Comparative Developmental Biology Research Articles

Embryology of the fat-tailed dunnart (Sminthopsis crassicaudata): A marsupial model for comparative mammalian developmental and evolutionary biology.

Marsupials are a diverse and unique group of mammals, but remain underutilized in developmental biology studies, hindering our understanding of mammalian diversity. This study focuses on establishing the fat-tailed dunnart (Sminthopsis crassicaudata) as an emerging laboratory model, providing reproductive monitoring methods and a detailed atlas of its embryonic development. We monitored the reproductive cycles of female dunnarts and established methods to confirm pregnancy and generate timed embryos. With this, we characterized dunnart embryo development from cleavage to birth, and provided detailed descriptions of its organogenesis and heterochronic growth patterns. Drawing stage-matched comparisons with other species, we highlight the dunnarts accelerated craniofacial and limb development, characteristic of marsupials. The fat-tailed dunnart is an exceptional marsupial model for developmental studies, where our detailed practices for reproductive monitoring and embryo collection enhance its accessibility in other laboratories. The accelerated developmental patterns observed in the Dunnart provide a valuable system for investigating molecular mechanisms underlying heterochrony. This study not only contributes to our understanding of marsupial development but also equips the scientific community with new resources for addressing biodiversity challenges and developing effective conservation strategies in marsupials.

Twenty years on from Developmental Plasticity and Evolution: middle-range theories and how to test them.

In Developmental Plasticity and Evolution, Mary-Jane West-Eberhard argued that the developmental mechanisms that enable organisms to respond to their environment are fundamental causes of adaptation and diversification. Twenty years after publication of this book, this once so highly controversial claim appears to have been assimilated by a wealth of studies on 'plasticity-led' evolution. However, we suggest that the role of development in explanations for adaptive evolution remains underappreciated in this body of work. By combining concepts of evolvability from evolutionary developmental biology and quantitative genetics, we outline a framework that is more appropriate to identify developmental causes of adaptive evolution. This framework demonstrates how experimental and comparative developmental biology and physiology can be leveraged to put the role of plasticity in evolution to the test.

Integrating Complex Life Cycles in Comparative Developmental Biology.

The goal of comparative developmental biology is identifying mechanistic differences in embryonic development between different taxa and how these evolutionary changes have led to morphological and organizational differences in adult body plans. Much of this work has focused on direct-developing species in which the adult forms straight from the embryo and embryonic modifications have direct effects on the adult. However, most animal lineages are defined by indirect development, in which the embryo gives rise to a larval body plan and the adult forms by transformation of the larva. Historically, much of our understanding of complex life cycles is viewed through the lenses of ecology and zoology. In this review, we discuss the importance of establishing developmental rather than morphological or ecological criteria for defining developmental mode and explicitly considering the evolutionary implications of incorporating complex life cycles into broad developmental comparisons of embryos across metazoans.

Modularity in the trilobite head consistent with the hypothesized segmental origin of the eyes.

The trilobite head served multiple functions and was composed of several fused segments. Yet, the underlying organization of the trilobite head, and whether patterns are conserved across trilobites, remains unclear. Modeling the head as being composed of modules, or subunits that vary and thus have the potential to evolve semi-independently can reveal underlying patterns of organization. Hypotheses of modular organization based on the comparative developmental biology of arthropods were evaluated using geometric morphometrics. Two-dimensional (semi)landmark datasets collected from the cranidia of two Ordovician trilobite species, Calyptaulax annulata (Phacopida) and Cloacaspis senilis (Olenida sensu Adrain, 2011) were analyzed. The degree and pattern of modularity were assessed using the covariance ratio (CR), which compares the covariation within putative modules to the covariation between them, and the fit of different models was compared using an effect size measure derived from the CR. When treating the eyes as a distinct module, the best modular hypothesis identified for C. annulata shows the eyes and anteriormost region of the head integrated as a single module. The best modular hypotheses for C. senilis are more complex but the eyes still covary mostly strongly with the anterior part of the head. The latter is also the case for all other well-supported models for both species. These results can be interpreted as a developmental signal corresponding to the anteriormost ocular segment of early arthropods that is retained throughout development, despite any likely selective pressures related to functional needs.

Genetics of Atavism

Atavisms have attracted people’s attention for a long time. First, atavisms excited their imagination and created fertile ground for myths and superstitions. With the development of science, atavisms became the subject of investigation, which soon provided evidence to support evolutionary theory. However, at the molecular level, the formation of atavisms remained insufficiently understood. Recent progress in comparative genomics and molecular developmental biology has helped in understanding the processes underlying the formation of one of the human atavisms: the vestigial tail.

Protocol for the in vitro rearing of Frieseomelitta varia workers (Hymenoptera: Apidae: Meliponini)

In vitro rearing protocols already established for honeybees are currently being adapted to assess the risk of pesticides, and to conduct comparative developmental biology studies on stingless bees. However, differences in critical life-history traits (development time and the type of larval nutrition leading to caste differentiation process) among social bees require the development of an in vitro rearing protocol for each species and caste. We generated a protocol to produce workers of Frieseomelitta varia (Lepeletier, 1836), a non-endangered and highly eusocial pollinator species with wide geographical distribution. We tested the viability of using either the eggs or the first instar larvae as the starting point for in-vitro transfer. In vitro rearing was performed in acrylic plates at 30 °C and 99% relative humidity during the larval feeding phase. The humidity was subsequently reduced to 75% during the following days of development. The experimental larvae were offered either 25 µL or 27 µL of larval food. The development time, emergence and mortality rates, and morphological parameters of the emerged workers were assessed. In the process of validating the protocol, the adults that emerged after in vitro rearing were compared with colony-reared adults. In our results, 27 µL of larval food allowed 90% of workers to emerge. No significant differences were found between the emerging workers reared in vitro and those reared in the colony. The described protocol is a useful method for rearing F. varia workers in vitro, which can be used for diverse types of experimental approaches.

Invention sharing is the mother of developmental biology.

Here we release Part 2 of this special issue, following Part 1 in October. In the preface of Part 1, we proposed the title “Invention sharing is the mother of developmental biology” as a novel adage in place of the classic one by Jonathan Swift, "Necessity is the mother of invention", to promote the formation of research platforms for comparative evolutionary developmental biology (Ogino et al., 2021). To further convince researchers of this title, Part 2 releases two method articles and two protocols as follows. Yamanaka et al. (2022) established culture and microinjection methods for embryos of false clownfish, one of the most popular anemonefish, and demonstrated the effectiveness of RNA injection into the yolk sac as a convenient misexpression tool. These methods are expected to open the way for reverse genetics in this and related anemonefish species, and may reveal the molecular mechanisms of sex change and brilliant color pattern formation. Tagami et al. (2022) showed that RNA interference (RNAi) effectively suppresses zygotic β-catenin activity after the mid-blastula transition (MBT) in the Xenopus embryo. In contrast with other model animals, the RNAi technique had not been successfully applied in Xenopus due to the lack of an essential component of the RISC complex, Argonaute2 (Ago2), at stages earlier than the MBT. However, this study revives it as a tool for gene knock-down in later stages. Tokanai et al. (2022) provide an easy and rapid staining protocol for confocal microscopic observation and reconstruction of three-dimensional images of echinoderm (sea lily, sea urchin, and sand dollar) larvae and juveniles. This protocol uses Nile blue and benzyl alcohol/benzyl benzoate (BABB) to obtain strong and stable fluorescent staining signals and clear opaque backgrounds, which could be applicable to other organisms with untransparent tissues. Hiraga et al. (2022) provide a high-throughput screening protocol for CRISPR/Cas9-mediated genome editing in a nematode Pristionchus pacificus. The use of co-injection markers that induce a certain “roller” phenotype and fluorescent protein expression allow researchers to perform quick visual screening of desired mutants and comparative studies with Caenorhabditis elegans. We hope the above articles convince the readers that sharing methods and protocols among researchers using different organisms would move science forward by inspiring new ambitious ideas and exciting collaborations.

Identifying Amygdala-Like Territories in Scyliorhinus canicula (Chondrichthyan): Evidence for a Pallial Amygdala

To identify the putative amygdalar complex in cartilaginous fishes, our first step was to obtain evidence that supports the existence of a pallial amygdala in the catshark Scyliorhinus canicula, at present the prevailing chondrichthyan model in comparative neurobiology and developmental biology. To this end, we analyzed the organization of the lateral walls of the telencephalic hemispheres of adults, juveniles, and early prehatching embryos by immunohistochemistry against tyrosine hydroxylase (TH), somatostatin (SOM), Pax6, serotonin (5HT), substance P (SP), and Met-enkephalin (MetEnk), calbindin-28k (CB), and calretinin (CR), and by in situ hybridization against regulatory genes such as Tbr1, Lhx9, Emx1, and Dlx2. Our data were integrated with those available from the literature related to the secondary olfactory projections in this shark species. We have characterized two possible amygdalar territories. One, which may represent a ventropallial component, was identified by its chemical signature (moderate density of Pax6-ir cells, scarce TH-ir and SOM-ir cells, and absence of CR-ir and CB-ir cells) and gene expressions (Tbr1 and Lhx9 expressions in an Emx1 negative domain, as the ventral pallium of amniotes). It is perhaps comparable to the lateral amygdala of amphibians and the pallial amygdala of teleosts. The second was a territory related to the pallial-subpallial boundary with abundant Pax6-ir and CR-ir cells, and 5HT-ir, SP-ir, and MetEnk-ir fibers capping dorsally the area superficialis basalis. This olfactory-related region at the neighborhood of the pallial-subpallial boundary may represent a subpallial amygdala subdivision that possibly contains migrated cells of ventropallial origin.

Invention sharing is the mother of developmental biology.

Invention sharing is the mother of developmental biology.

Pluripotent pig embryonic stem cell lines originating from in vitro-fertilized and parthenogenetic embryos.

Here, we report in vivo developmental competent pig embryonic stem cells (ESCs) originating from in vitro-fertilized and parthenogenetic embryos cultured in chemically defined culture media. The pig ESC lines expressed pluripotency markers and were capable of forming teratomas following subcutaneous injection into nude mice. These cell lines would be a useful resource for comparative developmental biology and agricultural biotechnology.

Disciplinary Fields in the Life Sciences: Evolving Divides and Anchor Concepts

Recent and ongoing debates in biology and in the philosophy of biology reveal widespread dissatisfaction with the current definitions or circumscriptions, which are often vague or controversial, of key concepts such as the gene, individual, species, and homology, and even of whole disciplinary fields within the life sciences. To some extent, the long growing awareness of these conceptual issues and the contrasting views defended in their regard can be construed as a symptom of the need to revisit traditional unchallenged partitions between the specialist disciplines within the life sciences. I argue here that the current relationships between anchor disciplines (e.g., developmental biology, evolutionary biology, biology of reproduction) and nomadic concepts wandering between them is worth being explored from a reciprocal perspective, by selecting suitable anchor concepts around which disciplinary fields can flexibly move. Three examples are offered, focusing on generalized anchor concepts of generation (redefined in a way that suggests new perspectives on development and reproduction), organizational module (with a wide-ranging domain of application in comparative morphology, developmental biology, and evolutionary biology) and species as unit of representation of biological diversity (suggesting a taxonomic pluralism that must be managed with suitable adjustments of current nomenclature rules).

A practical staging atlas to study embryonic development of Octopus vulgaris under controlled laboratory conditions

BackgroundOctopus vulgaris has been an iconic cephalopod species for neurobiology research as well as for cephalopod aquaculture. It is one of the most intelligent and well-studied invertebrates, possessing both long- and short-term memory and the striking ability to perform complex cognitive tasks. Nevertheless, how the common octopus developed these uncommon features remains enigmatic. O. vulgaris females spawn thousands of small eggs and remain with their clutch during their entire development, cleaning, venting and protecting the eggs. In fact, eggs incubated without females usually do not develop normally, mainly due to biological contamination (fungi, bacteria, etc.). This high level of parental care might have hampered laboratory research on the embryonic development of this intriguing cephalopod.ResultsHere, we present a completely parameter-controlled artificial seawater standalone egg incubation system that replaces maternal care and allows successful embryonic development of a small-egged octopus species until hatching in a laboratory environment. We also provide a practical and detailed staging atlas based on bright-field and light sheet fluorescence microscopy imaging for precise monitoring of embryonic development. The atlas has a comparative section to benchmark stages to the different scales published by Naef (1928), Arnold (1965) and Boletzky (2016). Finally, we provide methods to monitor health and wellbeing of embryos during organogenesis.ConclusionBesides introducing the study of O. vulgaris embryonic development to a wider community, this work can be a high-quality reference for comparative evolutionary developmental biology.

The OCIAD protein family: comparative developmental biology and stem cell application.

Over the last two decades, an exponential growth in technologies and techniques available to biologists has provided mind-boggling quantities of data and led to information overload. Yet, answers to fundamental questions such as "how are we made?" and "what keeps us ticking?" remain incomplete. Developmental biology has provided elegant approaches to address such questions leading to enlightening insights. While several important contributions to developmental biology have come from India over the decades, this area of research remains nascent. Here, we review the journey in India, from the discovery of the ociad gene family to decoding its role in development and stem cells. We compare analysis in silico, in vivo and ex vivo, with developmental models such as Drosophila, mouse and stem cells that gave important insight into how these clinically significant genes function.

How Weird is The Worm? Evolution of the Developmental Gene Toolkit in Caenorhabditis elegans.

Comparative developmental biology and comparative genomics are the cornerstones of evolutionary developmental biology. Decades of fruitful research using nematodes have produced detailed accounts of the developmental and genomic variation in the nematode phylum. Evolutionary developmental biologists are now utilising these data as a tool with which to interrogate the evolutionary basis for the similarities and differences observed in Nematoda. Nematodes have often seemed atypical compared to the rest of the animal kingdom—from their totally lineage-dependent mode of embryogenesis to their abandonment of key toolkit genes usually deployed by bilaterians for proper development—worms are notorious rule breakers of the bilaterian handbook. However, exploring the nature of these deviations is providing answers to some of the biggest questions about the evolution of animal development. For example, why is the evolvability of each embryonic stage not the same? Why can evolution sometimes tolerate the loss of genes involved in key developmental events? Lastly, why does natural selection act to radically diverge toolkit genes in number and sequence in certain taxa? In answering these questions, insight is not only being provided about the evolution of nematodes, but of all metazoans.

Differentially and Co-expressed Genes in Embryo, Germ-Line and Somatic Tissues of Tribolium castaneum.

Transcriptomic studies of Tribolium castaneum have led to significant advances in our understanding of co-regulation and differential expression of genes in development. However, previously used microarray approaches have covered only a subset of known genes. The aim of this study was to investigate gene expression patterns of beetle embryo, germ-line and somatic tissues. We identified 12,302 expressed genes and determined differentially expressed up and down-regulated genes among all samples. For example, 1624 and 3639 genes were differentially increased in expression greater than or equal to twofold change (FDR < 0.01) in testis vs. ovary (virgin female) and ovary vs. embryo (0-5 hr), respectively. Of these, many developmental, somatic and germ-line differentially expressed genes were identified. Furthermore, many maternally deposited transcripts were identified, whose expression either decreased rapidly or persisted during embryogenesis. Genes with the largest change in expression were predominantly decreased during early embryogenesis as compared to ovary or were increased in testis compared to embryo. We also identify zygotic genes induced after fertilization. The genome wide variation in transcript regulation in maternal and zygotic genes could provide additional information on how the anterior posterior axis formation is established in Tribolium embryos as compared to Drosophila. Together, our data will facilitate studies of comparative developmental biology as well as help identify candidate genes for identifying cis-elements to drive transgenic constructs.

Prospects for a General Theory of Evolutionary Novelty.

Novelty is a topic of broad interest, with two distinct approaches within evolutionary biology. The dominant approach since Darwin has been transformationist, with novelty arising through gradual changes in morphology. The Modern Synthesis emphasized the importance of ecological opportunity rather than the source of variation, and this view has many adherents today. Yet, since well before Darwin, an alternative view has held that novelties could arise by rapid changes and many not necessarily be connected to ecological opportunity. The rise of comparative evolutionary developmental biology since 1990 has led to a resurgence of these arguments. Many case studies have documented novelties and there have been rigorous efforts to define the attributes of novelty, but there have been few attempts at a more general model. In contrast, studies of technological innovation have been replete with qualitative models since the 1930s. In this article I consider several possibilities for constructing a general model of novelty and innovation: (1) A general formal theory. (2) Commonalities between different levels, such as genes and morphology, but with sufficient differences between domains that any formal theory would be level specific. (3) Commonalities across levels but for various reasons developing a formal theory even within domains is improbable. A final alternative is that novelty and innovation may be so deeply historical that any general framework is impossible. I conclude that a common conceptual framework can be developed and serve as the foundation for simulation studies, but the importance of feedbacks and potentiating factors renders a formal model implausible.

Manipulation of Developmental Function in Turtles with Notes on Alligators.

Reptiles have great taxonomic diversity that is reflected in their morphology, ecology, physiology, modes of reproduction, and development. Interest in comparative and evolutionary developmental biology makes protocols for the study of reptile embryos invaluable resources. The relatively large size, seasonal breeding, and long gestation times of turtles epitomize the challenges faced by the developmental biologist. We describe protocols for the preparation of turtle embryos for ex ovo culture, electroporation, in situ hybridization, and microcomputed tomography. Because these protocols have been adapted and optimized from methods used for frog, chick, and mouse embryos, it is likely that they could be used for other reptilian species. Notes are included for alligator embryos where appropriate.

Morphological, cellular and molecular characterization of posterior regeneration in the marine annelid Platynereis dumerilii

Regeneration, the ability to restore body parts after an injury or an amputation, is a widespread but highly variable and complex phenomenon in animals. While having fascinated scientists for centuries, fundamental questions about the cellular basis of animal regeneration as well as its evolutionary history remain largely unanswered. Here, we present a study of regeneration of the marine annelid Platynereis dumerilii, an emerging comparative developmental biology model, which, like many other annelids, displays important regenerative abilities. When P. dumerilii worms are amputated, they are able to regenerate the posteriormost differentiated part of their body and a stem cell-rich growth zone that allows the production of new segments replacing the amputated ones. We show that posterior regeneration is a rapid process that follows a well reproducible path and timeline, going through specific stages that we thoroughly defined. Wound healing is achieved one day after amputation and a regeneration blastema forms one day later. At this time point, some tissue specification already occurs, and a functional posterior growth zone is re-established as early as three days after amputation. Regeneration timing is only influenced, in a minor manner, by worm size. Comparable regenerative abilities are found for amputations performed at different positions along the antero-posterior axis of the worm, except when amputation planes are very close to the pharynx. Regenerative abilities persist upon repeated amputations without important alterations of the process. We also show that intense cell proliferation occurs during regeneration and that cell divisions are required for regeneration to proceed normally. Finally, 5-ethynyl-2’-deoxyuridine (EdU) pulse and chase experiments suggest that blastemal cells mostly derive from the segment immediately abutting the amputation plane. The detailed characterization of P. dumerilii posterior body regeneration presented in this article provides the foundation for future mechanistic and comparative studies of regeneration in this species.

Resurrected Protein Interaction Networks Reveal the Innovation Potential of Ancient Whole-Genome Duplication.

The evolution of plants is characterized by whole-genome duplications, sometimes closely associated with the origin of large groups of species. The gamma (γ) genome triplication occurred at the origin of the core eudicots, which comprise ∼75% of flowering plants. To better understand the impact of whole-genome duplication, we studied the protein interaction network of MADS domain transcription factors, which are key regulators of reproductive development. We reconstructed, synthesized, and tested the interactions of ancestral proteins immediately before and closely after the triplication and directly compared these ancestral networks to the extant networks of Arabidopsis thaliana and tomato (Solanum lycopersicum). We found that gamma expanded the MADS domain interaction network more strongly than subsequent genomic events. This event strongly rewired MADS domain interactions and allowed for the evolution of new functions and installed robustness through new redundancy. Despite extensive rewiring, the organization of the network was maintained through gamma. New interactions and protein retention compensated for its potentially destructive impact on network organization. Post gamma, the network evolved from an organization around the single hub SEP3 to a network organized around multiple hubs and well-connected proteins lost, rather than gained, interactions. The data provide a resource for comparative developmental biology in flowering plants.

Inventory and Assessment of theGorilla gorilla(Savage, 1847) Skeletal Collection Housed at the Yale Peabody Museum of Natural History

Museum collections are critical resources for examination of comparative anatomy, developmental biology, and life history hypotheses. Skeletal collections provide insight into spatiotemporal, species, population, and individual variation associated with environmental, social, and epidemiological history. For endangered species such as great apes, these collections provide data that are nearly impossible to replicate today. In this second in a series of articles reviewing the great ape holdings of the Peabody Museum of Natural History at Yale University, we describe the Gorilla gorilla skeletal collection, comprising 16 individuals: 13 males, 2 females, and 1 of indeterminate sex. Evidence of socioecology, interindividual violence, hunting, captive management, predation, senescence, and infectious disease are present within the skeletal material. The collection exhibits possible taxonomic diversity as well. Our evaluation of the Yale Peabody Museum collection provides a baseline for future research and testable hypotheses for alternate techniques, such as isotopic analyses of dental calculus and noninvasive genetic testing. Museum collections continue to provide new insights into taxonomic and individual variation and environmental context, and ultimately allow for comparisons between modern and historical environmental and behavioral variables.