Assessing the single cell and tissue response to altering Her6 dynamics by protein destabilisation in the zebrafish telencephalon

Abstract

HES1 is an oscillatory transcriptional inhibitor whose expression dynamics are critical for its role in maintaining neural progenitors in mammalian Central nervous system (CNS) development. Here, I have taken advantage of the optical and developmental properties of the zebrafish telencephalon to study and functionally assess the dynamic expression of Her6 , a zebrafish orthologue of HES1, by combining single cell and tissue level analysis which had never been done for HES1. her6 expression did not overlap with young elavl3-expressing neurons or early fate determination markers ngn1 and ascl1, supporting its function as an early progenitor regulator. Following detailed characterisation, I used the Her6-Venus (HV) endogenous knock-in (KI) line (Soto et al., 2020) to study Her6 protein dynamics between 20-30hpf. Her6 expression was heterogenous in tissue snapshots, explained by its short and long term temporal fluctuations in single cells. In 5-37% of cells, the short term fluctuations were oscillatory with 1.9 hour period and 1.4 fold-change. The long term trends in single cells were predominantly declining, consistent with Her6 down-regulation over time. To understand the role of these dynamics, I compared Her6 behaviour in HV with Her6-Venus-PEST (HVP) KI line where Her6 was destabilised and expected to have altered dynamics. The levels of Her6 expression were lower in most HVP cells but unexpectedly, also more heterogenous, as some cells retained the ability to express normal Her6 levels. Also, the differences in Her6 expression levels between neighbouring cells were higher in HVP. Mathematical modelling of these observations showed that HVP traits could only be replicated in presence of cell coupling and Notch-like lateral inhibition. I propose that Her6 pattern in HVP can be explained by indirect strengthening of lateral inhibition as a result of weakened Her6 auto-inhibition due to its low levels. This highlights the importance of combined cell and tissue analysis in interpreting oscillator behaviour. In HVP, Her6 oscillated in a larger proportion of cells (32.5-67.9%) with higher fold-change (1.9) but unaltered periodicity (~2.1 hours). But it was also expressed in fewer cells and downregulated slightly more rapidly. Even though the total number of proliferative cells in telencephalon was not affected, there was a presumptive shift in the mode of cell divisions in Her6+ cells from self-renewal towards asymmetric. This was consistent with prolonged or increased expression of differentiation genes in HVP. As a whole, these findings provide further support for the involvement of HES/Her oscillations in the transition between progenitor to neuronal commitment in the CNS.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Sharrocks (Supervisor) & Nancy Papalopulu (Supervisor)