Sex-specific developmental gene expression atlas unveils dimorphic gene networks in C. elegans.

Sex-specific traits and behaviors emerge during development by the acquisition of unique properties in the nervous system of each sex. However, the genetic events responsible for introducing these sex-specific features remain poorly understood. In this study, we create a comprehensive gene expression atlas of pure populations of hermaphrodites and males of the nematode Caenorhabditis elegans across development. We discover numerous differentially expressed genes, including neuronal gene families like transcription factors, neuropeptides, and G protein-coupled receptors. We identify INS-39, an insulin-like peptide, as a prominent male-biased gene expressed specifically in ciliated sensory neurons. We show that INS-39 serves as an early-stage male marker, facilitating the effective isolation of males in high-throughput experiments. Through complex and sex-specific regulation, ins-39 plays pleiotropic sexually dimorphic roles in various behaviors, while also playing a shared, dimorphic role in early life stress. This study offers a comparative sexual and developmental gene expression database for C. elegans. Furthermore, it highlights conserved genes that may underlie the sexually dimorphic manifestation of different human diseases.

Natural probiotics improve heritable sterility.

Disrupting the small RNA pathway and chromatin-modifying enzymes in C. elegans often leads to a mortal germline (Mrt) phenotype, characterized by progressive sterility observed over multiple generations at elevated temperature. This phenotype arises from the inheritance of aberrant epigenetic memory across generations. In this issue of EMBO Reports, Frézal and colleagues reported that, while in standard laboratory environment C. elegans wild isolates exhibit the Mrt phenotype, sterility does not occur when the worms are exposed to naturally associated bacteria and microsporidia. Excitingly, diet-induced epigenetic memory may persist for multiple generations. This suggests intriguing diet-gene interactions in modulating nongenetic inheritance, potentially shaping the evolutionary trajectory of the animals.

Nucleus-independent transgenerational small RNA inheritance in Caenorhabditis elegans.

In Caenorhabditis elegans worms, epigenetic information transmits transgenerationally. Still, it is unknown whether the effects transfer to the next generation inside or outside of the nucleus. Here, we use the tractability of gene-specific double-stranded RNA-induced silencing to demonstrate that RNA interference can be inherited independently of any nuclear factors via mothers that are genetically engineered to transmit only their ooplasm but not the oocytes' nuclei to the next generation. We characterize the mechanisms and, using RNA sequencing, chimeric worms, and sequence polymorphism between different isolates, identify endogenous small RNAs which, similarly to exogenous siRNAs, are inherited in a nucleus-independent manner. From a historical perspective, these results might be regarded as partial vindication of discredited cytoplasmic inheritance theories from the 19th century, such as Darwin's "pangenesis" theory.

AI and the Doctor Dolittle challenge.

Talking to animals is a fundamental human desire. The emergence of powerful AI algorithms, and specifically Large Language Models, has driven many to suggest that we are on the verge of fulfilling this wish. A few large scientific consortia have been formed around this topic and several commercial entities even offer such services. We frame the task of communicating with animals as 'The Doctor Dolittle challenge' and identify three main obstacles on the route to doing so. First, although generative AI models can create novel animal communication samples, it is very difficult to determine their context, and we will forever be biased by our human umwelt when doing so. Second, using AI to extract context in an unsupervised manner must be validated through controlled experiments aiming to measure the animals' response. This is difficult, and moreover, AI algorithms tend to cling on to any available information and are thus prone to finding spurious correlations. And third, animal communication focuses on a restricted set of contexts, such as alarm and courtship, highly limiting our ability to communicate regarding other contexts. Nevertheless, using the tremendous power of novel AI methods to decipher and mimic animal communication is both fascinating and important. We thus define the criteria for passing the Doctor Dolittle challenge and call upon scientists to take on the mission.

RNAlysis: analyze your RNA sequencing data without writing a single line of code.

BACKGROUND: Among the major challenges in next-generation sequencing experiments are exploratory data analysis, interpreting trends, identifying potential targets/candidates, and visualizing the results clearly and intuitively. These hurdles are further heightened for researchers who are not experienced in writing computer code since most available analysis tools require programming skills. Even for proficient computational biologists, an efficient and replicable system is warranted to generate standardized results. RESULTS: We have developed RNAlysis, a modular Python-based analysis software for RNA sequencing data. RNAlysis allows users to build customized analysis pipelines suiting their specific research questions, going all the way from raw FASTQ files (adapter trimming, alignment, and feature counting), through exploratory data analysis and data visualization, clustering analysis, and gene set enrichment analysis. RNAlysis provides a friendly graphical user interface, allowing researchers to analyze data without writing code. We demonstrate the use of RNAlysis by analyzing RNA sequencing data from different studies using C. elegans nematodes. We note that the software applies equally to data obtained from any organism with an existing reference genome. CONCLUSIONS: RNAlysis is suitable for investigating various biological questions, allowing researchers to more accurately and reproducibly run comprehensive bioinformatic analyses. It functions as a gateway into RNA sequencing analysis for less computer-savvy researchers, but can also help experienced bioinformaticians make their analyses more robust and efficient, as it offers diverse tools, scalability, automation, and standardization between analyses.

The third barrier to transgenerational inheritance in animals: somatic epigenetic resetting.

After early controversy, it is now increasingly clear that acquired responses to environmental factors may perpetuate across multiple generations-a phenomenon termed transgenerational epigenetic inheritance (TEI). Experiments with Caenorhabditis elegans, which exhibits robust heritable epigenetic effects, demonstrated small RNAs as key factors of TEI. Here, we discuss three major barriers to TEI in animals, two of which, the "Weismann barrier" and germline epigenetic reprogramming, have been known for decades. These are thought to effectively prevent TEI in mammals but not to the same extent in C. elegans. We argue that a third barrier-that we termed "somatic epigenetic resetting"-may further inhibit TEI and, unlike the other two, restricts TEI in C. elegans as well. While epigenetic information can overcome the Weismann barrier and transmit from the soma to the germline, it usually cannot "travel back" directly from the germline to the soma in subsequent generations. Nevertheless, heritable germline memory may still influence the animal's physiology by indirectly modifying gene expression in somatic tissues.

Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs.

In nematodes and kinetoplastids, mRNA processing involves a trans-splicing step through which a short sequence from a snRNP replaces the original 5' end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods. Here we use Oxford Nanopore's long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms. We demonstrate that spliced leader (SL) sequences at the 5' end of the mRNAs affect library preparation and generate sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we find evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These mRNAs all share the capacity to generate a 5' terminal hairpin structure mimicking the SL structure and offering a mechanistic explanation for their non conformity. Altogether, our data provide a comprehensive quantitative analysis of SL usage in C. elegans.

ZNFX-1 keeps RNAi in the loop.

In a recent issue of Nature Cell Biology, Ouyang et al. examined the dynamics of double-stranded-RNA-induced gene-silencing across the Caenorhabditis elegans germline and in different subcellular locations. They distinguished among several small RNA amplification loops which complement each other and only together achieve full gene expression inhibition.

Transgenerational inheritance of sexual attractiveness via small RNAs enhances evolvability in C. elegans.

It is unknown whether transient transgenerational epigenetic responses to environmental challenges affect the process of evolution, which typically unfolds over many generations. Here, we show that in C. elegans, inherited small RNAs control genetic variation by regulating the crucial decision of whether to self-fertilize or outcross. We found that under stressful temperatures, younger hermaphrodites secrete a male-attracting pheromone. Attractiveness transmits transgenerationally to unstressed progeny via heritable small RNAs and the Argonaute Heritable RNAi Deficient-1 (HRDE-1). We identified an endogenous small interfering RNA pathway, enriched in endo-siRNAs that target sperm genes, that transgenerationally regulates sexual attraction, male prevalence, and outcrossing rates. Multigenerational mating competition experiments and mathematical simulations revealed that over generations, animals that inherit attractiveness mate more and their alleles spread in the population. We propose that the sperm serves as a "stress-sensor" that, via small RNA inheritance, promotes outcrossing in challenging environments when increasing genetic variation is advantageous.

Plant and animal small RNA communications between cells and organisms.

Since the discovery of eukaryotic small RNAs as the main effectors of RNA interference in the late 1990s, diverse types of endogenous small RNAs have been characterized, most notably microRNAs, small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). These small RNAs associate with Argonaute proteins and, through sequence-specific gene regulation, affect almost every major biological process. Intriguing features of small RNAs, such as their mechanisms of amplification, rapid evolution and non-cell-autonomous function, bestow upon them the capacity to function as agents of intercellular communications in development, reproduction and immunity, and even in transgenerational inheritance. Although there are many types of extracellular small RNAs, and despite decades of research, the capacity of these molecules to transmit signals between cells and between organisms is still highly controversial. In this Review, we discuss evidence from different plants and animals that small RNAs can act in a non-cell-autonomous manner and even exchange information between species. We also discuss mechanistic insights into small RNA communications, such as the nature of the mobile agents, small RNA signal amplification during transit, signal perception and small RNA activity at the destination.

Can brain activity transmit transgenerationally?

Memories encoded in the parent's brain should not be able to transfer to the progeny. This assumption, which is compatible with the tenets of modern neuroscience and genetics, is challenged by new insights regarding inheritance of transgenerational epigenetic responses. Here we reflect on new discoveries regarding "molecular memories" in light of older and scandalous work on "Memory transfer" spearheaded by James V. McConnell and Georges Ungar. While the history of this field is filled with controversies, mechanisms for transmission of information across generations are being elucidated in different organisms. Most strikingly, it is now clear that in Caenorhabditis elegans nematodes, somatic responses can control gene activity in descendants via heritable small RNA molecules, and that this type of inheritance is tightly regulated by dedicated machinery. In this perspective we will focus mostly on studies conducted using C. elegans, and examine recent work on the connection between small RNAs in the nervous system and germline. We will discuss the evidence for the inheritance of brain-orchestrated behavior, and its possible significance.

Stress resets ancestral heritable small RNA responses.

Transgenerational inheritance of small RNAs challenges basic concepts of heredity. In Caenorhabditis elegans nematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self-genes from non-self-elements. Carryover of aberrant heritable small RNA responses was shown to be maladaptive and to lead to sterility. Here, we show that various types of stress (starvation, high temperatures, and high osmolarity) induce resetting of ancestral small RNA responses and a genome-wide reduction in heritable small RNA levels. We found that mutants that are defective in various stress pathways exhibit irregular RNAi inheritance dynamics even in the absence of stress. Moreover, we discovered that resetting of ancestral RNAi responses is specifically orchestrated by factors that function in the p38 MAPK pathway and the transcription factor SKN-1/Nrf2. Stress-dependent termination of small RNA inheritance could protect from run-on of environment-irrelevant heritable gene regulation.

Systematic analysis of long intergenic non-coding RNAs in C. elegans germline uncovers roles in somatic growth.

Long intergenic non-coding RNAs (lincRNAs) are transcripts longer than 200 nucleotides that are transcribed from non-coding loci yet undergo biosynthesis similar to coding mRNAs. The disproportional number of lincRNAs expressed in testes suggests that lincRNAs are important during gametogenesis, but experimental evidence has implicated very few lincRNAs in this process. We took advantage of the relatively limited number of lincRNAs in the genome of the nematode Caenorhabditis elegans to systematically analyse the functions of lincRNAs during meiosis. We deleted six lincRNA genes that are highly and dynamically expressed in the C. elegans gonad and tested the effects on central meiotic processes. Surprisingly, whereas the lincRNA deletions did not strongly impact fertility, germline apoptosis, crossovers, or synapsis, linc-4 was required for somatic growth. Slower growth was observed in linc-4-deletion mutants and in worms depleted of linc-4 using RNAi, indicating that linc-4 transcripts are required for this post-embryonic process. Unexpectedly, analysis of worms depleted of linc-4 in soma versus germline showed that the somatic role stems from linc-4 expression in germline cells. This unique feature suggests that some lincRNAs, like some small non-coding RNAs, are required for germ-soma interactions.

Transgenerational Inheritance: That Pathogen Gut Feeling.

Recognizing and remembering dangerous pathogens is of the utmost importance for an animal's survival. Nematodes use a digested bacterial small RNA molecule as a cue of pathogenicity. Inheritance of this RNA even protects the progeny from infection.

Germ Granules Allow Transmission of Small RNA-Based Parental Responses in the "Germ Plasm".

In the recent decade small RNA-based inheritance has been implicated in a variety of transmitted physiological responses to the environment. In Caenorhabditis elegans, heritable small RNAs rely on RNA-dependent RNA polymerases, RNA-processing machinery, chromatin modifiers, and argonauts for their biogenesis and gene-regulatory effects. Importantly, many of these factors reside in evolutionary conserved germ granules that are required for maintaining germ cell identity and gene expression. Recent literature demonstrated that transient disturbance to the stability of the germ granules leads to changes in the pools of heritable small RNAs and the physiology of the progeny. In this piece, we discuss the heritable consequences of transient destabilization of germ granules and elaborate on the various small RNA-related processes that act in the germ granules. We further propose that germ granules may serve as environment sensors that translate environmental changes to inheritable small RNA-based responses.

Three Rules Explain Transgenerational Small RNA Inheritance in C. elegans.

Experiences trigger transgenerational small RNA-based responses in C. elegans nematodes. Dedicated machinery ensures that heritable effects are reset, but how the responses segregate in the population is unknown. We show that isogenic individuals differ dramatically in the persistence of transgenerational responses. By examining lineages of more than 20,000 worms, three principles emerge: (1) The silencing each mother initiates is distributed evenly among her descendants; heritable RNAi dissipates but is uniform in every generation. (2) Differences between lineages arise because the mothers that initiate heritable responses stochastically assume different "inheritance states" that determine the progeny's fate. (3) The likelihood that an RNAi response would continue to be inherited increases the more generations it lasts. The inheritance states are determined by HSF-1, which regulates silencing factors and, accordingly, small RNA levels. We found that, based on the parents' inheritance state, the descendants' developmental rate in response to stress can be predicted.