Periodic Reporting for period 1 - EvoCellFate (Evolution of cell fate decision during development)
Periodo di rendicontazione: 2017-11-01 al 2019-10-31
Understanding the mechanisms underlying phenotypic diversification in evolution is a central challenge in modern biology. To understand the basis of biodiversity, we need to know how molecular and genetic processes control development and shape phenotypic evolution. A key consequence of the architecture of developmental mechanisms is to constraint and biases the evolutionary process, thus shaping the evolution of morphological diversity and possibly producing different evolutionary trajectories in different taxa (evolutionary trends;1,2). The mutational process generates molecular variation that influences phenotypic variation through the genotype-phenotype map, and this phenotypic variation is then subjected to selection and drift. Importantly, even if mutation is random at the genotype level (in first approximation), it does not necessarily result in isotropic exploration of phenotype space. Therefore, understanding the mutationally accessible phenotypic spectrum (mutational variance) is central to investigating the possible evolutionary routes of phenotypic change.
The nematode vulva is ideal to study the evolution of developmental mechanisms because of its defined and homologous cellular framework, its particularly well-understood developmental system, the fast development of C. elegans (a generation in 3 days) and the known nematode diversity (3, 4). Nematode vulva development occurs from a subset of six ventral epidermal blast cells, named P3.p to P8.p. The pattern of vulva cell fates is conserved and invariant within the Caenorhabditis genus, except for P3.p that can adopt two different fates that are highly variable within and between species. The fast evolution of P3.p fate matches the mutational variance properties of this cell when the organism is subjected to random mutation in the laboratory(7). In contrast, the Oscheius genus of nematodes displays a completely different pattern of natural variation in cell lineages during vulva development(5). In Oscheius, the P3.p cell has an almost invariant cell fate, and the patterns of cell division of P4.p and P8.p are highly polymorphic between and within Oscheius species. How do the natural patterns of evolution of homologous cells become so distinct between nematode genera?
The project is organized in two main objectives. The first objective was to identify the genetic and molecular elements that cause high mutational variance of P3.p cell fate variation in two Caenorhabditis species and to identify the genetic basis of natural variation in P3.p cell fate decision between wild isolates of C. elegans. The second objective was to investigate whether biases in developmental mutability could be the basis for the evolution of evolutionary trends between different genera.
The topic of cell fate decision in stem cell biology(9) and developmental biology(10) has been highly investigated. However, the role of spontaneous mutations and natural genetic variation in cell fate decision has been largely dismissed. Additionally, this question is not just relevant for the field of evolution of development, but fundamental to understand the role of human genetic diversity in diseases such as cancer(11).
The nematode vulva is ideal to study the evolution of developmental mechanisms because of its defined and homologous cellular framework, its particularly well-understood developmental system, the fast development of C. elegans (a generation in 3 days) and the known nematode diversity (3, 4). Nematode vulva development occurs from a subset of six ventral epidermal blast cells, named P3.p to P8.p. The pattern of vulva cell fates is conserved and invariant within the Caenorhabditis genus, except for P3.p that can adopt two different fates that are highly variable within and between species. The fast evolution of P3.p fate matches the mutational variance properties of this cell when the organism is subjected to random mutation in the laboratory(7). In contrast, the Oscheius genus of nematodes displays a completely different pattern of natural variation in cell lineages during vulva development(5). In Oscheius, the P3.p cell has an almost invariant cell fate, and the patterns of cell division of P4.p and P8.p are highly polymorphic between and within Oscheius species. How do the natural patterns of evolution of homologous cells become so distinct between nematode genera?
The project is organized in two main objectives. The first objective was to identify the genetic and molecular elements that cause high mutational variance of P3.p cell fate variation in two Caenorhabditis species and to identify the genetic basis of natural variation in P3.p cell fate decision between wild isolates of C. elegans. The second objective was to investigate whether biases in developmental mutability could be the basis for the evolution of evolutionary trends between different genera.
The topic of cell fate decision in stem cell biology(9) and developmental biology(10) has been highly investigated. However, the role of spontaneous mutations and natural genetic variation in cell fate decision has been largely dismissed. Additionally, this question is not just relevant for the field of evolution of development, but fundamental to understand the role of human genetic diversity in diseases such as cancer(11).
For the first objective, we combined mutation accumulation lines, whole-genome sequencing, genetic linkage analysis of the phenotype in recombinant lines, and candidate testing through mutant and CRISPR genome editing to identify causal mutations and the corresponding loci underlying the high mutational variance of P3.p. We identify and validate molecular lesions responsible for changes in this cell’s phenotype during a mutation accumulation experiment. We find that these loci do not present any characteristics of a high mutation rate, are scattered across the genome and belong to distinct biological pathways. This work is currently under revision in a renowned high-impact journal and is published as a pre-print article in the openly accessible archive bioRxiv.
Furthermore, we have collected preliminary data to test whether changes in gene expression in candidate loci are correlated in distinct patterns of P3.p division frequency in C. elegans wild isolates. For this, we conducted RNA expression analyses using single-molecule fluorescent in situ hybridisation technique to quantify the number of mRNA molecules of Hox genes and Wnt ligands in C. elegans wild isolates. Our preliminary data shows a correlation on the number of some Wnt ligand mRNAs and the frequency of P3.p division among wild isolates. We are currently validating and expanding our data set, and in the future we will investigate the causative nature of the association between levels of Wnt gene expression and cell-fate variability.
For objective two, we used a mutagenesis approach with the mutagen N-ethyl-N-nitrosourea to mutagenize two wild isolates of C. elegans, C. briggsae, O. tipulae and O. onirici. We generated eight panels of random mutant lines to a total of 553 lines. Second, we performed whole genome sequencing in a subset of 40 random mutant lines to estimate mutation rates, mutation spectrums and distributions along the genome. We are currently conducting the bioinformatic genome-wide analysis. Third, we are presently phenotyping the vulva cell fate differentiation of the random mutant lines to quantify the mutability of vulva precursor cell fates across micro and macro- evolutionary scales. The progress of this work has been presented in the format of Poster presentation in two international scientific meetings – 2018 EMBO Workshop on “C. elegans development, cell biology and gene expression” in Barcelona, Spain; 2019 “22nd International C. elegans Conference” in Los Angeles, USA -, in two internal departmental seminars and in several informal meetings.
Furthermore, we have collected preliminary data to test whether changes in gene expression in candidate loci are correlated in distinct patterns of P3.p division frequency in C. elegans wild isolates. For this, we conducted RNA expression analyses using single-molecule fluorescent in situ hybridisation technique to quantify the number of mRNA molecules of Hox genes and Wnt ligands in C. elegans wild isolates. Our preliminary data shows a correlation on the number of some Wnt ligand mRNAs and the frequency of P3.p division among wild isolates. We are currently validating and expanding our data set, and in the future we will investigate the causative nature of the association between levels of Wnt gene expression and cell-fate variability.
For objective two, we used a mutagenesis approach with the mutagen N-ethyl-N-nitrosourea to mutagenize two wild isolates of C. elegans, C. briggsae, O. tipulae and O. onirici. We generated eight panels of random mutant lines to a total of 553 lines. Second, we performed whole genome sequencing in a subset of 40 random mutant lines to estimate mutation rates, mutation spectrums and distributions along the genome. We are currently conducting the bioinformatic genome-wide analysis. Third, we are presently phenotyping the vulva cell fate differentiation of the random mutant lines to quantify the mutability of vulva precursor cell fates across micro and macro- evolutionary scales. The progress of this work has been presented in the format of Poster presentation in two international scientific meetings – 2018 EMBO Workshop on “C. elegans development, cell biology and gene expression” in Barcelona, Spain; 2019 “22nd International C. elegans Conference” in Los Angeles, USA -, in two internal departmental seminars and in several informal meetings.
The rapid evolution of a trait in a group of organisms - an evolutionary trend – has been attributed in some instances to their mutational variance (7, 11, 12), the propensity of a phenotype to change under mutation. However, the causes of high mutational variance are still elusive. In some case, fast morphological evolution was shown to depend on the high mutation rate of one or few underlying loci with short tandem repeats (e.g. 13, 14). Our data shows a novel mechanism underlying high mutational variance of a morphological trait: a broad mutational target, where many different loci involved in different biological pathways contribute to a variation of a trait. This result has important implication for the study of mechanisms underlying phenotypic evolution and for human disease such as cancer biology (15).
In objective two, we expect to discover that evolutionary trends can be shaped by evolution of mutational variances, showing that macro- and micro-evolution are constrained on the developmental genetic architecture of traits.
References
1. Arthur (2004) Evo & Dev 282–288
2. Lynch (2007) Nat Rev Genet 803–813
3. Félix (2002) Phys Biol 045001
4. Sternberg (2005) WormBook 1–28
5. Delattre & Felix (2001) Cur Bio 631–643
6. Pénigault & Félix (2011) Dev bio 419–427
7. Braendle et al. (2010) PLoS Gen e1000877
8. Wu & Belmonte (2016) Cell 1572–1585
9 Balázsi et al. (2011) Cell 910–925
10. Merlo & Maley (2010) J Clin Inves 401–403.
11. Houle et al. (2017) Nature 1–16
12. Farhadifar et al. (2016) Genetics 1859–1870.
13. Fondon & Garner (2004) PNAS 18058–18063
14. Chan et al. (2010) Science 302–305
15. Shpak & Lu (2016) A R Eco Evo & Syst 25–49
In objective two, we expect to discover that evolutionary trends can be shaped by evolution of mutational variances, showing that macro- and micro-evolution are constrained on the developmental genetic architecture of traits.
References
1. Arthur (2004) Evo & Dev 282–288
2. Lynch (2007) Nat Rev Genet 803–813
3. Félix (2002) Phys Biol 045001
4. Sternberg (2005) WormBook 1–28
5. Delattre & Felix (2001) Cur Bio 631–643
6. Pénigault & Félix (2011) Dev bio 419–427
7. Braendle et al. (2010) PLoS Gen e1000877
8. Wu & Belmonte (2016) Cell 1572–1585
9 Balázsi et al. (2011) Cell 910–925
10. Merlo & Maley (2010) J Clin Inves 401–403.
11. Houle et al. (2017) Nature 1–16
12. Farhadifar et al. (2016) Genetics 1859–1870.
13. Fondon & Garner (2004) PNAS 18058–18063
14. Chan et al. (2010) Science 302–305
15. Shpak & Lu (2016) A R Eco Evo & Syst 25–49