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Biologists have long been puzzled by the different rates of morphological evolution of animal species. Why do some taxa undergo rapid morphological changes while others, such as the coelacanth, can keep very similar morphologies over hundreds of millions of years? Is this correlated with the rates of genome divergence, or does this involve specific mechanisms to buffer or enhance the impact of genetic divergence? Our group is exploring the complex relationships between genotype and phenotype at the crossroad of developmental biology, computational biology, evolutionary biology and cell biology.

Lemaire fig1As a model system we use the embryos of tunicates because of the anatomical and genomic simplicity of these marine invertebrates, which are closely related to vertebrates (Left). We are focusing our work on two tunicate taxa: the ascidians and the thaliaceans.

Ascidian embryos develop, like most nematodes, with a fixed cell lineage that is remarkably well conserved between all studied species, even in those with genomes that have extensively diverged during 500 million years of parallel evolution. Ascidians thus offer a unique opportunity to decipher the developmental programme of a chordate at a cellular level of resolution, and to understand how genomic plasticity is compatible with morphological conservation. This latter phenomenon is referred to as Developmental Systems Divergence (DSD) and may explain why the modelling of human diseases in animal models is not always successful.

By contrast, the embryos of thaliaceans, which are considered paraphyletic to ascidians, have undergone a radical morphological change associated with an ecological transition from sessility to pelagy.

  • To identify genome-scale and gene regulatory network features that may explain the striking evolutionary stability of ascidian morphogenesis and how thaliaceans escaped it, we combine advanced quantitative imaging with genome sequencing, epigenetics, gene expression and cis-regulatory analyses. Our major aims are:
  • To elucidate the tunicate phylogeny and the evolution of the repertoire of developmental regulatory proteins in ascidians and thaliaceans.
  • To quantify ascidian morphogenesis and its evolution through advanced light-sheet microscopy and computational image analysis.
  • To sequence and assemble the genomes of closely or distantly related ascidian species
  • To unravel the evolution of the transcriptional programme and gene regulatory networks that drive embryogenesis between closely or distantly related ascidian species.
  • To identify the forces explaining the extreme evolutionary stability of ascidian morphogenesis at the systems level: relative impact of developmental constraints, selection and drift.
  • To develop ANISEED, an advanced model organism database system for the multi-scale integration of heterogeneous datasets describing developmental programmes at the genetic and cellular levels.

Our group is a member of the Institute for Computational Biology of Montpellier, of the EpiGenMed Laboratory of Excellence and of the Morphoscope project.

Our work is supported by the CNRS, the ANR, the Fondation pour la Recherche Médicale and the Fondation Bettencourt Schueller. We have strong collaborations with the INRIA Virtual plants group, the Phylogeny and Molecular Evolution group at the Institute for Evolutionary Sciences of Montpellier and the Dynamics of cell growth and tissue architecture group at EMBL.

 


Research group

► Matija BROZOVIC
(Research Assistant) +33 (0)4 34 35 95 59
► Christelle DANTEC
(Research Assistant) +33 (0)4 34 35 95 60
► Justine DARDAILLON
(Research Assistant) +33 (0)4 34 35 95 60
► Julien LAUSSU
(Post-Doc) +33 (0)4 34 35 95 63
► Patrick LEMAIRE Group Leader
(Staff Scientist) +33 (0)4 34 35 94 00
► Alicia MADGWICK
(PhD Student) +33 (0)4 34 35 95 63
► Jacques PIETTE
(Staff Scientist) +33 (0)4 34 35 95 63
  • M. Brozovic, C. Martin, C. Dantec, D. Dauga, M. Mendez, P. Simion, M. Percher, B. Laporte, C. Scornavacca, S. Fujiwara, M. Gineste, E. Lowe, J. Piette, Y. Sasakura, N. Takatori, TC. Brown, F. Delsuc, C.Gissi, E. Douzery, A. McDougall, H. Nishida, H. Sawada, B. Salla, H. Yasuo, P. Lemaire (2016). ANISEED 2015: a digital framework for the comparative developmental biology of ascidians. Nucleic Acid Research. Database issue in press.
  • J. Piette, P. Lemaire (2015). Thaliaceans, the neglected pelagic relatives of ascidians: a developmental and evolutionary enigma. The Quarterly review of biology 90(2):117-145. pubmed
  • P. Lemaire, J. Piette (2015). Tunicates: exploring the sea shores and roaming the open ocean. A tribute to Thomas Huxley. Open Biol 5, 150053. pubmed
  • A. Stolfi, Y. Sasakura, D. Chalopin, Y. Satou, L. Christiaen, C. Dantec, T. Endo, M. Naville, H. Nishida, BJ. Swalla, JN. Volff, A. Vosboboynik, D. Dauga, P. Lemaire (2015). Guidelines for the nomenclature of genetic elements in Tunicate genomes. Genesis 53, 1-14. pubmed
  • A. Roure, P. Lemaire, S. Darras (2014). An Otx/nodal regulatory signature for posterior neural developmenet in Ascidians. PloS Genetics, 10,e1004548. pubmed
  • L. Guignard, C. Godin, UM. Fiuza, L. Hufnagel, P. Lemaire, G. Malandain (2014). Spatio-temporal registration of embryo images. Proceedings of the IEEE International Symposium on Biomedical Imaging, Beijing.
  • R. Sanges, Y. Hadzhiev, M. Gueroult-Bellone, A. Roure, M. Ferg, N. Meola, G. Amore, S. Basu, E.R. Brown, M. De Simone, F. Petrera, D. Licastro, U. Strähle, S. Banfi, P. Lemaire, E. Birney, F. Müller and E. Stupka (2013). Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development. Nucleic Acids Res. 41:3600-18. pubmed

  • A. Jolma, J. Yan, T. Whitington, J. Toivonen, K.R. Nitta, P. Rastas, E. Morgunova, M. Enge, M. Taipale, G. Wei, K. Palin, J.M. Vaquerizas, R. Vincentelli, N.M. Luscombe, T.R. Hughes, P. Lemaire, E. Ukkonen, T. Kivioja and J. Taipale (2013). DNA-binding specificities of human transcription factors. Cell 152:327-39. pubmed

  • A. Pasini, R. Manenti, U. Rothbächer and P. Lemaire (2012). Antagonizing retinoic acid and FGF/MAPK pathways control posterior body patterning in the invertebrate chordate Ciona intestinalis. PLoS ONE 7:e46193. pubmed

  • P. Lemaire (2011). Evolutionary crossroads in developmental biology: the tunicates. Development 138:2143-52. pubmed

  • F.B. Robin, D. Dauga, O. Tassy, D. Sobral, F. Daian and P. Lemaire (2011). Imaging of fixed ciona embryos for creating 3D digital replicas. Cold Spring Harb Protoc 2011:1247-50. pubmed

  • F.B. Robin, D. Dauga, O. Tassy, D. Sobral, F. Daian and P. Lemaire (2011). Time-lapse imaging of live Phallusia embryos for creating 3D digital replicas. Cold Spring Harb Protoc 2011:1244-6. pubmed

  • F.B. Robin, D. Dauga, O. Tassy, D. Sobral, F. Daian and P. Lemaire (2011). Creating 3D digital replicas of ascidian embryos from stacks of confocal images. Cold Spring Harb Protoc 2011:1251-61. pubmed

  • K. Sherrard, F. Robin, P. Lemaire and E. Munro (2010). Sequential activation of apical and basolateral contractility drives ascidian endoderm invagination. Curr. Biol. 20:1499-510. pubmed

  • O. Tassy, D. Dauga, F. Daian, D. Sobral, F. Robin, P. Khoueiry, D. Salgado, V. Fox, D. Caillol, R. Schiappa, B. Laporte, A. Rios, G. Luxardi, T. Kusakabe, J.S. Joly, S. Darras, L. Christiaen, M. Contensin, H. Auger, C. Lamy, C. Hudson, U. Rothbächer, M.J. Gilchrist, K.W. Makabe, K. Hotta, S. Fujiwara, N. Satoh, Y. Satou and P. Lemaire (2010). The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program. Genome Res. 20:1459-68. pubmed

  • P. Khoueiry, U. Rothbächer, Y. Ohtsuka, F. Daian, E. Frangulian, A. Roure, I. Dubchak and P. Lemaire (2010). A cis-regulatory signature in ascidians and flies, independent of transcription factor binding sites. Curr. Biol. 20:792-802. pubmed

  • D. Sobral, O. Tassy and P. Lemaire (2009). Highly divergent gene expression programs can lead to similar chordate larval body plans. Curr. Biol. 19:2014-9. pubmed

  • H. Auger, C. Lamy, M. Haeussler, P. Khoueiry, P. Lemaire and J.S. Joly (2009). Similar regulatory logic in Ciona intestinalis for two Wnt pathway modulators, ROR and SFRP-1/5. Dev. Biol. 329:364-73. pubmed

  • P. Lemaire (2009). Unfolding a chordate developmental program, one cell at a time: invariant cell lineages, short-range inductions and evolutionary plasticity in ascidians. Dev. Biol. 332:48-60. pubmed

  • C. Lamy and P. Lemaire (2008). [Ascidian embryos: from the birth of experimental embryology to the analysis of gene regulatory networks]. Med Sci (Paris) 24:263-9. pubmed

  • J. Matsumoto, Y. Katsuyama, Y. Ohtsuka, P. Lemaire and Y. Okamura (2008). Functional analysis of synaptotagmin gene regulatory regions in two distantly related ascidian species. Dev. Growth Differ. 50:543-52. pubmed

  • O. Luu, M. Nagel, S. Wacker, P. Lemaire and R. Winklbauer (2008). Control of gastrula cell motility by the Goosecoid/Mix.1/ Siamois network: basic patterns and paradoxical effects. Dev. Dyn. 237:1307-20. pubmed

  • Y. Satou, K. Mineta, M. Ogasawara, Y. Sasakura, E. Shoguchi, K. Ueno, L. Yamada, J. Matsumoto, J. Wasserscheid, K. Dewar, G.B. Wiley, S.L. Macmil, B.A. Roe, R.W. Zeller, K.E. Hastings, P. Lemaire, E. Lindquist, T. Endo, K. Hotta and K. Inaba (2008). Improved genome assembly and evidence-based global gene model set for the chordate Ciona intestinalis: new insight into intron and operon populations. Genome Biol. 9:R152. pubmed

  • P. Lemaire, W.C. Smith and H. Nishida (2008). Ascidians and the plasticity of the chordate developmental program. Curr. Biol. 18:R620-31. pubmed

  • E. W. Deutsch, C. A. Ball, J. J. Berman, G. S. Bova, A. Brazma, R. E. Bumgarner, D. Campbell, H. C. Causton, J. H. Christiansen, F. Daian, D. Dauga, D. R. Davidson, G. Gimenez, Y. A. Goo, S. Grimmond, T. Henrich, B. G. Herrmann, M. H. Johnson, M. Korb, J. C. Mills, A. J. Oudes, H. E. Parkinson, L. E. Pascal, N. I. Pollet, J. Quackenbush, M. Ramialison, M. Ringwald, D. Salgado, S. A. Sansone, G. Sherlock, C. J. Stoeckert, J. Swedlow, R. C. Taylor, L. Walashek, A. Warford, D. G. Wilkinson, Y. Zhou, L. I. Zon, A. Y. Liu, and L. D. True (2008). Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). Nature Biotechnology. 26:305-12.

  • U. Rothbächer, V. Bertrand, C. Lamy and P. Lemaire (2007). A combinatorial code of maternal GATA, Ets and beta-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm. Development 134:4023-32. pubmed

  • H. Bielen, S. Oberleitner, S. Marcellini, L. Gee, P. Lemaire, H.R. Bode, R. Rupp and U. Technau (2007). Divergent functions of two ancient Hydra Brachyury paralogues suggest specific roles for their C-terminal domains in tissue fate induction. Development 134:4187-97. pubmed

  • A. Roure, U. Rothbächer, F. Robin, E. Kalmar, G. Ferone, C. Lamy, C. Missero, F. Mueller and P. Lemaire (2007). A multicassette Gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos. PLoS ONE 2:e916. pubmed

  • P. Lemaire (2006). Q & A: an interview with Patrick Lemaire. Curr. Biol. 16:R143-4. pubmed

  • C. Lamy, U. Rothbächer, D. Caillol and P. Lemaire (2006). Ci-FoxA-a is the earliest zygotic determinant of the ascidian anterior ectoderm and directly activates Ci-sFRP1/5. Development 133:2835-44. pubmed

  • A. Pasini, A. Amiel, U. Rothbächer, A. Roure, P. Lemaire and S. Darras (2006). Formation of the ascidian epidermal sensory neurons: insights into the origin of the chordate peripheral nervous system. PLoS Biol. 4:e225. pubmed

  • E. Munro, F. Robin and P. Lemaire (2006). Cellular morphogenesis in ascidians: how to shape a simple tadpole. Curr. Opin. Genet. Dev. 16:399-405. pubmed

  • P. Lemaire (2006). Developmental biology. How many ways to make a chordate? Science 312:1145-6. pubmed

  • O. Tassy, F. Daian, C. Hudson, V. Bertrand and P. Lemaire (2006). A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis. Curr. Biol. 16:345-58. pubmed

OUR QUESTIONS AND MODEL ORGANISMS

Developmental Systems Divergence.
It is seducing to think that when phenotypic traits are similar, they are obtained through the action of similar (or homologous) molecular cascades. This hypothesis is indeed a central argument for using animal models to understand human disease: as the physiology of homologous rodent and human organs is very similar, we expect that the same genetic pathways may act in both and that their deregulation may lead to similar phenotypes. This happens to be true in many cases. Yet, as many as 20% of essential human genes have no obvious phenotype when mutated in laboratory mice, suggesting that an ancestral biological process, conserved between species, may come under divergent molecular controls in the course of evolution.

This process is referred to as Developmental Systems Divergence, and is frequently observed when studying the evolution of developmental regulatory logics. It reflects a previously unsuspected level of flexibility in evolutionary trajectories. There is currently no satisfactory explanation on why some processes remain under their ancestral control mechanism, while others come under divergent control.

The tunicates, our surprising marine cousins

Tunicates are the sister group of vertebrates, a shared ancestry reflected by the small tadpole larvae most species produce. Whereas the affinities of tunicates with vertebrates have been well established by phylogenomic studies using nuclear and mitochondrial DNA data, the interrelationships among the major tunicate clades are more uncertain. Divergence times are unknown and even the tree topology remains somewhat uncertain. The standard view divides tunicates into ascidians (sessile adults), thaliaceans (planktonic adults) and larvaceans (tadpole-like adults) (Figure 1). Ascidians are classified according to their branchial sac structure into stolidobranchs, phlebobranchs, and aplousobranchs. Molecular analyses recover close affinities between thaliaceans and aplousobranchs or phlebobranchs, suggesting the paraphyletic nature of ascidians (Figure 1).
 

Lemaire fig1b 400
Figure 1: Cladogram of the major Tunicate lineages (yellow box) in the context of the bilaterian animal phylogeny. The relative position of thaliaceans, aplousobranchs and phlebobranchs is not resolved.

Ascidians constitute the largest and most studied tunicate group (Figure 1). They provide an extreme case of evolutionary conservation of embryogenesis. In all analysed species, ascidian embryos develop in a stereotyped manner with invariant embryonic cell lineages, thus allowing the study of developmental mechanisms at a subcellular level of resolution. Remarkably, the embryonic cell lineages of Ciona intestinalis and Halocynthia roretzi, two species separated by more than 400 million years of divergent evolution, are nearly identical. Even the shape, position and surface of contacts between embryonic cells appear well conserved. The stereotyped development of ascidians, based on invariant cell lineages, may in part explain this extreme level of embryonic morphology conservation. Nematodes and annelids, however, also develop with invariant cell lineages, but with lesser conservation of these lineages.

Although their embryonic morphologies are impressively conserved, ascidians show the highest level of intra-specific polymorphism within animals (up to 12.4% of polymorphism in Ciona intestinalis). Consistently two genomes of the genus Ciona (C. intestinalis type A subspecies and Ciona savignyi) show striking divergence in spite of high morphological similarity at the adult stage.
Surprisingly, the pattern of morphological conservation observed in ascidians does not extend to thaliaceans, which are considered paraphyletic to ascidians. Their major ecological transition from a sessile ancestor to their pelagic (planktonic) lifestyle was accompanied by a radical change in embryonic morphologies. Many thaliaceans have lost the tadpole stage found in all other chordates, and may also have departed from the stereotyped cell lineage used by ascidians. Among chordates, the group formed by ascidians and thaliaceans thus shows both the highest evolutionary conservation of embryonic morphologies (within ascidians) and the most radical deviation from the prototypical chordate morphologies (in thaliaceans).

OUR PROJECTS:

Our approach is to move step by step from genome sequence to transcriptional regulation, the assembly of Gene Regulatory Networks and their control of cell fates and shapes by fully exploiting the on-going revolutions in high throughput sequencing and live imaging.

A refined and quantitative tunicate phylogeny (Collaboration with the Phylogeny and Molecular Evolution group at the Institute for Evolutionary Sciences of Montpellier)

Tunicate phylogeny remains somewhat controversial and this may cause difficulties in the subsequent comparison of the developmental strategies used by ascidian species and thaliaceans. In particular, currently we do not have any reliable estimate of the divergence times between the ascidian species we sequenced. Also, the paraphyly of ascidians and thaliaceans remains a working hypothesis that needs to be confirmed.

We have generated transcriptomic data for two thaliacean species (a salp and a doliolid) and have generated sets of gene models for several ascidian species (see below). We are now using these data to build a refined and quantitative tunicate phylogeny. These data will also be used to assess the evolution of the repertoire of regulatory genes among tunicates and to identify rapidly diverging thaliacean genes that may play a role in the striking divergence of the thaliacean developmental program from that of their benthic ascidian-like ancestors.

Quantification of the ascidian morphogenetic programme (Collaboration with the INRIA Virtual Plants group and the Dynamics of cell growth and tissue architecture group at EMBL).

We are using advanced live light-sheet microscopy to quantify the morphogenetic programme of ascidian embryos at subcellular resolution and to study its variability within and between species. In parallel, we are developing a computational framework to automatically segment, digitalize and statistically analyse the geometry of developing embryos and its dynamics.
We are using these technological breakthroughs to analyse, with cellular resolution, the geometric variability (shape, organization, contacts between cells) within and between species. Our aim is to relate patterns of variability and constraints to known embryological mechanisms (e.g., gastrulation, cell-cell communication…) and to evolutionary concepts, such as the developmental hourglass according to which early and late embryogenesis are less constrained than mid embryogenesis.

Evolution of the ascidian genomes

We have recently sequenced, assembled and annotated the genomes of several ascidian species to increase the range of phylogenetic distances relative to the previously sequenced Ciona genomes: Ciona intestinalis type B (<50 million years from Ciona intestinalis type A), Phallusia mammillata and P. fumigata (~300 million years from Ciona intestinalis) and Halocynthia roretzi and H. aurantium (>400 million years from Ciona intestinalis). These genomes show a remarkable level of coding and non-coding divergence, as well as profound architectural reorganizations that disrupt the syntenic relationships and the gene order on chromosomes.

Evolution of the ascidian transcriptional programme and gene regulatory networks

We are testing various hypotheses to explain how divergent genomes can support very similar morphogenetic programmes. Our work indicates that most genes show conserved expression patterns between species in spite of extensive genomic divergence such that non-coding sequences cannot be aligned at orthologous loci.

We found in isolated examples that this conservation of expression profiles is facilitated by the flexibility of the architecture of cis-regulatory sequences, which allows for extensive transcription factor binding site turnover. We are now testing this hypothesis at the genome scale.

In parallel, we found that a substantial number of genes show divergent expression profiles, indicating a rewiring of gene regulatory networks, and evidencing a high level of Developmental Systems Divergence. We are now reconstructing the early developmental regulatory networks in Phallusia mammillata to better understand how they evolved and how they are coupled to the cellular networks that drive morphogenesis and in particular gastrulation.

Forces that explain the extreme evolutionary stability of ascidian morphogenesis (Collaboration with the Phylogeny and Molecular Evolution group at the Institute for Evolutionary Sciences of Montpellier and the INRIA Virtual Plants group)

Several evolutionary hypotheses could explain morphological stasis, including stabilizing selection and developmental constraints. To test these hypotheses, we are first adopting a micro-evolutionary approach based on the sequencing of the genome of multiple Ciona and Phallusia individuals, in the hope of identifying selection signatures on coding and non-coding sequences. In parallel, we are imaging and reconstructing many individual Phallusia embryos to identify the most constrained and divergent morphological features. We will then develop a statistical test to infer signatures of selection at the morphological level in these embryos.

Lemaire Fig2                         Lemaire Fig3

Maximal projection of confocal imaging of a Ciona intestinalis late gastrula embryo in which the cortical actin network was fluorescently labelled.

 

 

 

 

 

 

 

 

 

Ciona intestinalis young gastrula imaged by confocal microscopy and segmented. The colour code indicates to the larval cell fates of each embryonic cell. Note the regularity of the embryo, which reflects its stereotyped and bilaterally symmetrical development.