Investigating the role of endothelin signalling system in the establishment of adult pigment progenitors in zebrafish development.

Student thesis: Doctoral ThesisPhD

Abstract

In this work, we present the characterisation of parade mutants, which disrupt activity of the Endothelin receptor Aa (EdnrAa), and which exhibit ectopic melanophores and iridophores in the medial trunk, ventral to the dorsal aorta (DA).

Our first studies were based upon the hypothesis of disruption of embryonic pigment development. Using a transgenic line (sox10:cre/hsp70:lox:dsRed:lox-LYN-GFP) that labels neural crest (NC)-derived cells, we showed that cell migration is not disrupted. Furthermore, quantification of other NC-derivatives (sensory, sympathetic, nor enteric neurons) revealed similar numbers in WT and ednraa mutant siblings, arguing against transdifferentiation to pigment cells. Instead, by immunodetection of Phospho-Histone-H3, we detected proliferation of NC-derived cells restricted to the vicinity of the DA of ednraa mutants.

Previously, a small molecule screen of 1396 compounds performed by a former PhD student of the lab showed that an inhibitor of Erb signalling, which is crucial for setting aside adult pigment stem cells (APSCs) in the embryo, rescued the ednraa mutant phenotype. Here we further investigate the requirement for Erb signalling in the ednraa phenotype. Strikingly, we have demonstrated that Erb signalling is required for the formation of ectopic pigment cells in ednraa mutant embryos in the same time-window in which APSCs are set aside. This strongly implicates APSCs as the source of the ectopic pigment cells. Based on these results, we proposed that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.

In order to identify what other components of the Endothelin signalling pathway contribute to Ednraa’s function in the pde phenotype, in collaboration with the Nuesslein-Volhard lab we generated null mutants for some of the endothelin system genes (edn1, edn2a edn3a, edn3b, ednraa, ednrab and ece1) using CRISPR/Cas9 targeted mutagenesis. Our analysis of the pigment phenotype of these mutants identified that edn1 and edn2a have redundant roles in embryonic iridophore development as single mutants of these genes display a strong reduction of iridophores in the dorsal and ventral stripes, while only edn2a mutants lack iridophores in the lateral patches. We also confirmed the previously reported phenotype of edn1 and edn2a mutants affecting the formation of ventral craniofacial cartilages. Furthermore, we determined that no single ligand mutant (edn1, edn2a, edn3a and edn3b) phenocopies the ednraa mutant, suggesting that more than one ligand is necessary for APSC development. Unexpectedly, we found that double mutants of ednraa; ednrab rescued the ednraa single mutant phenotype (no ectopic pigment cells), suggesting that ednrab has a role on APSC biology.

To further test our model, we investigated whether formation of ectopic chromatophores shares other molecular traits with APSC development. Formation of APSCs does not require activity of mitfa; similarly, we determined that formation of ectopic cells in ednraa mutants does not require the early activity of this gene, supporting our hypothesis that ectopic pigment cells in ednraa mutant are derived from a source different to embryonic pigment cells. Secondly, we began to test whether signalling pathways known to regulate stem cells in other models are required in the formation of ectopic pigment cells in ednraa mutants. For instance, we showed that the ednraa mutant phenotype depends upon Wnt signalling and can be phenocopied by chemical inhibition of Notch signalling.

Our revised model proposes that APSC associated with a niche consisting of the ventral spinal nerve projections, is regulated (indirectly) through ednraa signalling from the dorsal aorta. Thus, we have found in the ednraa mutant an exciting opportunity for in vivo study of adult pigment stem cell biology.
Date of Award13 Feb 2019
LanguageEnglish
Awarding Institution
  • University of Bath
SupervisorRobert Kelsh (Supervisor)

Keywords

  • Zebrafish
  • Neural Crest
  • Stem Cells
  • Endothelin Signalling
  • Pigment Cells

Cite this

Investigating the role of endothelin signalling system in the establishment of adult pigment progenitors in zebrafish development.
Camargo Sosa, K. (Author). 13 Feb 2019

Student thesis: Doctoral ThesisPhD