Towards a cellular and molecular understanding of human hindbrain developmental disorders

Background:

The hindbrain (brainstem and cerebellum) regulates vital body functions and has a central role in motor coordination and cognitive and linguistic processing. Whilst progress has been made with regards to the diagnosis of hindbrain developmental disorders, their pathological basis remains poorly understood. The aim of this proposal is to investigate the cellular and molecular mechanisms underlying normal human hindbrain development and to elucidate the developmental origins of hindbrain disorders. Historically, studies on normal and abnormal hindbrain development have used animal models, in particular, the mouse as a model organism. However, our recent work has identified several critical differences between human and mouse development. These include spatiotemporally expanded primary and secondary progenitor zones and human-specific neural progenitor types.1 We also discovered that these unique progenitor zones are specifically affected in Dandy-Walker malformation.1,2 Down syndrome (DS), is a common but complex chromosomal disorder (prevalence 1:800 livebirths) with myriad clinical phenotypes including mild to severe intellectual disability, speech impairment, epilepsy, and autism. Pontine hypoplasia and cerebellar dysmorphology (hypoplasia and heterotopias) are common neuroimaging findings in DS.3 This study will test the hypothesis that the hindbrain hypoplasia observed in DS results from the disruption of normal developmental programs in human-specific progenitor zones. The development of the human brainstem in healthy children and in those with DS remains largely uncharacterised.

Aims/Objectives:

1. To determine if human-specific hindbrain progenitor zones are altered in DS in terms of proliferation rate and cell type. (0-18 months)

2. To model normal human cerebellar development and malformations thereof using 3D cerebellar organoids from normal and DS patient-derived induced pluripotent stem cells (iPSC). (12-24 months)

3. Develop live-imaging in hindbrain organoids and organotypic slice cultures of human hindbrain. (12-36 months).

Methods:

Aim1 will determine the appropriate range of cell types found in the proliferative zones and their respective proliferative rates in healthy and DS hindbrains provided by the Human Developmental Biology Resource. This is essential in order to define and distinguish the cellular and molecular mechanisms involved in hindbrain development in healthy children and in children with DS. This part of the study will combine histological analysis (immunohistochemistry and in situ hybridization) with single-cell RNA sequencing (validated by histological approaches).4 Aim2 will expand this work to three-dimensional human cerebellar cell cultures – i.e., ‘organoids’ of normal and DS-derived iPSCs. Several normal and DS-derived iPSC lines are available in the Alexandre Lab. Whilst animal models of cerebellar development lack critical transient proliferative zones, organoids can recapitulate in full the early stages of human cerebellar development: generating the majority of cerebellar neuronal and progenitor populations (unpublished data & Silva et al.).5 Aim3. Live-imaging of individual cells in organotypic slice culture of hindbrain and hindbrain organoids. This is critical for evaluation of dynamic processes such as neurogenesis and cell migratory behaviour in normal and DS-derived cells.

This study will investigate the underlying causes of hindbrain hypoplasia, a common phenotype shared among several hindbrain disorders. This is a critical step to develop future therapeutic and develop translational lines of research. All techniques are available in the supervisory (Dr Alexandre and Professor Greene) and collaborative teams (collaborators: Dr Haldipur and Professor Millen from Seattle Children’s Research Institute, US).

References:

1.  Haldipur et al. Science. 2019;366(6464):454-460. 2.  Haldipur et al. Acta Neuropathol. 2021;142(4):761-776. 3.  Rodrigues M et al. Insights Imaging. 2019;10(1):52.  4.  Aldinger KA et al. Nat Neurosci. 2021;24(8):1163-1175. 5. Silva TP et al. J Vis Exp. 2020;(160):10.3791/61143.