Use of genetic engineering (by CRISPR/Cas9) to study ascidians nervous system formation.

The development of the central nervous system (CNS) depends on complex gene regulatory networks orchestrating the specification, patterning and differentiation of neural cell types. We would like to take advantage of the unique characteristics of a simple chordate, the ascidian Ciona robusta, to investigate the molecular mechanisms underlying cell specification within CNS, with a cellular resolution unprecedented in chordate models. The Ciona anterior sensory vesicle, considered to be homologous to the forebrain of vertebrates, contains two distinct pigment cell-containing sensory organs, the otolith and the ocellus. The otolith is a single cell containing a melanin granule and involved in negative geotactism while the ocellus in a complex organ involved in light detection. Transcriptome profiling of pigmented cells, isolated by Fluorescence-Activated Cell Sorting (FACS), allowed us to unravel a plethora of genes, possibly involved in the differentiation of single cells inside the nervous system.

Our group is currently using CRISPR/Cas9 technologies to knock out a number of these genes with the final goal to understand how gene activity can instruct each developing brain cell to move around, change shape, and connect to other cells inside the Ciona sensory vesicle with special attention to pigment cell differentiation.

The student will contribute to research activities within this main project and her/his work will involve:

  • In silico identification of sgRNA for CRISPR/Cas9
  • Preparation of construct for CRISPR/Cas9 interference with gene function.
  • Handling of Ciona embryos including gamete collection, set up of embryo cultures and fixation at different developmental and postembryonic stages after treatments.
  • In vivo electroporation of Ciona fertilized eggs to introduce interfering construct into the live animals.
  • Phenotypic analyses by light and confocalmicroscopy of treated larvae
  • Colorimetric and two-colour fluorescent whole mount in-situ hybridisation.
  • Embryo imaging by DIC, epifluorescence and confocal microscopy.
  • RNA extraction, purification and cDNA synthesis from normal and treated embryos.
  • Identification of sgRNA for CRISPR/Cas9
Language requirements: 
English
Specific competences required : 
No additional skills required
Safety issues: 
The students will be exposed to low levels of a few toxic chemicals routinely used in molecular biology labs. No need of vaccinations, no diving, no other safety issues involved.
Accommodation possibilities : 
There is no accommodation at the Stazione Zoologica. Single rooms in a shared apartment in Naples cost around 300-400 euro. Bed and Breakfast rooms cost around 30€ per day.
Additional costs to be covered by the student: 
No