Author response: Notch controls the cell cycle to define leader versus follower identities during collective cell migration Article Swipe

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, such 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. In conclusion, TNC migratory identities are defined through the interaction of Notch signalling and cell cycle progression. Editor's evaluation Using a combination of in vivo and in silico approaches, the authors have demonstrated how cell-fate decisions are orchestrated at the level of leader vs. follower cells in collective cell migration of trunk neural crest cells. They highlight the role of Notch signaling and cell cycle progression, showing how these traits differ between the leader and follower cells. The findings are of wide interest, as collective cell migration is a fundamental process critical for embryonic development as well as invasion of various cancers. https://doi.org/10.7554/eLife.73550.sa0 Decision letter eLife's review process Introduction The harmonious coupling of cell proliferation with migration is fundamental for the normal growth and homeostasis of multicellular organisms. A prominent consequence of the dysregulation of these processes is cancer. Uncontrolled cell proliferation leads to primary tumours, and the acquisition of migratory capacities leads to the formation of secondary tumours, the most common cause of cancer deaths. Metastatic cells can migrate collectively, which endows them with more aggressive behaviours (Nagai et al., 2020). Collective cell migration refers to the movement of a group of cells that maintain contact and read guidance cues cooperatively (Rorth, 2009). This mechanism has been studied in several contexts, such as wound healing, angiogenesis, and neural crest (NC) migration. However, how cell proliferation impacts collective cell migration, and vice versa, remains largely unknown. The molecular signals that may couple these two fundamental processes remain equally unclear. The NC is a mesenchymal cell population that arises early in development and migrates throughout the body, giving rise to a variety of cell types (neurons, glia, pigment cells, etc.). The NC's stereotypical migratory behaviour (Gammill and Roffers-Agarwal, 2010) and similarity to metastatic cells (Maguire et al., 2015) make this cell type an ideal model to study the mechanisms of collective cell migration in vivo. Our previous work has shown that zebrafish trunk neural crest (TNC) migrate collectively forming single-file chains (Richardson et al., 2016). One cell at the front of the chain, the leader, is the only cell capable of instructing directionality to the group, while follower cells trail the leader. This division of roles into leaders and followers has been observed in other collectively migrating systems (Theveneau and Linker, 2017). Moreover, histopathological studies from cancer samples and cell lines show clear morphological and molecular differences between the invasive front, leaders, and the lagging cells, followers (Pandya et al., 2017). One outstanding question from these studies is what are the signals that determine leader versus follower migratory identities? Notch signalling is a cell-cell communication pathway that directly translates receptor activation at the membrane into gene expression changes. Notch receptors are activated by membrane-bound ligands of the Delta/Serrate/Lag2 family. Upon ligand binding, Notch receptors are cleaved by γ-secretases releasing their intracellular domain (NICD). Subsequently, NICD translocates to the nucleus, binds the CBF1/Su(H)/Lag-1 complex, and initiates transcription (Bray, 2016). Among the direct Notch targets are members of the Hes gene family, which encode transcriptional repressors able to antagonise the expression of specific cell fate determinants and Notch ligands, generating a negative feedback loop in which cells with high Notch receptor activity downregulate the expression of Notch ligands, and cannot activate the pathway in their neighbours. Hence, adjacent cells interacting through the Notch pathway typically end up with either low or high levels of Notch activity and adopt distinct fates, a mechanism known as lateral inhibition (Lewis, 1998). Interestingly, Notch signalling has also been implicated in cell migration (Giniger, 1998; Leslie et al., 2007; Timmerman et al., 2004) and promotes invasive behaviours during cancer progression (Reichrath and Reichrath, 2012). Furthermore, lateral inhibition is implicated in the allocation of migratory identities during angiogenesis (Phng and Gerhardt, 2009), trachea formation in Drosophila (Caussinus et al., 2008), and in cell culture (Riahi et al., 2015). Whether Notch signalling plays a similar role in the context of mesenchymal cell migration is unknown. Notch signalling is required for NC induction (Cornell and Eisen, 2005), and its components and activity remain present in migrating NC (Liu et al., 2015; Rios et al., 2011). Nevertheless, the role of Notch during NC migration remains unclear. Cardiac NC are reported to develop normally under lack of Notch signalling (High et al., 2007). However, using different genetic tools, it has been shown that both gain and loss of Notch function led to the lack of NC derivatives (Mead and Yutzey, 2012). Moreover, in Xenopus the loss of Notch effectors leads to aberrant NC migration (Vega‐López et al., 2015). The Notch pathway has not only been implicated in cell fate allocation, but it is also important for cell proliferation. Depending on the context, Notch can inhibit or promote cell cycle progression (Campos et al., 2002; Carlson et al., 2008; Devgan et al., 2005; Fang et al., 2017; Georgia et al., 2006; Mammucari et al., 2005; Nguyen et al., 2006; Nicoli et al., 2012; Noseda et al., 2004; Ohnuma et al., 1999; Park et al., 2005; Patel et al., 2016; Rangarajan et al., 2001; Riccio et al., 2008; Zalc et al., 2014). Indeed, Notch target genes include important cell cycle regulators such as CyclinD1, p21 and MYC (Campa et al., 2008; Guo et al., 2009; Joshi et al., 2009; Palomero et al., 2006; Ronchini and Capobianco, 2001). Using a combination of in vivo and in silico approaches, we have established that differences in Notch activity between premigratory TNC select the leader cell. Cells with high levels of Notch signalling adopt a leader identity, while cells that lack Notch activity become followers. Our data show that a single progenitor cell in the premigratory area divides asymmetrically, giving rise to a large prospective leader and smaller follower cell. We propose that this original small asymmetry generates differences in Notch activity between TNC that are thereafter enhanced by cell-cell communication through Notch lateral inhibition. Differences in Notch activity in turn drive distinct cell cycle progression patterns and regulate the expression of phox2bb. Leader cells undergo the G1/S transition faster and remain in S-phase for longer than follower cells. Moreover, continuous progression through the cell cycle is required for TNC migration. Taken together, our results support a model in which the interaction between Notch and the cell cycle defines leader and follower migratory behaviours. Results Notch signalling is required for TNC migration NC cells are induced at the border of the neural plate early during development. The prospective NC expresses Notch components, and Notch activity is required for NC induction (Cornell and Eisen, 2005). Our analysis reveals that Notch components remain expressed in NC after induction, suggesting that Notch signalling may also be involved in later aspects of NC development (Figure 1—figure supplement 1). Moreover, analysis of the Notch activity reporter line 12xNRE:egpf (Moro et al., 2013) shows that Notch signalling levels vary widely between premigratory TNC (Figure 1), suggesting that Notch may play a role after TNC induction. To explore the role of Notch in TNC development, we first aimed to define the stage at which NC induction becomes independent of Notch signalling. To this end, we treated embryos with the γ-secretase inhibitor DAPT (Richter et al., 2017) and assessed expression of NC marker. Our results showed that Notch inhibition impairs TNC induction up to 11 hours post-fertilization (hpf; Figure 2) and confirmed previous reports that induction of the cranial and vagal NC populations is independent of Notch signalling (Cornell and Eisen, 2000). Next, we analysed the effect of Notch inhibition at 12 hpf on the development of TNC derivatives. We found a reduction in all TNC derivatives (neurons, glia, and pigment cells; Figure 3A–F) upon Notch inhibition, suggesting that Notch activity is important in a process subsequent to induction, yet prior to differentiation. We next explored whether TNC migration is affected by Notch inhibition. Analysis of crestin expression showed a reduction in the number of TNC cell chains formed and in their ventral advance upon DAPT treatment (Figure 3G–J), which likely explains the lack of TNC derivatives at later stages. We then asked whether these results are due to a delay or a halt of migration. To this end, embryos were treated with DAPT from 12 hpf for 6–12 hr and processed for crestin expression. Decreased numbers of migratory chains were observed at all timepoints, but as embryos developed new chains were formed, indicating that the blockade of Notch signalling delays TNC migration (Figure 3K). Comparable results were obtained by inhibiting Notch genetically in embryos where the dominant-negative form of Suppressor of Hairless is under the control of a heat shock element (Latimer et al., 2005; hs:dnSu(H); Figure 3L). We reasoned that if Notch inhibition delays the onset of TNC migration, its overactivation might lead to TNC migrating earlier, leading to an increased number of chains. To test this, we induced NICD expression in all tissues by heat shock of hs:Gal4;UAS:NICD embryos (Scheer and Campos-Ortega, 1999). To our surprise, Notch gain of function (GOF) and loss of function (LOF) resulted in almost identical phenotypes, both showing a similar reduction of TNC chain numbers (Figure 3L). Taken together, these results show that precise regulation of Notch signalling levels is required for TNC migration. Figure 1 with 1 supplement see all Download asset Open asset Trunk neural crest (TNC) present different levels of Notch activity. (A, E) Images of two different Notch reporter 12xNRE:egfp embryos (18 hpf) stained for sox10 (magenta) and GFP (green) RNAs, and nuclei stained with DAPI (blue). (B) Enlargement of the anterior area in (A). (C) Enlargement of the more posterior area in (A). (D) Enlargement of the anterior most posterior area in (A). (F) Enlargement of the outlined area in (E). Anterior to the left, dorsal top. White lines show approximate cell boundaries. Figure 2 Download asset Open asset Trunk neural crest (TNC) induction is independent of Notch signalling after 12 hpf. (A) crestin in situ hybridisation in wildtype (WT) embryo at 18 hpf. (B, C) crestin in situ hybridisation in DAPT-treated embryos: (B) reduced or (C) absent TNC. (D) Quantification of the crestin expression phenotypes upon DAPT treatment (phenotypes: WT, black; reduced, orange; absent, red; 30% epiboly n = 38, 75% epiboly n = 32, 11 hpf n = 35, 12 hpf n = 39). (E–J) In situ hybridisation for neural crest (NC) markers in representative control (DMSO) and DAPT-treated embryos from 12 to 16 hpf. (E, F) crestin (DMSO n = 32, DAPT n = 38), (G, H) foxd3 (DMSO n = 16, DAPT n = 35), and (I, J) sox10 (DMSO n = 27, DAPT n = 29). Anterior to the left, dorsal top. Figure 3 Download asset Open asset Notch signalling is required for trunk neural crest (TNC) migration and derivatives formation. (A, B) Glial marker mbp in situ hybridisation upon (A) control (DMSO; n = 15) and (B) DAPT (n = 20) treatment from 12 hpf. (C, D) Neuronal marker bdh in situ hybridisation upon (C) control (DMSO; n = 25) and (D) DAPT (n = 18) treatment from 12 hpf. (E, F) Pigmentation upon (E) control (DMSO; n = 40) and (F) DAPT (n = 52) treatment from 12 hpf. (G, H) Neural crest marker crestin in situ hybridisation upon (G) control (DMSO) and (H) DAPT treatment from 12 to 18 hpf. (I, J) crestin in situ hybridisation upon (I) control (DMSO) and (J) DAPT treatment from 12 to 24 hpf. (K) Quantification of migratory chain formation upon control (DMSO) and DAPT treatment from 12 to 18 hpf (DMSO n = 98; DAPT n = 126), 20 hpf (DMSO n = 111; DAPT n = 109), and 24 hpf (DMSO n = 42; DAPT n = 61). (L) Quantification of migratory chain formation in control (HS:Gal4; n = 516), Notch loss of function (LOF) (HS:dnSu(H); n = 220), and gain of function (GOF) conditions (HS:Gal4xUAS:NICD; n = 142) heat shocked at 11 hpf and analysed at 18 hpf. Mann–Whitney U-test, control vs. LOF ****p<0.0001, control vs. GOF **p=0.0020. Anterior to the left, dorsal top, except in (C, D) anterior left, ventral view. Arrowheads indicate gene expression. All treatments performed from 12 hpf. In vivo Notch activity allocates TNC migratory identity Interestingly, Notch signalling is required during collective migration to define distinct identities (Phng and Gerhardt, 2009; Caussinus et al., 2008; Riahi et al., 2015). To test whether Notch plays a similar role in TNC migration, we performed live-imaging analysis of TNC migration under lack (inhibition and LOF) or overactivation (GOF) of Notch signalling (Figure 4, Figure 4—videos 1 and 2). Our previous work defined a leader as the cell that retains the front position of the chain throughout migration, advancing faster and in a more directional manner than followers (Richardson et al., 2016). Under Notch inhibition (treatment with γ-secretase inhibitor Compound E; Richter et al., 2017), TNC remain motile with a single cell initiating the movement of the chain, but in contrast to control treatment (DMSO) the leader cell is unable to retain the front position and is overtaken by one or several followers (Figure 4A and C, Figure 5A and B, Figure 4—video 1). The overtaking follower cell, in turn, is not always able to retain the front position and can be overtaken by cells further behind in the chain. This loss of group coherence corresponds with a reduction in ventral advance, with most leader cells unable to move beyond the neural tube/notochord boundary (NT/not; Figure 4C, Figure 5A and C). This behaviour leads to an accumulation of cells at the NT/not, where some cells repolarise moving anterior or posteriorly and crossing the somite boundary and, in some cases, joining adjacent chains. Analysis of single-cell tracking showed that under Notch inhibition leader cells also have decreased speed and directionality (Figure 5D and E). Similar results were observed when Notch inhibition was achieved genetically by driving overexpression of dnSu(H) through heat shock in the entire embryo (not shown; hs:dnSu(H) line). Together, these results strongly suggest that upon lack of Notch signalling the TNC population is formed solely by follower cells that are unable to coordinate the movement of the group. Nevertheless, Notch signalling is important for the development of tissues surrounding TNC that act as a substrate for migration, raising the possibility that Notch signalling does not act cell-autonomously in TNC and instead the phenotypes observed are simply the consequence of somite and/or neural tube malformations. However, this appears unlikely as somite development (formation, patterning, and differentiation) and neuron formation are not affected by Notch inhibition at the axial level analysed (Figure 4—figure supplement 1). Next, we directly tested whether Notch signalling is autonomously required in TNC by inhibiting Notch activity exclusively in NC at the time of migration. To this end, we generated a new UAS:dnSu(H) line and crossed it with Sox10:Kalt4 fish (Alhashem et al., 2021). In the resultant embryos, all NC express Gal4 fused to the oestrogen receptor binding region (Gal4-ER) and are fluorescently labelled by nuclear-RFP. Under normal conditions, Gal4-ER is maintained inactive in the cytoplasm, whilst upon addition of tamoxifen, Gal4-ER is translocated to the nucleus activating transcription from the UAS:dnSu(H) transgene (Figure 4—figure supplement 2). We found that autonomous inhibition of Notch signalling in NC phenocopies the chemical inhibition. Leader cells are unable to retain the front position, being overtaken by followers, and ventral advance is reduced with cells accumulating at the NT/not boundary (Figure 4D, Figure 5A–C, Figure 4—video 2). Moreover, leader cells adopt followers' migratory parameters, showing decreased speed and directionality (Figure 5D and E), confirming that Notch activity is autonomously required in TNC for identity allocation, and suggest that in the absence of Notch signalling a homogenous group of followers is established. In view of these results, we hypothesised that a homogeneous group of leaders would be formed upon Notch overactivation. Using a similar strategy, Notch overactivation was induced in the whole embryo (not shown, hs:Gal4;UAS:NICD; Scheer and Campos-Ortega, 1999), or exclusively in NC (Sox10:Kalt4;UAS:NICD), and migration was analysed by live imaging. Similar results were obtained in both experimental conditions: group coherence is lost, leader cells are overtaken by followers, and ventral advance is impaired (Figure 4F, Figure 5A–C, Figure 4—video 2). Interestingly, in Notch GOF conditions follower cells adopt leaders' characteristics, moving with increased speed, but all cells in the chain follow less directional trajectories, which hinders the ventral advance of the group (Figure 5D and E), indicating that all cells in the chain migrate as leaders. Next, we tested whether the behavioural changes observed upon Notch alterations were mirrored by molecular changes by using the leader marker phox2bb. In control conditions, phox2bb transcripts are highly enriched in the leader cells from early stages of migration (Figure 6A, B, and G; Alhashem et al., 2022a). Consistent with expectations, upon Notch overactivation phox2bb is expressed by all the cells in the chain (Figure 6C, D, and G), while its expression is absent when Notch is inhibited (Figure 6E–G). These data show that Notch activity controls phox2bb expression and allocates TNC migratory identity. Figure 4 with 5 supplements see all Download asset Open asset Notch activity allocates trunk neural crest (TNC) migratory identity. (A) Selected frames from in vivo imaging of Sox10:Kalt4 control (DMSO treated) embryos. (B) Selected frames from control simulation with 1:3 leader/follower ratio. (C) Selected frames from in vivo imaging under Notch-inhibited condition, Sox10:Kalt4 embryos treated with CompE. (D) Selected frames from in vivo imaging of Notch loss of function (LOF) condition, Sox10:Kalt4; UAS:dnSu(H) embryos. (E) Selected frames from all followers simulation. (F) Selected frames from in vivo imaging of Notch gain of function (GOF) condition Sox10:Kalt4; UAS:NICD embryos. (G) Selected frames from all leaders simulation. Magenta tracks and green arrowheads indicate leaders; green arrows and cyan tracks follower cells. Asterisks indicate cells crossing somite borders. White line marks dorsal midline. Anterior to the left, dorsal up. Time in Figure 5 Download asset Open asset Trunk neural crest (TNC) migration in vivo and in (A) position of cell in model and in vivo under different In silico results in in vivo data in model and dorsal of the premigratory area and NT/not boundary Anterior left, dorsal up. (B) Quantification of leader overtaking in vivo and in Leader overtaken by a single follower is overtaken = leader overtaken by more than one follower cell is overtaken (C) Quantification of the ventral advance of cells in vivo and in (D) Quantification of cell speed in vivo and in (E) Quantification of cell directionality in vivo and in Leader cells in followers in Magenta and cyan lines indicate the for leaders and followers analysis in Figure Download asset Open asset Notch signalling controls phox2bb expression leader cells. (A, B) Images of phox2bb expression in control embryos (C, D) Images of phox2bb expression under Notch gain of function (GOF) conditions UAS:NICD (E, F) Images of phox2bb expression in Notch inhibition conditions E). Magenta and cyan arrowheads indicate leaders and followers (G) Quantification of phox2bb expression in control (n = Notch GOF (n = and Notch inhibition conditions (n = control vs. GOF ****p<0.0001, control vs. inhibition In our in vivo and molecular data show that Notch signalling is required autonomously in TNC for migratory identity TNC with high levels of Notch express phox2bb and become leaders, while cells with low Notch activity migrate as followers. of Notch signalling lead to a homogeneous TNC group with a single migratory identity that is unable to undergo collective migration. Taken together, these data suggest Notch lateral inhibition as the mechanism for TNC migratory identity In silico modelling that more than one leader is required for TNC migration Our in vivo analysis shows that upon both Notch inhibition and overactivation TNC are unable to undergo collective migration due to lack of group the other our molecular analysis shows that upon Notch inhibition an group is while Notch overactivation leads to the formation of an group. To gain a of these results, we an in silico a element model of TNC migration. Cells were as moving into a and with control cell movement in the contact inhibition of and define movement directionality and group while cell as cell while a element was to the (Figure A on in vivo was developed to how with different mechanisms chain behaviours. The were chain a of is between adjacent cells; single migration for at of the followers undergo while leaders retain the front position, and the chain advance to the end of the migratory (Figure Using this analysis and a modelling we to in vivo TNC migration with the form of the only in an to the of mechanisms We first chains of homogeneous cells and all We found combination able to all confirming our previous findings that cell is required for TNC migration (Figure et al., 2016). from other systems et al., et al., and and 2013) led to that differences in the response between cells may be at we chains in which only cells of different identities present Figure These several but chains are unable to the end of the migratory (Figure Figure 4—video Next, we and cell for leader cells. Interestingly, the model is only able to control conditions when the between leaders and follower is for all Nevertheless, it is unable to Notch GOF and LOF phenotypes (Figure Our previous results show that differences in Notch signalling migratory suggesting that lateral inhibition may be the mechanism at To explore whether different of lateral inhibition may the model to Notch conditions and different of leader/follower cells were We first tested a this chain moving beyond the end of the pathway (Figure Figure 4—video Interestingly, we found that several from the and 1:3 leader/follower were able to in vivo control condition, as well as the loss of group coherence and ventral advance observed in Notch GOF leader and LOF follower Figure and Figure Figure 4—video In these the that all in vivo followers at the low while leaders' or high cell or low and all Nevertheless, all these the leader with enhanced migratory Figure Download asset Open asset In silico modelling of trunk neural crest (TNC) migration. 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Leader cells from the division of a progenitor cell is a prominent leader from follower cells. are almost as as followers during migration and this is migration (Richardson et al., suggesting that arises at or Interestingly, cell as an important in our in silico to more leader/follower behaviours. To the of these we whether leader and follower cells a common and at which differences in become To this end, we embryos. The reporter and