Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - COLOUR PATTERN (Morphogenesis and Molecular Regulation of Colour Patterning in Birds)

Teaser

Report period: from 01/11/2016 to 31/10/2017Problem/issue being addressed:Our laboratory focuses on the processes of formation and evolution of natural patterns (i.e. visible arrangement of characters across bodies and tissues). These patterns result from the establishment of...

Summary

Report period: from 01/11/2016 to 31/10/2017

Problem/issue being addressed:

Our laboratory focuses on the processes of formation and evolution of natural patterns (i.e. visible arrangement of characters across bodies and tissues). These patterns result from the establishment of discrete domains within developing organs, in a process that involves the integration of positional cues by cells that consequently differentially express target genes and adopt specific fates. This creates spatial heterogeneity in the tissue by producing compartments and localised boundaries. A major challenge has thus been to understand how patterns can arise from initially homogeneous structures in a reproducible manner, while allowing apparent complexity and diversity. We set to identify molecules acting as positional cues, characterise the morphogenetic response (cell / tissue rearrangements) to these cues, and how such pattern-forming processes constrain (or promote) the evolution of natural patterns. We use an innovative approach that combines two predictive tools (1) natural variation (as the identification of common attributes of patterns allows drawing developmental predictions) and (2) mathematical modelling of both pattern emergence and variation. Predictions are then tested experimentally in vivo. To implement this method, we take advantage of birds, which are versatile developmental models, and the color and feather pattern, an emblematic and visual study system.


Importance for society:

This work is important for the scientific community as it addresses one of the longest standing questions in developmental biology (i.e., how is positional information provided within developing tissues). In addition, because it relies on variation in ecologically-relevant phenotypes in natural populations, it work will shed light on the developmental constraints shaping the evolution of adaptive traits in nature, a major question in evolutionary biology. Finally, its impact goes beyond the scientific community: colour pattern fascinates the general public, both for its natural diversity and ecological significance.


Overall objectives:
Our aims are to characterise skin organisation (i.e., pre-pattern), study the developmental basis of patterning strategies, identify positional factors, and test their mode of action for two chosen systems: large color domains in Estrildid finches and periodic striped patterns in Galliform birds

Work performed

Report period: from 01/11/2016 to 31/10/2017

Aim1: Characterise color pre-pattern formation and the source of positional cues forming large color domains
As planned in the DoA, we had achieved at the end of the previous report period the description of the color and feather pattern in adult and juvenile zebra finches and linked these to distinct domains in the dorsal and ventral embryonic skin, separated in three (anterior, saddle, posterior) and four (chest, flank, main, posterior) reproducible domains, respectively. We had obtained RNA samples from each of these putative domains prior to feather follicle and pigment appearance. During the second reporting period, we extracted RNA from these samples and performed RNAseq experiments in collaboration with the genomics plateform of the Ecole Normale Supérieure (Paris). Despite the relatively low coverage and annotation of the zebra finch genome, we obtained high quality scores and found several hundred genes differentially expressed between putative color regions (we compared domains both individually, and by grouping ventral and dorsal regions). This work was performed in collaboration with the Genosplice company (Station F, Paris). Along the antero-posterior (AP) axis, most genes showing differential expression appeared in both dorsal and ventral analyses; the striking majority belonged to the homeobox family (known for its role in providing compartment identity and establishing boundaries along the AP axis). Along the dorso-ventral (DV) axis, many genes belonged to developmental pathways (e.g., Wnt pathway). Overall, we identified 25 first candidates based on spatial expression differences and overall strong expression levels. We currently perform expression analyses. So far, we found 5 candidates marking putative domains along the AP axis (namely Hoxa4, Ism1, and Pitx2 in the saddle, Hoxa7 in the anterior region, and Irx1 in the posterior ventral region), and along the DV axis (namely Wnt5a, Sox2, and Osr2 in the flank, Zic3 in the dorsum, and agouti in the ventral main region). Note that we had previously identified agouti through an in situ screen, and used it as a positive control. We will finish the analysis of remaining candidates both in the zebra finch and in sister species (see Aim2) to build a comprehensive reference map of developmentally expressed genes in putative color domains. We envision the publication of a manuscript in 2018.

Aim2: Identify the molecular events establishing the pre-pattern of large color domains.
As mentioned in the previous report, the selection of 4 parental species was completed. As then planned, we extended thorough phenotypic analyses to the Gouldian finch (based on its relevant pattern along the AP axis) and now use it together with the 3 previously selected species (Owl finch, Zebra finch and Long-tailed finch) for comparative expression experiments. We analyzed the expression of previously mentioned candidates (identified through RNA seq analysis of putative color regions in all 4 selected species. We found that the expression of Osr2, Agouti and Irx1 change between species and while they do not entirely spatially correlate with pigment boundaries, their expression pattern is compatible with observed phenotypic variation. These results and those of Aim1 are being combined for the preparation, as previously mentioned, of a publication in 2018. We currently work to investigate the function of these candidates through CRISPr-Cas9 mediated gene editing (construct delivery will be performed using electroporation and viral infection on developing skin explants; which we have successfully produced in finches comparably to Galliforms).
Work on Aim3: Characterise the morphogenetic events underlying the response to positional cues in periodic color patterns

Aim3: Characterisation of stripe patterning dynamics
As planed in the DoA and previous report, we described the color and feather pattern in 11 Galliform and 3 Ratite species. We had

Final results

Our work has so far provided important advances to the state of the art by showing that:
- Large colour domains result from the coordinated expression of developmental genes in a timely fashion (Aim 1 and 2)
- Periodic patterns result from both early developmental landmarks that constrain their evolution and local dynamics occurring late within developing tissues (Aim 3 and 4)
- Colour and skin patterns are triggered by initial conditions present in the naive skin and relayed by the progression of competence fronts (Aim 3)
- Diversity in colour and skin pattern is largely linked to morphogenetic events and biophysical mechanics in the cutaneous tissue (Aim 3 and 4)

By exploring the developmental pathways we identified through unbiased RNA seq experiments, we expect to uncover the nature of molecular factors providing positional information to large colour domains.
By performing tissue lineage experiments in Galliforms and Ratites, we expect to uncover the source and molecular nature of factors acting as developmental landmarks shaping periodic patterning.
By generating model-based predictions on spatio-temporal events involved in skin pattern establishment, we expect to draw testable hypotheses on the factors involved in the onset and progression of competence fronts in the developing skin.
By modelling, imaging and consequently functionally testing the establishment of feather follicles in various species, we expect to uncover the mechanical events that control their size and spacing in the skin.