Gene-drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years [AU please provide a real estimate, this seems vague]. We describe CRISPR-Cas9 endonuclease constructs that function as gene-drive systems in Anopheles gambiae, the main vector for malaria [AU:OK?]. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene-drive constructs designed to target and edit each gene [AU:OK?]. For each locus targeted we observed strong gene drive at the molecular level, with transmission rates to progeny of 91 to 99.6%. Population modelling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to control suppress mosquito populations to levels that do not support malaria transmission.

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens.

The epidermis of Caenorhabditis elegans is an essential tissue for survival as it contributes to the formation of the cuticle barrier, as well as facilitates developmental progression and animal growth. Most of the epidermis consists of the hyp7 hypodermal syncytium, the nuclei of which are largely generated by the seam cells that exhibit stem cell-like behaviour during development. How the seam cell progenitors differ transcriptionally from the differentiated hypodermis is poorly understood. Here, we introduce Targeted DamID (TaDa) in C. elegans as a method for identifying genes expressed within a tissue of interest without cell isolation. We show that TaDa signal enrichment profiles can be used to identify genes transcribed in the epidermis and use this method to resolve differences in gene expression between the seam cells and the hypodermis. We finally predict and functionally validate new transcription and chromatin factors acting in seam cell development. These findings provide insights into cell-type-specific gene expression profiles likely associated with epidermal cell fate patterning.

Populations often display consistent developmental phenotypes across individuals despite inevitable biological stochasticity. Nevertheless, developmental robustness has limits, and systems can fail upon change in the environment or the genetic background. We use here the seam cells, a population of epidermal stem cells in Caenorhabditis elegans, to study the influence of temperature change and genetic variation on cell fate. Seam cell development has mostly been studied so far in the laboratory reference strain (N2), grown at 20° temperature. We demonstrate that an increase in culture temperature to 25° introduces variability in the wild-type seam cell lineage, with a proportion of animals showing an increase in seam cell number. We map this increase to lineage-specific symmetrization events of normally asymmetric cell divisions at the fourth larval stage, leading to the retention of seam cell fate in both daughter cells. Using genetics and single-molecule imaging, we demonstrate that this symmetrization occurs via changes in the Wnt asymmetry pathway, leading to aberrant Wnt target activation in anterior cell daughters. We find that intrinsic differences in the Wnt asymmetry pathway already exist between seam cells at 20° and this may sensitize cells toward a cell fate switch at increased temperature. Finally, we demonstrate that wild isolates of C. elegans display variation in seam cell sensitivity to increased culture temperature, although their average seam cell number is comparable at 20°. Our results highlight how temperature can modulate cell fate decisions in an invertebrate model of stem cell patterning.

Individual cells and organisms experience perturbations from internal and external sources, yet manage to buffer these to produce consistent phenotypes, a property known as robustness. While phenotypic robustness has often been examined in unicellular organisms, it has not been sufficiently studied in multicellular animals. Here, we investigate phenotypic robustness in Caenorhabditis elegans seam cells. Seam cells are stem cell-like epithelial cells along the lateral edges of the animal, which go through asymmetric and symmetric divisions contributing cells to the hypodermis and neurons, while replenishing the stem cell reservoir. The terminal number of seam cells is almost invariant in the wild-type population, allowing the investigation of how developmental precision is achieved. We report here that a loss-of-function mutation in the highly conserved N-acetyltransferase nath-10/NAT10 increases seam cell number variance in the isogenic population. RNA-seq analysis revealed increased levels of mRNA transcript variability in nath-10 mutant populations, which may have an impact on the phenotypic variability observed. Furthermore, we found disruption of Wnt signaling upon perturbing nath-10 function, as evidenced by changes in POP-1/TCF nuclear distribution and ectopic activation of its GATA transcription factor target egl-18. These results highlight that NATH-10/NAT-10 can influence phenotypic variability partly through modulation of the Wnt signaling pathway.

Transcription factors are key players in gene networks controlling cell fate specification during development. In multicellular organisms, they display complex patterns of expression and binding to their targets, hence, tissue specificity is required in the characterization of transcription factor–target interactions. We introduce here targeted DamID (TaDa) as a method for tissue-specific transcription factor target identification in intact Caenorhabditis elegans animals. We use TaDa to recover targets in the epidermis for two factors, the HES1 homolog LIN-22, and the NR5A1/2 nuclear hormone receptor NHR-25. We demonstrate a direct link between LIN-22 and the Wnt signaling pathway through repression of the Frizzled receptor lin-17 . We report a direct role for NHR-25 in promoting cell differentiation via repressing the expression of stem cell–promoting GATA factors. Our results expand our understanding of the epidermal gene network and highlight the potential of TaDa to dissect the architecture of tissue-specific gene regulatory networks.

A fundamental question in medical genetics is how the genetic background modifies the phenotypic outcome of mutations. We address this question by focusing on the seam cells, which display stem cell properties in the epidermis of Caenorhabditis elegans. We demonstrate that a putative null mutation in the GATA transcription factor egl-18, which is involved in seam cell fate maintenance, is more tolerated in the CB4856 isolate from Hawaii than the lab reference strain N2 from Bristol. We identify multiple quantitative trait loci (QTLs) underlying the difference in phenotype expressivity between the two isolates. These QTLs reveal cryptic genetic variation that reinforces seam cell fate through potentiating Wnt signalling. Within one QTL region, a single amino acid deletion in the heat shock protein HSP-110 in CB4856 is sufficient to modify Wnt signalling and seam cell development, highlighting that natural variation in conserved heat shock proteins can shape phenotype expressivity.

23Populations often display consistent developmental phenotypes across individuals 24 despite the inevitable biological stochasticity. Nevertheless, developmental robustness 25 has limits and systems can fail upon change in the environment or the genetic 26 background. We use here the seam cells, a population of epidermal stem cells in 27 Caenorhabditis elegans, to study the influence of temperature change and genetic 28 variation on cell fate. Seam cell development has mostly been studied so far in the lab 29 reference strain (N2), grown at 20° temperature. We demonstrate that an increase in 30 culture temperature to 25°, introduces variability in the wild-type seam cell lineage with a 31 proportion of animals showing an increase in seam cell number. We map this increase to 32 lineage-specific symmetrisation events of normally asymmetric cell divisions at the final 33 larval stage, leading to the retention of seam cell fate in both daughter cells. Using 34 genetics and single molecule imaging, we demonstrate that this symmetrisation occurs 35 via changes in the Wnt asymmetry pathway, leading to aberrant Wnt target activation in 36 anterior cell daughters. We find that intrinsic differences in the Wnt asymmetry pathway 37 already exist between seam cells at 20° and this may sensitise cells towards a cell fate 38 switch at increased temperature. Finally, we demonstrate that wild isolates of C. elegans 39 display variation in seam cell sensitivity to increased culture temperature, although seam 40 cell numbers are comparable when raised at 20°. Our results highlight how temperature 41 can modulate cell fate decisions in an invertebrate model of stem cell patterning. 42 43 44 45 46 47 48 During development, organisms must withstand environmental and genetic perturbations 49 to produce consistent phenotypes (Felix and Barkoulas 2012). These phenotypes are 50 often a product of complex developmental events that require a tight balance between cell 51 division and cell differentiation (Soufi and Dalton 2016). A key example is stem cell 52 divisions, consisting of highly controlled asymmetric and symmetric patterns, which are 53 vital for generating cell diversity, as well as maintaining cell numbers in tissues and organs 54 (Morrison and Kimble 2006; Knoblich 2008). Developmental robustness has inherent 55 limits and certain perturbations can push a system outside its buffering zone (Braendle 56 and Felix 2008; Barkoulas et al. 2013). In these cases, it is also important to understand 57 how systems fail by investigating how perturbations precisely modulate developmental 58 processes. Here we address the question of how changes in environmental temperature 59 can affect cell fate outcomes using the nematode C. elegans as a model system. While it 60 is well known that increasing or decreasing environmental temperature can change the 61 development speed in C. elegans, the effect of temperature on specific cell division and 62 fate acquisition events is less well understood. The C. elegans adult hermaphrodite 63 co...