Investigate the mechanisms that regulate human hindbrain development

Background:
Hindbrain (cerebellum and brainstem) malformations have impact on motor and mental health, with hypoplasia being a common feature linking a number of disorders including Down syndrome(1) and autism. While there is a wealth of knowledge on the mechanisms involved in hindbrain development in animal models, little is known about that developmental changes that take place in humans. Therefore, the underlying causes of human hindbrain malformation remain also ignored.

We recently published the first comprehensive histological and molecular analyses of human cerebellar development. We discovered that neural progenitor zones in human cerebellum are spatiotemporally expanded when compared to rodent, avian and fish models (Haldipur el al., 2019, Science)(2). Both human cerebellar primary progenitor zones (ventricular zone - VZ and rhombic lip - RL) develop a substructure that contains certain progenitor types (basal Radial Glia-like cells, bRG-like) that are absent in mice(2) but can be found in the developing human cerebral cortex(3) where they contribute to brain gyrification and expansion. Understanding the organisation of progenitors zones during human hindbrain development is essential to understand the origin of hindbrain malformations such as Down Syndrome.

Aims/objectives:
This project aims to characterise neural progenitor population and dynamics during human hindbrain development in normal and Down Syndrome hindbrain. The objectives are to:

1. Characterise the main progenitor zones during hinbrain development.
2. Determine the cellular identity of hindbrain progenitors by comparing cell morphology, molecular identity and self-renewal ability of hindbrain neural progenitor and compare to well known progenitors in the cerebral cortex.
3. Investigate whether the cellular composition and organisation of progenitor zones are disrupted in the Down Syndrome hindbrain.

Methods:
This project combines the expertise of Alexandre and Livesey labs in human cortical and hindbrain development and organotypic slice culture, live-imaging, histological analysis. Single cell RNA sequencing analysis will be performed in collaboration with Kathy Millen and Parthiv Haldipur (Seattle’s Children Research Institute, USA).

• Immunohistochemistry and in situ hybridization assays will be used to characterise the progenitor zones and cell types in the developing human cerebellum and brain stem.
• DiI, GFP focal electroporation or lentiviral transfection of single cells in organotypic slices(2) or primary cell cultures combined with live-imaging or immunohistochemistry will determine the morphology, molecular identity and progenitor behaviours.
• BrdU/EdU pulses at 30min and 48h (in whole mount and slice cultures) will also contribute to evaluate self-renewing ability of neural progenitors.
• Single cell RNA sequencing analysis will help determine the molecular signatures and developmental trajectories of hindbrain progenitors(4).
• Tissue for histological analysis has been acquired. Fresh tissue for future experiments wil be provided by Human Developmental Biology Resource (HDBR- London (UCL GOS ICH) and Newcastle.

References:
1.            Fujii Y. et al. Brain Dev 3, 298-305 (2017)
2.            Haldipur P et al. Science 266, 454-460 (2019)
3.            Hansen et al. Nat. Neurosci. 13, 690-699 (2010)
4.            Aldinger KA et al, submitted to Dev Cell.