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An evolutionary perspective enhances our understanding of biological mechanisms. Comparison of sex determination and X-chromosome dosage compensation mechanisms between the closely related nematode species Caenorhabditis briggsae (Cbr) and Caenorhabditis elegans (Cel) revealed that the genetic regulatory hierarchy controlling both processes is conserved, but the X-chromosome target specificity and mode of binding for the specialized condensin dosage compensation complex (DCC) controlling X expression have diverged. We identified two motifs within Cbr DCC recruitment sites that are highly enriched on X: 13 bp MEX and 30 bp MEX II. Mutating either MEX or MEX II in an endogenous recruitment site with multiple copies of one or both motifs reduced binding, but only removing all motifs eliminated binding in vivo. Hence, DCC binding to Cbr recruitment sites appears additive. In contrast, DCC binding to Cel recruitment sites is synergistic: mutating even one motif in vivo eliminated binding. Although all X-chromosome motifs share the sequence CAGGG, they have otherwise diverged so that a motif from one species cannot function in the other. Functional divergence was demonstrated in vivo and in vitro. A single nucleotide position in Cbr MEX can determine whether Cel DCC binds. This rapid divergence of DCC target specificity could have been an important factor in establishing reproductive isolation between nematode species and contrasts dramatically with the conservation of target specificity for X-chromosome dosage compensation across Drosophila species and for transcription factors controlling developmental processes such as body-plan specification from fruit flies to mice.

An evolutionary perspective enhances our understanding of biological mechanisms. Comparison of sex determination and X-chromosome dosage compensation mechanisms between the closely related nematode speciesC. briggsae(Cbr) andC. elegans(Cel) revealed that the genetic regulatory hierarchy controlling both processes is conserved, but the X-chromosome target specificity and mode of binding for the specialized condensin dosage compensation complex (DCC) controlling X expression have diverged. We identified two motifs withinCbrDCC recruitment sites that are highly enriched on X: 13-bp MEX and 30-bp MEX II. Mutating either MEX or MEX II in an endogenous recruitment site with multiple copies of one or both motifs reduced binding, but only removing all motifs eliminated bindingin vivo. Hence, DCC binding toCbrrecruitment sites appears additive. In contrast, DCC binding toCelrecruitment sites is synergistic: mutating even one motifin vivoeliminated binding. Although all X-chromosome motifs share the sequence CAGGG, they have otherwise diverged so that a motif from one species cannot function in the other. Functional divergence was demonstratedin vivoandin vitro. A single nucleotide position inCbrMEX can determine whetherCelDCC binds. This rapid divergence of DCC target specificity could have been an important factor in establishing reproductive isolation between nematode species and contrasts dramatically with conservation of target specificity for X-chromosome dosage compensation acrossDrosophilaspecies and for transcription factors controlling developmental processes such as body-plan specification from fruit flies to mice.

To fully appreciate genetics, one must understand the link between genotype (DNA sequence) and phenotype (observable characteristics). Advances in high-throughput genomic sequencing technologies and applications, so-called "-omics," have made genetic sequencing readily available across fields in biology from applications in non-traditional study organisms to precision medicine. Thus, understanding these tools is critical for any biologist, especially those early in their career. This comprehensive review discusses the chronological development of different sequencing methods, the bioinformatics steps to analyzing this data, and social and ethical issues raised by these techniques that must be discussed and evaluated, including anticipatory guides and discussion questions for active engagement in the classroom. Additionally, the Supporting Information includes a case study to apply technical and ethical concepts from the text.

Proper germ cell sex determination inCaenorhabditisnematodes 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. InC. elegans, one of these selfing species, the global sex-determining genetra-2is regulated in germ cells by a conserved RBP, GLD-1, via the 3’ untranslated region (3’UTR) of its transcript. AC. 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 ingld-1andfog-2null 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 (includingtra-2)is unchanged infog-2mutants. Epitope tagging reveals abundant TRA-2 expression in somatic tissues, but an undetectably low level in wild-type germ cells. Loss ofgld-1function elevates germline TRA-2 expression to detectable levels, but loss offog-2function does not. A simple quantitative model oftra-2activity constrained by these results can successfully sort genotypes into normal or feminized groups. Surprisingly,fog-2andgld-1activity enable the sperm fate even when GLD-1 cannot bind to thetra-23’ UTR. This suggests the GLD-1-FOG-2 complex regulates uncharacterized sites withintra-2, or other mRNA targets. Finally, we quantify the RNA-binding capacities of dominant missense alleles of GLD-1 that act genetically as “hyperrepressors” oftra-2activity. These variants bind RNA more weaklyin vitrothan does wild-type GLD-1. These results indicate thatgld-1andfog-2regulate germline sex via multiple interactions, and that our understanding of the control and evolution of germ cell sex determination in theC. eleganshermaphrodite is far from complete.

To fully appreciate genetics, one must understand the link between genotype (DNA sequence) and phenotype (observable characteristics). Advances in high-throughput genomic sequencing technologies and applications, so-called "-omics," have made genetic sequencing readily available across fields in biology from applications in non-traditional study organisms to precision medicine. Thus, understanding these tools is critical for any biologist, especially those early in their career. This comprehensive review discusses the chronological development of different sequencing methods, the bioinformatics steps to analyzing this data, and social and ethical issues raised by these techniques that must be discussed and evaluated, including anticipatory guides and discussion questions for active engagement in the classroom. Additionally, the Supporting Information includes a case study to apply technical and ethical concepts from the text.

Disrupting a gene to determine its effect on an organism's phenotype is an indispensable tool in molecular biology. Such techniques are critical for understanding how a gene product contributes to the development and cellular identity of organisms. The explosion of genomic sequencing technologies combined with recent advances in genome-editing techniques has elevated the possibilities of genetic manipulations in numerous organisms in which these experiments were previously not readily accessible or possible. Introducing the next generation of molecular biologists to these emerging techniques is key in the modern biology classroom. This comprehensive review introduces undergraduates to CRISPR/Cas9 editing and its uses in genetic studies. The goals of this review are to explain how CRISPR functions as a prokaryotic immune system, describe how researchers generate mutations with CRISPR/Cas9, highlight how Cas9 has been adapted for new functions, and discuss ethical considerations of genome editing. Additionally, anticipatory guides and questions for discussion are posed throughout the review to encourage active exploration of these topics in the classroom. Finally, the supplement includes a study guide and practical suggestions to incorporate CRISPR/Cas9 experiments into lab courses at the undergraduate level. © 2018 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 46(2):195-205, 2018.

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.

Dosage compensation, the process by which the expression of X-linked genes are equalized between males, which have a single X chromosome and females, which have two, is essential in all heterogametic organisms. In C. elegans, dosage compensation is a complex process that is regulated by the developmental switch gene, xol-1. To better our understanding of the evolution of dosage compensation in nematodes, we use C. briggsae which has diverged from C. elegans ~15-30 million years ago, as a comparative model organism. In both species, loss of xol-1 results in a male specific lethality phenotype. We exploited this phenotype in C. briggsae and performed a classic genetic suppressor screen and identified nine suppressor mutations that are likely to represent components in the C. briggsae dosage compensation pathway.

As undergraduates, science students receive the educational foundation required for their future STEM careers. They build their knowledge base of basic science concepts, as well as learn hands-on skills that will help them in their future fields [1,2]. Often, this education is transmitted through lectures and teaching labs, where students play a passive role. However, undergraduate research experiences can serve as a more enriching alternative to such methods by combining biological concepts with laboratory skills in the context of novel scientific discovery [1,3]. Due to this, many undergraduate courses are incorporating discovery-based research into their curricula.

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y1009 and Y1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

The Caenorhabditis elegans FGF receptor, EGL-15, is alternatively-spliced to yield two major isoforms that differ in their extracellular domains. The EGL-15(5A) isoform is necessary for the gonadal chemoattraction of the migrating sex myoblasts (SMs), while the EGL-15(5B) isoform is required for viability. Here we show that 5A is predominantly expressed in the M lineage, which gives rise to the migrating SMs and their sex muscle descendants, while 5B is predominantly expressed in the hypodermis. Tissue-specific expression, however, explains only part of the functional differences between these two receptor isoforms. 5A can carry out the reciprocal essential function of 5B when expressed in the hypodermis, but 5B is incapable of carrying out SM chemoattraction. Our data, therefore, indicate that the structural differences in these two isoforms contribute to their functional differences. Two lines of evidence indicate that the 5B isoform also plays a role in SM migration, implicating it in the repulsion that is observed when the chemoattraction is compromised. Thus, structural differences in the extracellular domains of these two isoforms can specify either attraction to or repulsion from the gonad.

Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.