Felix M-A et al. (2000) European Worm Meeting "Vulva development in oscheius Sp."

Louvet-Vall S, Felix M-A, Dichtel ML

[European Worm Meeting, 2000]

The vulva patterning mechanism is not conserved in all nematodes. We have started a genetic analysis in Oscheius/Dolichorhabditis CEW1, and about 60 vulva mutants have been isolated in an F2 Egl screen. The vulva lineage in this species is simple: P(5-7).p form the vulva; the central cell, P6.p, divides three times and then acquires the inner fate characterized by a "tttt" lineage. After two divisions, P5.p and P7.p acquire the outer vulval fate characterized by a "uuuu" lineage. P4.p and P8.p belong to the competence group but in normal development they divide two times and then acquire a non-vulval fate characterized by a "ssss" lineage. We found several mutants affecting the number of vulva cell divisions, irrespective of fate induction. 1) Some have defects only in P4.p and P8.p cells which divide too few times. Laser ablation studies in these mutants indicate that the correct number of divisions of P(5-7).p depends on a gonadal signal; in one mutant, the vulva fate and the number of divisions can be uncoupled while they are coupled for the other mutants [unpublshed data, Mark Viney and Paul W. Sternberg]. Moreover, it seems that the defect of division of P4.p and P8.p is correlated to a difference of these cells to respond to the anchor cell signal. It is interesting to note that in some natural populations of Oscheius Dolichorhabditis sp., P4.p and P8.p divide also too few times. 2) In other mutants, the defect of division affects all P(4-8).p which divide too few times. They appear to omit the second cell cycle in all lineages. 3) Some other mutants show an excess of P(5-7).p divisions: we observe a 3rd round of division for P5.p and P7.p and a 4th round in the P6.p lineage. All these phenotypes are unknown in C. elegans. We found also many mutants with a defect in the competence group and/or centering of the vulva. 4) In some, P(4-8).p behave like non-competent Pn.ps (lin-39 or bar-1 phenotype in C. elegans). 5) The competence group is enlarged to P3.p. 6) The competence group is enlarged to P9.p but some cells within the group are non-competent. Moreover, the vulva is miscentered on P5.p (this phenotype is unknown in C. elegans). 7) The vulva is miscentered on P7.p without modification of the competence group. This spectrum of vulva mutations is very different from that of C. elegans and also suggests that the relation between vulval cell cycle and fate differs from that of C. elegans.

Felix M-A et al. (1997) International C. elegans Meeting "Evolutionary variations in nematode vulva patterning mechanisms"

Viney ME, Sternberg PW, Felix M-A

[International C. elegans Meeting, 1997]

To understand how developmental mechanisms vary during evolution, we compared vulva development of different nematode species and have initiated genetic analyses in one species other than C. elegans. During vulva development, Pn.p precursor cells are aligned in the ventral cord and acquire one of three fates in a symmetric pattern centered around the gonadal anchor cell: non-vulval fates at the periphery, outer and inner vulval fates towards the center. In C. elegans, the pattern is centered on P6.p. The three fates are specified around the same time by cell interactions: a graded induction by the anchor cell and lateral signaling of P6.p to P5.p and P7.p. We find that in three other nematode species spanning two families, the centered pattern is obtained by two temporally distinct inductions. The first induction specifies vulval vs. non-vulval fates, the second, localized to a subset of the daughters of the vulva precursors, specifies inner vs. outer vulval fates. In Panagrolaimus, the pattern is centered between P6.p and P7.p, the vulva is formed from four precursor cells (P5.p to P8.p) and the inner vulval fate is shared between P6.pp and P7.pa. In Oscheius and Rhabditella, the pattern is centered on P6.p as in C. elegans, but a late induction by the anchor cell on P6.pa and P6.pp is required for the proper specification of inner fates. Variation of the spatio-temporal connectivity of cell interactions may thus occur but nonetheless result in the same global pattern of cell fates. To understand the molecular nature of the 2 signals from the anchor cell and their relation with the pathways known in C. elegans, we have started a genetic and molecular analysis of 2-step vulva induction in Oscheius n. sp. CEW1. So far, we have isolated in a random F2 screen several vulva mutations and 35 marker mutations (a spectrum similar to C. elegans). The first characterized vulva mutation (sy447) affects the first induction only: the outermost cells adopt the outer vulval fate instead of a non-vulval fate. We are also developing molecular tools in this species, such as a cDNA library and transformation, and cloning homologs of molecules involved in vulva induction in C. elegans, such as LIN-3 and Ras.

Felix M-A et al. (1998) European Worm Meeting "Genetic screen for vulva mutants in Oscheius sp. CEW1"

Felix M-A, Viney ME, Sternberg PW, Dichtel ML

[European Worm Meeting, 1998]

To understand how developmental mechanisms vary during evolution, we compare vulva development of different nematode species. We previously found an evolutionary variation in the cell interactions patterning the vulva precursor cells (Felix & Sternberg, Development 124, 253). In C. elegans, the three fates of the Pn.p precursor cells are specified around the same time by a graded induction by the anchor cell and lateral signaling among the precursors. In several other species, the same centered pattern is obtained by two nested inductions by the anchor cell: the first induction specifies vulval vs. non-vulval fates, the second later specifies inner vs. outer vulval fates in the Pn.p daughters. We have started a genetic analysis of vulva development in a species with 2-step vulva induction, Oscheius / Dolichorhabditis sp. CEW1. In a F2 screen, we have isolated so far vulva mutants and about 60 markers. The spectrum of markers is similar to C. elegans (Dpy, Unc, Rol, Sma, Lon, Con). The spectrum of vulva mutations however appears different. As in C. elegans, we find mutants of Pn.p cell generation and gonad development, but surprisingly none or very few Vulvaless mutants defective in vulval induction. We found so far two Hyperinduced mutants affecting the first induction only, in which P4.p and P8.p adopt an outer vulval fate and participate in the main vulval invagination. Categories of mutants that are well represented are: 1. Mutants in which P(4-8).p (the competence group) behave like non-competent Pn.ps. 2. Mutants in which conversely P3.p follows the lineage of P4.p. 3. Mutants with an excessive number of vulval divisions, either with an abnormal third round of division for P5.p and P7.p, or with a fourth round in the P6.p lineage. 4. Mutants with a lesser number of divisions, either of all P(4-8).p, or specifically of P4.p and P8.p. Laser ablation studies in a mutant of the latter class (sy543) indicate that the number of divisions of P(5-7).p depends on a gonadal signal, but can be uncoupled from fate specification. This suggests that the relation between cell cycle and vulval cell fate differs from that found in C. elegans. We are currently screening for more vulva mutants.

Felix M-A et al. (1995) International C. elegans Meeting "Symmetry breakage in the development of one-armed gonads in nematodes"

Felix M-A, Sternberg PW

[International C. elegans Meeting, 1995]

The 2-armed gonad of C. elegans hermaphrodites is built symmetrically from 2 somatic precursors, Z1 and Z4. Asymmetric one-armed gonads seem to have evolved repeatedly in nematodes: the posterior arm is either reduced to a post-vulval sac (Panagrellus, Panagrolaimus, Cephalobus and Acrobeloides spp.) or totally eliminated and the vulva shifted posteriorly (Mesorhabditis sp. and Diplogastrellus sp.). We study the development of these one-armed gonads to understand how asymmetry and diversification arise in evolution.

Felix M-A et al. (1996) West Coast Worm Meeting "EVOLUTIONARY VARIATIONS IN NEMATODE GONAD DEVELOPMENT AND VULVA PATTERNING MECHANISMS"

Felix M-A, Sternberg PW

[West Coast Worm Meeting, 1996]

To understand how developmental mechanisms vary during evolution, we compare the development of different nematode species. Whereas the hermaphrodite gonad of C. elegans has two symmetric arms (didelphy), the female/hermaphrodite gonad of many other species features a single anterior arm (monodelphy). In C. elegans, the two arms develop symmetrically from two somatic precursor cells, Z1 (anterior) and Z4 (posterior). In monodelphic species, Z1 and Z4 have different fates: this is an example of evolutionary diversification. Reduction of the posterior arm correlates with cell death in the lineage of Z4 in most species. The specific fate of Z4 progeny is induced by Z1 (or its daughters) in Cephalobus sp. PS1197. In the uterus primordium in C. elegans, symmetric lateral signalling between Z1.ppp and Z4.aaa renders them equally likely to become the anchor cell (AC). In the different monodelphic species, AC specification is biased, or fully fixed, to a descendant of either Z1, or Z4. Replacement regulation upon anchor cell ablation is conserved in some species, but lost in others, leading to a mosaic-type development. Differentiation between Z1 and Z4 is thus manifest at this later stage in the breakage of symmetry of cell interactions in the ventral uterus. During vulva development, precursor cells acquire one of three distinct fates in a symmetric pattern centered around the AC. Non-vulval fates are at the periphery, outer and inner vulval fates are towards the center. In C. elegans, specific cell interactions generate the pattern centered on P6.p: a graded induction by the AC and lateral signalling among the vulva precursors. The three fates are specified around the same time. We find that in three other nematode species (Panagrolaimus, Oscheius and Rhabditella spp.) spanning two families, the centered pattern is obtained by two temporally distinct inductions. The first induction specifies vulval fates, the second induction, localized to a subset of the daughters of the vulva precursors, specifies the inner fates. In Panagrolaimus, the anchor cell is positioned between P6.p and P7.p, and the 'TTTT' inner fate is shared between their 2 central daughters, P6.pp and P7.pa. Extreme variation of developmental mechanism at the level of the spatio-temporal connectivity of cell interactions may thus occur but nonetheless result in the same global pattern of cell fates. To understand the molecular nature of the 2 signals from the AC and the relation with the molecular pathways known in C. elegans., we are starting a genetic and molecular analysis of vulva induction in Oscheius n. sp. CEW1.

Remy JJ et al. (2000) European Worm Meeting "Does olfactory imprinting exist in nematodes?"

Felix M-A, Remy JJ

[European Worm Meeting, 2000]

Environmental sensory signals present during critical developmental periods can influence and partially fix patterns of sensitivities at the adult stage. Imprinting of early olfactive environment is well documented in a number of animal species. Nematodes have complex olfactory systems, and highly rely on their chemical sense for survival. In C. elegans, it involves 11 pairs of morphologically different amphid neurons, and each pair is thought to express a limited number of chemoreceptors out of several hundred members. As demonstrated for the AWA and AWB neurons, which respectively control attraction or repulsion to volatil odorants, identity of the neuron in which a given receptor is expressed direct worms chemotactic behaviour toward the ligand of this receptor. Genetic analysis on the chemoreceptor transducing pathways demonstrated activity dependence of sensory neurons morphofunctionality during development, and a growing number of evidences support the idea of a relatively high degree of plasticity in the nematode chemosensory system. For example, in adults, reversible adaptative desensitization after prolonged exposure to some odorants as well as odorant discrimination vary with environment signals and previous experience. It seems however that adult nematodes cannot be sensitized to odorants even through associative learning protocols. We want to adress the question of a possible influence of environmental olfactory stimuli on chemoreceptor genes expression in functionally distinct sensory neurons. Receptors are thought to be expressed very early during development, as transcripts are present even before hatching. By exposing worms to odorants at different stages of development, and measuring modifications of chemotactic responses in the adults, we hope to be able to define a critical period during which chemosensitivity could be experimentally manipulated. This olfactive "learning" process by early imprinting could then be further pharmacologically and genetically analysed.

Delattre M et al. (1998) European Worm Meeting "Evolution of vulval development and P11/12 cell migration in nematodes."

Felix M-A, Delattre M

[European Worm Meeting, 1998]

In nematodes, the vulva is formed by a set of precursors called Pn.p cells. In C. elegans, each Pn.p cell is specified by an inductive signal coming from a specialized cell of the gonad, the anchor cell, and a lateral signaling between the Pn.p cells. In other nematode species, like Oscheius sp. CEW1 (family Rhabditidae), patterning of the Pn.p cells is specified not by several simultaneous cellular interactions but by two successive inductions by the gonad or the anchor cell (AC). The complexity of the vulva patterning can therefore be achieved by very distinct networks of cellular interactions. Our aim is to understand changes in the mechanisms of vulva patterning at the molecular level, wich means understanding the molecular nature of the two signals coming from the AC and of the intracellular pathways transducing them in the recieving cells, in CEW1. Are the two signals identical, or different forms of the same molecule, or distinct molecules? We are cloning, in CEW1, homologues of genes known to be involved in C. elegans vulval patterning genes of the lin-3/let-23 signalling pathway. We first focus on lin-3, the inductive signal from the AC and let-60, a Ras molecule highly conserved during evolution. These molecules will be cloned by PCR with degenerated primers or by hybridization at low stringency using C. elegans gene as probe. The P precursors cells are responsible for the postembryonic development of the ventral nerve cord. At hatching, the 12 cells P1-P12 are disposed laterally along the antero-posterior body axis in six left/right pairs. They start to migrate ventrally several hours after hatching to enter the ventral cord. For most left/right pairs, the antero-posterior order is random (Sulston and Horvitz, 1977). But in the P11/12 pair in C. elegans, the left cell always migrates anteriorly and becomes P11, whereas the right cell becomes P12. In Panagrellus redivivus (family Panagrolaimidae), conversely, the right cell becomes P11 (Sternberg and Horvitz, 1982). We wonder if there is a switch in P11/12 cell migration during evolution of nematodes, particulary in the order Rhabditida. Do species with P11/12 random migration exist ? We are now looking at P11/12 cell migration in different species. It seems that in Panagrolaimus, a species near Panagrellus, migration of P11/12 is actually random, at least variable. Our aim is to look at more species in the family Panagrolaimidae and at species with sinistral population, to understand if P11/12 migration is linked with asymmetry of the whole body .

Dubois, C. et al. (2017) International Worm Meeting "Efficient identification of candidate gene mutations by mapping-by-sequencing in the non-model nematode Oscheius tipulae."

Koutsovoulos, G., Besnard, F., Vargas-Velazquez, A., Dubois, C., Felix, M-A., Blaxter, M.

[International Worm Meeting, 2017]

Oscheius tipulae is a common free-living nematode in the same clade as the parasitic taxa Heterorhabditis and strongylids, and closer to the model species Caenorhabditis elegans than the outgroup Pristionchus pacificus. This hermaphroditic species is thus informative for comparative genetics, developmental and evolutionary studies. However, the genetic toolbox for non-model organisms such as O. tipulae is still underdeveloped. In model species with fully assembled and annotated genomes, mapping-by-sequencing has become a standard method to map and identify phenotype-causing mutations. Candidate variants are pinpointed using a cross to a divergent mapping strain and sequencing of a pool of mutant segregants (e.g. ref. 1). Chemical mutagenesis (EMS) performed about 15-20 years ago by Marie-Anne Felix's lab on the O. tipulae strain CEW1 generated several vulval development and other morphological mutants, including dumpy, roller and uncoordinated phenotypes (refs 2-6). Using the mapping-by-sequencing approach, a draft genome sequence for O. tipulae CEW1 and crosses with the divergent wild isolate JU170, we have identified for the first time relevant candidate genes for these phenotypes in O. tipulae. Our success suggests that a draft assembly with multiple scaffolds per chromosome is sufficient to perform mapping-by-sequencing. Other recent genetics tools such as CRISPR/Cas9 system have been developed and widely used on Caenorhabditis species to perform targeted mutagenesis, and we propose to confirm candidate genes by developing CRISPR/Cas9 system on O. tipulae. We argue here that the mapping-by-sequencing approach and genomic analysis tools can be easily used in non-model organisms. This brings non-model species firmly into genomics-enabled science, and provides tools to investigate the biology of non-model species and improve our understanding of evolution and developmental mechanisms. (1) Doitsidou et al. PLOS One 2011; (2) Felix et al. Nematology, 2000; (3) Dichtel et al. Genetics 2001; (4) Louvet-Vallee et al. Genetics 2003; (5) Dichtel-Danjoy et al. Dev Biol 2004; (6) Felix, Oscheius tipulae, WormBook, 2006.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Picao Osorio, J. et al. (2019) International Worm Meeting "The role of developmental genetic architecture in shaping evolutionary trends."

Felix, M-A, Braendle, C., Picao Osorio, J.

[International Worm Meeting, 2019]

Random mutation of the genotype does not generate random phenotypic variation because development biases the mutationally inducible phenotypic spectrum. Therefore, understanding such biases in the introduction of phenotypic variation is essential to reveal which phenotypes can be explored and selected in the evolutionary process. Whether such developmental genetic biases in the construction of phenotypic variation influence evolutionary trends is poorly understood. Here we address this problem using the homologous cellular framework of vulval precursor cells in two clades of nematodes that have divergent evolutionary trajectories of cell fate variation. In Caenorhabditis species, among the six vulva precursor cells (VPC) termed P3.p to P8.p, only P3.p cell fate shows significant evolutionary variation within and among species. In contrast, in Oscheius species (sister genus) evolutionary variation of cell fates is highest in different precursor cells, i.e. P4.p and P8.p. We will probe the role of developmental constraints in shaping evolutionary trends of nematode vulva cell fate variation. For this, we will access the mutationally accessible phenotypic spectrum (i.e. mutational variance) using a combination of random mutagenesis and quantitative genetics. First, we have generated eight panels of random mutant lines in wild isolates of Caenorhabditis and Oscheius to quantify the mutability of VPC fates across micro and macro-evolutionary scales. Second, we will compare this mutational variance with the natural cell fate variation at different evolutionary scales. This project will lead us to causally connect developmental mutability and evolutionary trends at the single-cell level.