Notch controls the cell cycle to define leader versus follower identities during collective cell migration

Abstract

Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, as cancer. How cell cycle progression affects migration, and vice-versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish Trunk Neural Crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. We propose that migratory behaviours are defined through the interaction of Notch signalling and cell cycle progression.

Trunk neural crest migratory position and asymmetric division predict terminal differentiation

“…The asymmetric division of leader cells may unequally distribute fate determination receptors and/or activity, such as Notch (see above), hence defining a distinct differentiation bias for each of the sibling cells. Furthermore phox2bb has been shown to regulate Notch receptor expression (Revet et al, 2008), and our data show that Notch signalling in turn controls phox2bb transcription in TNC (Alhashem et al, 2021a). Hence, it is possible that Notch and phox2bb establish a positive forward loop that reinforces each other's expression only in the leader cell.…”

“…Moreover, Notch activity controls the expression of signalling components that orchestrate the mitotic spindle polarity and orientation, α-adaptin and Numb (Charnley et al, 2020). Hence, it is possible that the asymmetric distribution of Notch signalling controls identity allocation during the leader’s division and maintains a single leader cell per chain, akin to its role in identity allocation before migration initiation (Alhashem et al, 2021a). The asymmetric division of the leader cells is also reminiscent of tip cell divisions during blood vessel formation (Costa et al, 2016).…”

“…Leader cells are larger, more elongated and polarized than followers. Leaders and followers present a similar total cell cycle span, but leaders present a long S-phase while followers spend most of the cycle in G 1 (Alhashem et al, 2021a). Together these features distinguish leaders from followers, and allow the collective migration of the group, but whether these traits impact terminal TNC differentiation is unknown.…”

Distinct pathways drive anterior hypoblast specification in the implanting human embryo

“…Following thaw, blastocysts were briefly treated with acidic Tyrode's solution (T1788, Sigma) to remove the zona pellucida and placed in pre-equilibrated human IVC1 in eight-well µ-slide tissue culture plates (80826, Ibidi) in approximately 400 µl volume per embryo per well. Half medium changes were done every 24 h. For small-molecule experiments, human IVC1 was supplemented with either 2 µM A83-01 (72022, STEMCELL Technologies) 80,81 , 25 ng ml −1 Activin-A (Qk001, QKINE) [82][83][84] , 200 nM LDN (S2618, SelleckChem) 85,86 , 50 ng ml −1 BMP6 (SRP3017, Sigma Aldrich) 85,86 , 20 µM DAPT (72082, STEMCELL Technologies) [87][88][89][90] , 10 µM Compound-E (ab142164, Abcam) [91][92][93] , 20 µM MK-0752 (S2660, Selleck Chemicals) [94][95][96] or dimethyl sulfoxide (DMSO) for 48 h. In all cases, these concentrations fall within a range of those used for either vertebrate embryos or complex human ES cell-derived models of the embryo. Within these ranges, a low-to-intermediate concentration was selected to avoid non-specific cytotoxic effects while still considering the higher concentration needed for embryo permeation compared with minimal 2D cell culture to achieve inhibitor action.…”