Establish an in vitro model to study formation and function of the nervous system in the pathological conditions of Neural Tube Defects
Neural tubedefects (NTDs) are severe defects of the brain and spinal cord occurring by theend of the first month of pregnancy, when the neural plate develops then closesinto a tube. The World Health Organization reports a prevalence of NTDs allover the world (e.g.: 3.3 to 27.9 births per 10,000 in USA). Because NTDsrepresent a global public health burden, strategic plans have focused on betterunderstanding these diseases, in order to develop efficient treatments in thefuture. One recent advance in understanding mammalian brain development comes fromstem cell based technology, aimed at reproducing brain tissue in vitro. Organoids of different brain identitieshave beengenerated. Whilethese organoids display microanatomy similar to the authentic brain, they lackoverall proper spatial patterning of a developing brain in vivo. To overcome these limitations, the Thisse lab hasundertaken an innovative approach with the goal of recapitulating in vitro the successive steps of normalembryonic development and generating an embryo mimetic-system (embryoid)containing the three germ layers and their tissue/organ derivatives. Theseembryoids are patterned along the anterior-posterior and dorsal-ventral axesand they reach a developmental stage equivalent to embryonic day E9.5 of themouse embryo. More importantly, this is the first in vitro system, built from embryonic stem cells (ESCs), in which aneural plate forms and fold to form a neural tube.
The goal of theproposed study is to take advantages of this innovative model of embryoidsgenerated in vitro to study NTD. To do so, we will deriveESCs from embryos lacking the protein-tyrosinekinase 7 (Ptk7), which is critical for the regulation of planar cell polarity(PCP) and implicated in human NTDs. Its absence leads to craniorachischisis, asevere NTD in which the neural tube fails to close from midbrain to low spine.
Successful completion of these studies will establish arobust in vitro model to study formation and function of the nervoussystem in the pathological conditions of NTD. Our results will have a major impact on the field ofmammalian embryology in general by creating an accessible platform for thedissection of the molecular and cellular mechanisms of mammalian CNSdevelopment. Creating in vitro models of neural development amenable to experimentalmanipulation and large-scale drug screening is critically important fordelineating the molecular basis of, and developing clinical therapies for neurological disorders. It will also lay the groundwork for the creation in the future of anautologous human nervous system model, using human induced pluripotent stem cells.
Create a model of mammalian neural tube defect in vitro
1) Ptk7 mutant ESCs will be derived from homozygous Ptk7-null mouse embryos. In addition,pathogenic Ptk7 point mutations identified in human NTDs will be generatedusing CRISPR-Cas9 mediated genome editing in mESCs. (Lu and Thisse labs)
2) Embryoids will be generated using either Ptk7mutant ESCs or wild-type ESCs. (Thisse lab)
3) Wild-typeand mutant embryoids will be analyzed for their folding, closure or absence ofclosure of their neural plate using immuno-histological analyzes. (Thisse andLu labs)
4) Wild-type andmutant embryoids will be analyzed for defects in PCP-dependentconvergent-extension of the neural plate through live cell imaging and tracing,and quantitative analysis of cell motility parameters pertinent toconvergence-extension, folding of the neural plate and closure of the neuraltube. (Keller lab)
30K will be usedfor salary support (Graduate Student: Jinwoo Lee, Post-Doctoral Fellow: Dr AndreLandin Malt). The rest of the funds (30K) will be used to acquire the necessaryreagents and to cover the cost of using facilities (e.g.: imaging core facility,mouse stem cell facility) for this project.