skills, and development of social interactions. Therefore, it is important to diagnose hearing loss soon after birth so that interventions can be provided as early as possible. Most newborns in the United States are screened for hearing deficits and commercially available next-generation sequencing hearing loss panels often can identify the causative gene, which may also identify congenital defects in other organs. One of the most prevalent autosomal dominant congenital hearing loss syndromes is branchio-oto-renal syndrome (BOR), which also presents with defects in craniofacial structures and the kidney. Currently, mutations in three genes, SIX1, SIX5, and EYA1, are known to be causative in about half of the BOR patients that have been tested. To uncover new candidate genes that could be added to congenital hearing loss genetic screens, we have combined the power of Drosophila mutants and protein biochemical assays with the embryological advantages of Xenopus, a key aquatic animal model with a high level of genomic similarity to human, to identify potential Six1 transcriptional targets and interacting proteins that play a role during otic development. We review our transcriptomic, yeast 2-hybrid, and proteomic approaches that have revealed a large number of new candidates. We also discuss how we have begun to identify how Six1 and co-factors interact to direct developmental events necessary for normal otic development.

Paradise fish (Macropodus opercularis) is an air-breathing freshwater fish species with a signature labyrinth organ capable of extracting oxygen from the air that helps these fish to survive in hypoxic environments. The appearance of this evolutionary innovation in anabantoids resulted in a rewired circulatory system, but also in the emergence of species-specific behaviors, such as territorial display, courtship and parental care in the case of the paradise fish. Early zoologists were intrigued by the structure and function of the labyrinth apparatus and a series of detailed descriptive histological studies at the beginning of the 20th century revealed the ontogenesis and function of this specialized system. A few decades later, these fish became the subject of numerous ethological studies, and detailed ethograms of their behavior were constructed. These latter studies also demonstrated a strong genetic component underlying their behavior, but due to lack of adequate molecular tools, the fine genetic dissection of the behavior was not possible at the time. The technological breakthroughs that transformed developmental biology and behavioral genetics in the past decades, however, give us now a unique opportunity to revisit these old questions. Building on the classic descriptive studies, the new methodologies will allow us to follow the development of the labyrinth apparatus at a cellular resolution, reveal the genes involved in this process and also the genetic architecture behind the complex behaviors that we can observe in this species.