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May 10, 2019

RPG Seminar Series (Speaker: Mr. LUI Jeffrey / Mr. LYU Quanwei)

Date: Friday, 10 May 2019

Venue: Seminar Room 1, G/F
Laboratory Block, Faculty of Medicine Building
21 Sassoon Road, Hong Kong

Time: 5:00 p.m.

Title: The Role of ephrin-A1/EphA4 signaling in Enteric Neural Crest Cell Migration
Speaker: Mr. LUI Jeffrey (MPhil candidate)

Enteric neural crest cells (ENCCs) migrate and colonize the gut during enteric nervous system (ENS) development, in which Ret and p75 support ENCC survival, proliferation, migration and differentiation. Cell-cell interaction is largely regulated by Eph/ephrin signaling that induces attraction or repulsion between cells, crucial to cell segregation and thus cell migration in nervous system development. However, the role of Eph/ephrin signaling in regulating ENCC migration remains unknown. Mutation of Ephs and ephrins have been reported in Hirschsprung disease (HSCR) patients, suggesting the involvement of Eph/ephrin signaling. I examined the role of ephrin-A1/EphA4 signaling using 293T cells to investigate their interaction and regulation of cell segregation. Immunoprecipitation shows that ephrin-A1 can interact with Ret and p75. Ephrin-A1 mutant cloned to mimic human mutation reported is also able to interact with Ret and p75, and perform ligand-receptor binding interaction with EphA4. Cell segregation assay using stably-transfected cells shows that ephrin-A1 mutant-expressing cells demonstrate abnormal cell segregation when co-culturing with EphA4-expressing cells. These findings suggest that ephrin-A1 mutant could disrupt cell segregation regulated by ephrin-A1/EphA4 signaling, while interaction of ephrin-A1 with Ret and p75 suggests that it potentially plays an important role in this cell-cell interaction to regulate ENCC migration.

Title: A knock-in strategy to study protein localization in human induced pluripotent stem cell (hiPSC)-derived neuron through genome editing
Speaker: Mr. LYU Quanwei (MPhil candidate)

The emergence of human induced pluripotent stem cells (hiPSCs) and their ability to differentiate into multiple subtypes of neuron present the attractive potential as a cellular model to study human brain disorders. Furthermore, the graft of hiPSC-derived neurons into the mouse brains potentially allows exploration of how their synaptic connections form in vivo. Many disorders on the central nervous system involve mis-localization of proteins in neurons. Here I use CRISPR-Cas9 to create a knock-in epitope tag to study protein localization in hiPSC-derived neurons. We have developed the differentiation protocol to generate cortical neurons that express synaptic markers and produce miniature excitatory postsynaptic current. As a proof-of-principle investigation, we test the possibility of labeling endogenous β-actin by adding a hemagglutinin (HA) tag to the N-terminal of b-actin through genome editing. After differentiation of the genome-edited hiPSCs into cortical neurons, the enrichment of b-actin in the growth cones and dendritic spines were visualized by immunostaining through anti-HA antibody. Our findings suggest the feasibility of this knock-in strategy to label synaptic proteins for the study of protein localization during synaptogenesis in hiPSC-derived cortical neurons. I will also discuss the use of this approach to create and screen for mutant iPSCs for the study of amyotrophic lateral sclerosis (ALS).