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.

Genetic tools for the study of the mangrove killifish, Kryptolebias marmoratus, an emerging vertebrate model for phenotypic plasticity.

Kryptolebias marmoratus (Kmar), a teleost fish of the order Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic-varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self-fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage-stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild-type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono- and bi-allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity.

Fisher vs. the Worms: Extraordinary Sex Ratios in Nematodes and the Mechanisms that Produce Them.

Parker, Baker, and Smith provided the first robust theory explaining why anisogamy evolves in parallel in multicellular organisms. Anisogamy sets the stage for the emergence of separate sexes, and for another phenomenon with which Parker is associated: sperm competition. In outcrossing taxa with separate sexes, Fisher proposed that the sex ratio will tend towards unity in large, randomly mating populations due to a fitness advantage that accrues in individuals of the rarer sex. This creates a vast excess of sperm over that required to fertilize all available eggs, and intense competition as a result. However, small, inbred populations can experience selection for skewed sex ratios. This is widely appreciated in haplodiploid organisms, in which females can control the sex ratio behaviorally. In this review, we discuss recent research in nematodes that has characterized the mechanisms underlying highly skewed sex ratios in fully diploid systems. These include self-fertile hermaphroditism and the adaptive elimination of sperm competition factors, facultative parthenogenesis, non-Mendelian meiotic oddities involving the sex chromosomes, and environmental sex determination. By connecting sex ratio evolution and sperm biology in surprising ways, these phenomena link two "seminal" contributions of G. A. Parker.

Evolution of sex ratio through gene loss.

The maintenance of males at intermediate frequencies is an important evolutionary problem. Several species of Caenorhabditis nematodes have evolved a mating system in which selfing hermaphrodites and males coexist. While selfing produces XX hermaphrodites, cross-fertilization produces 50% XO male progeny. Thus, male mating success dictates the sex ratio. Here, we focus on the contribution of the male secreted short (mss) gene family to male mating success, sex ratio, and population growth. The mss family is essential for sperm competitiveness in gonochoristic species, but has been lost in parallel in androdioecious species. Using a transgene to restore mss function to the androdioecious Caenorhabditis briggsae, we examined how mating system and population subdivision influence the fitness of the mss+ genotype. Consistent with theoretical expectations, when mss+ and mss-null (i.e., wild type) genotypes compete, mss+ is positively selected in both mixed-mating and strictly outcrossing situations, though more strongly in the latter. Thus, while sexual mode alone affects the fitness of mss+, it is insufficient to explain its parallel loss. However, in genetically homogenous androdioecious populations, mss+ both increases male frequency and depresses population growth. We propose that the lack of inbreeding depression and the strong subdivision that characterize natural Caenorhabditis populations impose selection on sex ratio that makes loss of mss adaptive after self-fertility evolves.

Still Searching for Specialized Ribosomes.

Reporting in Developmental Cell, Cenik et al. (2019) show that the maternal ribosome supply is sufficient for C. elegans embryonic development, arguing against tissue-specific specialized ribosomes in this context. Examination of ribosomal requirement with the genetic tool kit presented in Artiles et al. (2019) suggests a checkpoint that prevents uncoordinated growth.

Multi-modal regulation of C. elegans hermaphrodite spermatogenesis by the GLD-1-FOG-2 complex.

Proper germ cell sex determination in Caenorhabditis nematodes requires a network of RNA-binding proteins (RBPs) and their target mRNAs. In some species, changes in this network enabled limited XX spermatogenesis, and thus self-fertility. In C. elegans, one of these selfing species, the global sex-determining gene tra-2 is regulated in germ cells by a conserved RBP, GLD-1, via the 3' untranslated region (3'UTR) of its transcript. A C. elegans-specific GLD-1 cofactor, FOG-2, is also required for hermaphrodite sperm fate, but how it modifies GLD-1 function is unknown. Germline feminization in gld-1 and fog-2 null mutants has been interpreted as due to cell-autonomous elevation of TRA-2 translation. Consistent with the proposed role of FOG-2 in translational control, the abundance of nearly all GLD-1 target mRNAs (including tra-2) is unchanged in fog-2 mutants. Epitope tagging reveals abundant TRA-2 expression in somatic tissues, but an undetectably low level in wild-type germ cells. Loss of gld-1 function elevates germline TRA-2 expression to detectable levels, but loss of fog-2 function does not. A simple quantitative model of tra-2 activity constrained by these results can successfully sort genotypes into normal or feminized groups. Surprisingly, fog-2 and gld-1 activity enable the sperm fate even when GLD-1 cannot bind to the tra-2 3' UTR. This suggests the GLD-1-FOG-2 complex regulates uncharacterized sites within tra-2, or other mRNA targets. Finally, we quantify the RNA-binding capacities of dominant missense alleles of GLD-1 that act genetically as "hyper-repressors" of tra-2 activity. These variants bind RNA more weakly in vitro than does wild-type GLD-1. These results indicate that gld-1 and fog-2 regulate germline sex via multiple interactions, and that our understanding of the control and evolution of germ cell sex determination in the C. elegans hermaphrodite is far from complete.

From "the Worm" to "the Worms" and Back Again: The Evolutionary Developmental Biology of Nematodes.

Since the earliest days of research on nematodes, scientists have noted the developmental and morphological variation that exists within and between species. As various cellular and developmental processes were revealed through intense focus on Caenorhabditis elegans, these comparative studies have expanded. Within the genus Caenorhabditis, they include characterization of intraspecific polymorphisms and comparisons of distinct species, all generally amenable to the same laboratory culture methods and supported by robust genomic and experimental tools. The C. elegans paradigm has also motivated studies with more distantly related nematodes and animals. Combined with improved phylogenies, this work has led to important insights about the evolution of nematode development. First, while many aspects of C. elegans development are representative of Caenorhabditis, and of terrestrial nematodes more generally, others vary in ways both obvious and cryptic. Second, the system has revealed several clear examples of developmental flexibility in achieving a particular trait. This includes developmental system drift, in which the developmental control of homologous traits has diverged in different lineages, and cases of convergent evolution. Overall, the wealth of information and experimental techniques developed in C. elegans is being leveraged to make nematodes a powerful system for evolutionary cellular and developmental biology.

How to Make a Billion Parasites.

Transmission of the human parasite Brugia malayi relies on the sustained production of larvae in blood. In this issue of Developmental Cell,Foray et al. (2018) use methods developed in the model nematode C. elegans to reveal how a symbiotic bacterium supports the female germ cell development underlying this massive fecundity.

Rapid genome shrinkage in a self-fertile nematode reveals sperm competition proteins.

To reveal impacts of sexual mode on genome content, we compared chromosome-scale assemblies of the outcrossing nematode Caenorhabditis nigoni to its self-fertile sibling species, C. briggsaeC. nigoni's genome resembles that of outcrossing relatives but encodes 31% more protein-coding genes than C. briggsaeC. nigoni genes lacking C. briggsae orthologs were disproportionately small and male-biased in expression. These include the male secreted short (mss) gene family, which encodes sperm surface glycoproteins conserved only in outcrossing species. Sperm from mss-null males of outcrossing C. remanei failed to compete with wild-type sperm, despite normal fertility in noncompetitive mating. Restoring mss to C. briggsae males was sufficient to enhance sperm competitiveness. Thus, sex has a pervasive influence on genome content that can be used to identify sperm competition factors.

Revisiting Suppression of Interspecies Hybrid Male Lethality in Caenorhabditis Nematodes.

Within the nematode genus Caenorhabditis, Caenorhabditis briggsae and C. nigoni are among the most closely related species known. They differ in sexual mode, with C. nigoni retaining the ancestral XO male-XX female outcrossing system, while C. briggsae recently evolved self-fertility and an XX-biased sex ratio. Wild-type C. briggsae and C. nigoni can produce fertile hybrid XX female progeny, but XO progeny are either 100% inviable (when C. briggsae is the mother) or viable but sterile (when C. nigoni is the mother). A recent study provided evidence suggesting that loss of the Cbr-him-8 meiotic regulator in C. briggsae hermaphrodites allowed them to produce viable and fertile hybrid XO male progeny when mated to C. nigoni Because such males would be useful for a variety of genetic experiments, we sought to verify this result. Preliminary crosses with wild-type C. briggsae hermaphrodites occasionally produced fertile males, but they could not be confirmed to be interspecies hybrids. Using an RNA interference (RNAi) protocol that eliminates any possibility of self-progeny in Cbr-him-8 hermaphrodites, we found sterile males bearing the C. nigoni X chromosome, but no fertile males bearing the C. briggsae X, as in wild-type crosses. Our results suggest that the apparent rescue of XO hybrid viability and fertility is due to incomplete purging of self-sperm prior to mating.

The Use of Group Activities in Introductory Biology Supports Learning Gains and Uniquely Benefits High-Achieving Students.

This study describes the implementation and effectiveness of small-group active engagement (GAE) exercises in an introductory biology course (BSCI207) taught in a large auditorium setting. BSCI207 (Principles of Biology III-Organismal Biology) is the third introductory core course for Biological Sciences majors. In fall 2014, the instructors redesigned one section to include GAE activities to supplement lecture content. One section (n = 198) employed three lectures per week. The other section (n = 136) replaced one lecture per week with a GAE class. We explored the benefits and challenges associated with implementing GAE exercises and their relative effectiveness for unique student groups (e.g., minority students, high- and low-grade point average [GPA] students). Our findings show that undergraduates in the GAE class exhibited greater improvement in learning outcomes than undergraduates in the traditional class. Findings also indicate that high-achieving students experienced the greatest benefit from GAE activities. Some at-risk student groups (e.g., two-year transfer students) showed comparably low learning gains in the course, despite the additional support that may have been afforded by active learning. Collectively, these findings provide valuable feedback that may assist other instructors who wish to revise their courses and recommendations for institutions regarding prerequisite coursework approval policies.

Fundamentals of Comparative Genome Analysis in Caenorhabditis Nematodes.

The genome of the nematode Caenorhabditis elegans was the first of any animal to be sequenced completely, and it remains the "gold standard" for completeness and annotations. Even before the C. elegans genome was completed, however, biologists began examining the generality of its features in the genomes of other Caenorhabditis species. With many such genomes now sequenced and available via WormBase, C. elegans researchers are often confronted with how to interpret comparative genomic data. In this article, we present practical approaches to addressing several common issues, including possible sources of error in homology annotations, the often complex relationships between sequence similarity, orthology, paralogy, and gene family evolution, the impact of sexual mode on genome assemblies and content, and the determination and use of synteny as a tool.

Evolutionary Dynamics of GLD-1-mRNA complexes in Caenorhabditis nematodes.

Given the large number of RNA-binding proteins and regulatory RNAs within genomes, posttranscriptional regulation may be an underappreciated aspect of cis-regulatory evolution. Here, we focus on nematode germ cells, which are known to rely heavily upon translational control to regulate meiosis and gametogenesis. GLD-1 belongs to the STAR-domain family of RNA-binding proteins, conserved throughout eukaryotes, and functions in Caenorhabditis elegans as a germline-specific translational repressor. A phylogenetic analysis across opisthokonts shows that GLD-1 is most closely related to Drosophila How and deuterostome Quaking, both implicated in alternative splicing. We identify messenger RNAs associated with C. briggsae GLD-1 on a genome-wide scale and provide evidence that many participate in aspects of germline development. By comparing our results with published C. elegans GLD-1 targets, we detect nearly 100 that are conserved between the two species. We also detected several hundred Cbr-GLD-1 targets whose homologs have not been reported to be associated with C. elegans GLD-1 in either of two independent studies. Low expression in C. elegans may explain the failure to detect most of them, but a highly expressed subset are strong candidates for Cbr-GLD-1-specific targets. We examine GLD-1-binding motifs among targets conserved in C. elegans and C. briggsae and find that most, but not all, display evidence of shared ancestral binding sites. Our work illustrates both the conservative and the dynamic character of evolution at the posttranslational level of gene regulation, even between congeners.

Mating damages the cuticle of C. elegans hermaphrodites.

Lifespan costs to reproduction are common across multiple species, and such costs could potentially arise through a number of mechanisms. In the nematode Caenorhabditis elegans, it has been suggested that part of the lifespan cost to hermaphrodites from mating results from physical damage owing to the act of copulation itself. Here, we examine whether mating damages the surface of the hermaphrodite cuticle via scanning electron microscopy. It is found that mated hermaphrodites suffered delamination of cuticle layers surrounding the vulva, and that the incidence of such damage depends on genetic background. Unmated hermaphrodites demonstrated almost no such damage, even when cultured in soil with potentially abrasive particles. Thus, a consequence of mating for C. elegans hermaphrodites is physical cuticle damage. These experiments did not assess the consequences of cuticle damage for lifespan, and the biological significance of this damage remains unclear. We further discuss our results within the context of recent studies linking the lifespan cost to mating in C. elegans hermaphrodites to male secretions.

Intense sperm-mediated sexual conflict promotes reproductive isolation in Caenorhabditis nematodes.

Conflict between the sexes over reproductive interests can drive rapid evolution of reproductive traits and promote speciation. Here we show that inter-species mating between Caenorhabditis nematodes sterilizes maternal individuals. The principal effectors of male-induced harm are sperm cells, which induce sterility and shorten lifespan by displacing conspecific sperm, invading the ovary, and sometimes breaching the gonad to infiltrate other tissues. This sperm-mediated harm is pervasive across species, but idiosyncrasies in its magnitude implicate both independent histories of sexually antagonistic coevolution within species and differences in reproductive mode (self-fertilizing hermaphrodites versus females) in determining its severity. Consistent with this conclusion, in androdioecious species the hermaphrodites are more vulnerable, the males more benign, or both. Patterns of assortative mating and a low incidence of invasive sperm occurring with conspecific mating are indicative of ongoing intra-specific sexual conflict that results in inter-species reproductive incompatibility.

Evolutionarily dynamic roles of a PUF RNA-binding protein in the somatic development of Caenorhabditis briggsae.

Gene duplication and divergence has emerged as an important aspect of developmental evolution. The genomes of Caenorhabditis nematodes encode an ancient family of PUF RNA-binding proteins. Most have been implicated in germline development, and are often redundant with paralogs of the same sub-family. An exception is Cbr-puf-2 (one of three Caenorhabditis briggsae PUF-2 sub-family paralogs), which is required for development past the second larval stage. Here, we provide a detailed functional characterization of Cbr-puf-2. The larval arrest of Cbr-puf-2 mutant animals is caused by inefficient breakdown of bacterial food, which leads to starvation. Cbr-puf-2 is required for the normal grinding cycle of the muscular terminal bulb during early larval stages, and is transiently expressed in this tissue. In addition, rescue of larval arrest reveals that Cbr-puf-2 also promotes normal vulval development. It is expressed in the anchor cell (which induces vulval fate) and vulval muscles, but not in the vulva precursor cells (VPCs) themselves. This contrasts with the VPC-autonomous repression of vulval development described for the Caenorhabditis elegans homologs fbf-1/2. These different roles for PUF proteins occur even as the vulva and pharynx maintain highly conserved anatomies across Caenorhabditis, indicating pervasive developmental system drift (DSD). Because Cbr-PUF-2 shares RNA-binding specificity with its paralogs and with C. elegans FBF, we suggest that functional novelty of RNA-binding proteins evolves through changes in the site of their expression, perhaps in concert with cis-regulatory evolution in target mRNAs.