The lateral line system is an essential sensory modality used by fishes and aquatic amphibians to sense hydrodynamic information. The system comprises distributed sense organs called neuromasts and their afferent nerves, organized into anterior lateral lines around the eye and jaw and posterior lateral lines (LL) on the trunk. At postembryonic stages, early forming neuromasts expand in size and sink into bony canals, while late-forming superficial neuromasts are added as the fish grows. Unlike the well-studied zebrafish posterior LL, details of anterior LL postembryonic development remain unknown. Here, we have characterized developmental mechanisms and innervation patterns driving expansion of the zebrafish anterior LL. Using tissue-clearing to observe neuromast and nerve markers through ontogeny, we demonstrate continuous neuromast addition in the anterior LL, with peak rates at larval stages of 7-10 mm standard length (SL). Lines of superficial neuromasts form parallel to existing lines of presumptive canal neuromasts as late as 7 mm SL, with new neuromasts added through migration of new primordia, budding, intercalation, and a novel "hybrid-origin" mechanism. Despite some canal lines being innervated by the anterodorsal ganglion, all superficial lines are innervated by the anteroventral ganglion. Anterior LL ganglion ablation reveals that denervation abrogates superficial neuromast formation-including via the hybrid-origin mechanism-and reduces growth of canal neuromasts. While the anterior and posterior LL use disparate developmental mechanisms, innervation is critical to the expansion of both. Our findings reveal a "developmental switch" at 7 mm SL, when innervation becomes necessary for a secondary phase of anterior LL development.

The origin of paired fins is an unresolved controversy in vertebrate evolutionary biology. Karl Gegenbaur famously proposed that paired fins evolved by the transformation of a gill arch, but this hypothesis remains largely unsupported by the fossil record. Also, seemingly at odds with gill arch-paired fin serial homology are their distinct embryonic origins from neural crest and lateral plate mesoderm, respectively. We have previously shown that skate gill arch skeletal elements may derive solely from neural crest, or from a mixture of neural crest and mesoderm, and we have shown that skate gill arches and jawed vertebrate fins/limbs share several molecular patterning mechanisms. Here, we show in the little skate (Leucoraja erinacea) that neural crest and lateral plate mesoderm-derived mesenchyme are developmentally equivalent and interchangeable in the pharyngeal arches and fin buds. Using heterotopic transplantation experiments, we find that neural crest cells can contribute to the typically mesoderm-derived fin skeleton, and that lateral plate mesodermal mesenchyme can contribute to the typically neural crest-derived jaw skeleton. These findings point to shared skeletogenic competence of neural crest and mesoderm-derived mesenchyme at the head-trunk interface. We argue that this shared competence accounts for the anatomical and developmental parallels of the gill arch and paired fin/limb skeletons and forms the basis of their serial homology.

BACKGROUND:Fate maps relate progenitor cell positions to later fates and locations of their progeny, revealing early embryonic organization. Previous zebrafish fate maps identified the origin of germ layers and derivative cell fates, but ventral gastrula progenitor domains were not fully resolved. In particular, the neural crest, a multipotent cell type central to vertebrate development and evolution, was not characterized in the zebrafish fate map. RESULTS:We generated a region-based fate map to resolve the origin of the neural crest and overall organization of the ventral gastrula. This fate map demonstrates that cranial neural crest and placodes arise from a broadly overlapping progenitor domain. Notably, trunk neural crest and neural tube always arise together, while cranial neural crest and neural tube can arise from segregated progenitor pools. A separate, far-ventral progenitor domain produces somites, spinal cord, neural crest, and epidermis in the posterior tail. CONCLUSIONS:We situate the neural crest in the fate map and clarify the early embryonic organization of the zebrafish ventral gastrula. Our fate map establishes multiple mixed-fate domains in the ventral gastrula and their contributions to the overall body plan. This fate map provides information for further investigation of early vertebrate development and neural crest biology.

The mechanosensory lateral line system of aquatic vertebrates comprises a superficial network of distributed sensory organs, the neuromasts, which are arranged over the head and trunk and innervated by lateral line nerves to allow detection of changes in water flow and pressure. While the well-studied zebrafish posterior lateral line has emerged as a powerful model to study collective cell migration, far less is known about development of the anterior lateral line, which produces the supraorbital and infraorbital lines around the eye, as well as mandibular and opercular lines over the jaw and cheek. Here we show that normal development of the zebrafish anterior lateral line system from cranial placodes is dependent on another vertebrate-specific cell type, the cranial neural crest. We find that cranial neural crest and anterior lateral lines develop in close proximity, with absence of neural crest cells leading to major disruptions in the overlying anterior lateral line system. Specifically, in the absence of neural crest neither supraorbital nor infraorbital lateral lines fully extend, such that the most anterior cranial regions remain devoid of neuromasts, while supernumerary ectopic neuromasts form in the posterior supraorbital region. Both neural crest and cranial placodes contribute neurons to the lateral line ganglia that innervate the neuromasts and in the absence of neural crest these ganglia, as well as the lateral line afferent nerves, are disrupted. Finally, we establish that as ontogeny proceeds, the most anterior supraorbital neuromasts come to lie within neural crest-derived frontal and nasal bones in the developing cranium. These are the same anterior supraorbital neuromasts that are absent or mislocated in specimens lacking neural crest cells. Together, our results establish that cranial neural crest and cranial placode derivatives function in concert over the course of ontogeny to build the complex cranial lateral line system.

Lateral lines are placodally derived mechanosensory systems on the heads and trunks of many aquatic vertebrates. There is evidence of lateral lines in the earliest known vertebrate fossils, and they exist in organisms with widely different craniofacial morphologies - including the presence or absence of jaws, external or internal nostrils, and variable positions of the cranial cartilages with respect to eyes and braincase. Consequently, the lateral lines make an ideal study system to understand how morphological variation in a deeply conserved sensory system responds to overall evolution of the head. However, palaeontological and developmental data have not been integrated to elucidate the history of this system in the context of evolving vertebrate crania. The emergence of new imaging techniques and molecular methods to study ontogeny in non-model systems provides unique opportunities for such a study. This review examines open questions in light of new fossil discoveries that have altered our understanding of vertebrate evolution as well as new insights on the development of non-model taxa. We find that the diversity of lateral lines is not the result of simplification from a complex ancestral condition as previously supposed. Rather, the anterior lateral line systems of living gnathostomes result from an evolutionary episode of reduction and reassembly, both preceding and overlapping the origin of jawed vertebrates. This event is coupled to a marked postorbital to orbital-preorbital shift in the territorial elaboration of the lateral line systems, and we argue that this spatial move likely signals functional change, coinciding with a major enhancement of the gnathostome vestibular system.

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 minutes. Figure 5 Download asset Open asset Trunk neural crest (TNC) migration measurements in vivo and in silico. (A) Final position of each cell in model simulations and in vivo experiments under different conditions. In silico results depicted in confined pathway, in vivo data graphed in model embryo, somites contour and dorsal midline (dark grey lines), edge of the premigratory area (dashed lines), and NT/not boundary (light grey lines). Anterior left, dorsal up. (B) Quantification of leader overtaking events in vivo and in silico. Leader overtaken by a single follower is overtaken = 1; leader overtaken by more than one follower cell is overtaken >1. (C) Quantification of the ventral advance of cells in vivo and in silico. (D) Quantification of cell speed in vivo and in silico. (E) Quantification of cell directionality in vivo and in silico. Leader cells in magenta, followers in cyan. Magenta and cyan dashed lines indicate the average values for leaders and followers respectively. Full statistical analysis in Supplementary file 1. Figure 6 Download asset Open asset Notch signalling controls phox2bb expression defining leader cells. (A, B) Images of phox2bb expression in control embryos (Sox10:Kalt4). (C, D) Images of phox2bb expression under Notch gain of function (GOF) conditions (Sox10:Kalt4; UAS:NICD embryos). (E, F) Images of phox2bb expression in Notch inhibition conditions (Compound E). Magenta and cyan arrowheads indicate leaders and followers respectively. (G) Quantification of phox2bb expression in control (n = 13), Notch GOF (n = 14), and Notch inhibition conditions (n = 11). Welch's t-test, Kalt4 control vs. GOF ****p<0.0001, DMSO control vs. inhibition ****p<0.0001. In summary, our in vivo and molecular data show that Notch signalling is required autonomously in TNC for migratory identity allocation. TNC with high levels of Notch express phox2bb and become leaders, while cells with low Notch activity migrate as followers. Alterations 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 responsible for TNC migratory identity acquisition. In silico modelling predicts 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 coherence. On the other hand, our molecular analysis shows that upon Notch inhibition an all-followers group is established, while Notch overactivation leads to the formation of an all-leaders group. To gain a better understanding of these paradoxical results, we took an in silico approach, developing a discrete element model of TNC migration. Cells were simulated as 2D particles moving into a constrained space and endowed with intrinsic motility. Four variables control cell movement in the model: contact inhibition of locomotion (CIL) and co-attraction (co-A) define movement directionality and group cohesion, while volume exclusion regulates cell overlap, intuitively understood as cell size, while a noise element (zeta) was added to the cell's trajectory (Figure 7A). A multi-objective scoring system, based on in vivo measurements, was developed to evaluate how close simulations with different underlying mechanisms matched chain behaviours. The scores were (1) chain cohesion, a maximum distance of 57 μm is allowed between adjacent cells; (2) single file migration for at least 80% of the simulation; (3) followers undergo rearrangements, while (4) leaders retain the front position, and (5) the chain should advance to the end of the migratory path (Figure 7B). Using this analysis and a parsimonious modelling approach, we attempted to match in vivo TNC migration with the simplest form of the model, only adding complexity incrementally in an effort to find the minimal set of predicted mechanisms required. We first simulated chains composed of homogeneous cells and systematically covaried all parameters. We found no parameter combination able to match all scores, confirming our previous findings that cell heterogeneity is required for TNC migration (Figure 7C; Richardson et al., 2016). Evidence from other systems (Astin et al., 2010; Bentley et al., 2014; Parkinson and Edwards, 1978; Theveneau and Mayor, 2013) led us to hypothesise that differences in the CIL response between cells may be at play. Thus, we simulated chains in which only cells of different identities present CIL (Diff CIL; Figure 7A). These simulations match several scores, but chains are unable to reach the end of the migratory path (Figure 7C, Figure 4—video 3). Next, we varied Diff CIL intensity, co-A, and cell size (volume exclusion) for leader cells. Interestingly, the model is only able to recapitulate control conditions when the difference between leaders and follower is maximal for all variables. Nevertheless, it is unable to recapitulate Notch GOF and LOF phenotypes (Figure 7C). Our previous results show that differences in Notch signalling establish migratory identities, suggesting that lateral inhibition may be the mechanism at play. To explore whether different outcomes of lateral inhibition may allow the model to simulate Notch altered conditions (GOF and LOF), different ratios of leader/follower cells were simulated. We first tested a 1:1 ratio, surprisingly this chain architecture over-migrates, moving beyond the end of the pathway (Figure 7C, Figure 4—video 3). Interestingly, we found that several parameter combinations from the 1:2 and 1:3 leader/follower ratios were able to recapitulate in vivo control condition, as well as the loss of group coherence and ventral advance observed in Notch GOF (all leader simulation) and LOF (all follower simulation; Figure 4B, E, and G, Figure 5, Figure 4—video 3). In these simulations, the six parameter combinations that match all in vivo scores had followers at the low setting, while leaders' CIL intensity took medium or high values, cell size took medium or low values, and co-attraction took all levels. Nevertheless, all these parameter combinations endow the leader with enhanced migratory behaviour. Figure 7 Download asset Open asset In silico modelling of trunk neural crest (TNC) migration. (A) Schematics of model parameters. Diff CIL: only leader/follower collisions induce repulsion and change of directionality. Intensity CIL: the leader's response upon collision is stronger than the follower's response. Co-A: co-attraction pulls together cells at a distance. Cell size: volume exclusion. (B) Schematics of simulations multi-objective scores. (C) Depiction of parameter space analysis showing the number of parameters sets that fulfilled each score when different variables were tested. One leader refers to chains with a single leader cell. 1:1, 1:2, and 1:3 refer to leader/follower ratios. (D) 3D plot of linear discriminant analysis (LDA). (E) LDA coefficients of in vivo data. A random dataset was used as control. (F) LDA coefficients of in silico data. A random dataset was used as control. Next, we used a linear discriminant analysis (LDA) to study which of the model parameters bear most weight in the definition of leader and follower identity. LDA is a dimensionality reduction method that projects the data onto a lower dimensional space minimizing the variation within classes (e.g. between leaders) and maximizing the variation between classes (leaders versus followers), allowing the hierarchical ordering of the factors that best explain the class separation. First, we used the in vivo data to determine whether leaders and followers were properly separated by LDA. A visual inspection of the data makes clear that LDA works well to classify migratory identities (Figure 7D). Moreover, the LDA shows that ventral distance is the most important variable separating leaders from followers, with speed and directionality playing a less dominant role (Figure 7E). Next, we used this method to assess the importance of each of the model parameters. CIL intensity appears to be the parameter that most differ between leader and follower cells, while heterogeneity in the other parameters is not essential (Figure 7F). Taken together, the in silico data confirms our previous conclusion that TNC chains are a heterogeneous group. Remarkably, it also predicts CIL intensity to be the most important distinction between leaders and followers. Finally, the model anticipates that TNC chains are formed of leaders and followers in a 1:2 or 1:3 ratio. Leader cells arise from the asymmetric division of a progenitor cell Cell size is a prominent characteristic distinguishing leader from follower cells. Leaders are almost twice as big as followers during migration and this difference is evident before migration initiation (Richardson et al., 2016), suggesting that size disparity arises at birth or shortly thereafter. Interestingly, differential cell size emerged as an important parameter in our in silico analysis, contributing to more realistic leader/follower coordination behaviours. To understand the origin of these size differences, we investigated whether leader and follower cells share a common progenitor, and at which point differences in size become apparent. To this end, we imaged FoxD3:mCherry;H2aFVA:H2a-GFP embryos. The FoxD3:mCherry reporter (Hochgreb-Hägele and B

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.

During vertebrate embryonic development complex morphogenetic events drive the formation of internal organs associated with the developing digestive tract. The foregut organs derive from hepatopancreatic precursor cells that originate bilaterally within the endoderm monolayer, and subsequently converge toward the midline where they coalesce to produce the gut tube from which the liver and pancreas form. The progenitor cells of these internal organs are influenced by the lateral plate mesoderm (LPM), which helps direct them towards their specific fates. However, it is not completely understood how the bilateral organ precursors move toward the embryonic midline and ultimately coalesce to form functional organs. Here we demonstrate that the zebrafish homeobox gene hoxb5b regulates morphogenesis of the foregut endoderm at the midline. At early segmentation stages, hoxb5b is expressed in the LPM adjacent to the developing foregut endoderm. By 24 hpf hoxb5b is expressed directly in the endoderm cells of the developing gut tube. When Hoxb5b function is disrupted, either by morpholino knockdown or sgRNA/Cas9 somatic disruption, the process of foregut morphogenesis is disrupted, resulting in a bifurcated foregut. By contrast, knockdown of the paralogous hoxb5a gene does not alter gut morphology. Further analysis has indicated that Hoxb5b knockdown specimens produce endocrine pancreas cell types, but liver cells are absent. Finally, cell transplantation experiments revealed that Hoxb5b function in the endoderm is not needed for proper coalescence of the foregut at the midline. Together, our findings imply that midline morphogenesis of foregut endoderm is guided by a hoxb5b-mediated mechanism that functions extrinsically, likely within the LPM. Loss of hoxb5b function prevents normal coalescence of endoderm cells at the midline and thus disrupts gut morphogenesis.

In vertebrate animals, motor and sensory efferent neurons carry information from the central nervous system (CNS) to peripheral targets. These two types of efferent systems sometimes bear a close resemblance, sharing common segmental organization, axon pathways, and chemical messengers. Here, we focus on the development of the octavolateral efferent neurons (OENs) and their interactions with the closely‐related facial branchiomotor neurons (FBMNs) in zebrafish. Using live‐imaging approaches, we investigate the birth, migration, and projection patterns of OENs. We find that OENs are born in two distinct groups: a group of rostral efferent neurons (RENs) that arises in the fourth segment, or rhombomere (r4), of the hindbrain and a group of caudal efferent neurons (CENs) that arises in r5. Both RENs and CENs then migrate posteriorly through the hindbrain between 18 and 48 hrs postfertilization, alongside the r4‐derived FBMNs. Like the FBMNs, migration of the r4‐derived RENs depends on function of the segmental identity gene hoxb1a; unlike the FBMNs, however, both OEN populations move independently of prickle1b. Further, we investigate whether the previously described “pioneer” neuron that leads FBMN migration through the hindbrain is an r4‐derived FBMN/REN or an r5‐derived CEN. Our experiments verify that the pioneer is an r4‐derived neuron and reaffirm its role in leading FBMN migration across the r4/5 border. In contrast, the r5‐derived CENs migrate independently of the pioneer. Together, these results indicate that the mechanisms OENs use to navigate the hindbrain differ significantly from those employed by FBMNs.

The neural crest (NC) is a transient multipotent cell population that migrates extensively to produce a remarkable array of vertebrate cell types. NC cell specification progresses in an anterior to posterior fashion, resulting in distinct, axial-restricted subpopulations. The anterior-most, cranial, population of NC is specified as gastrulation concludes and neurulation begins, while more posterior populations become specified as the body elongates. The mechanisms that govern development of the more posterior NC cells remain incompletely understood. Here, we report a key role for zebrafish Cdx4, a homeodomain transcription factor, in the development of posterior NC cells. We demonstrate that cdx4 is expressed in trunk NC cell progenitors, directly binds NC cell-specific enhancers in the NC GRN, and regulates expression of the key NC development gene foxd3 in the posterior body. Moreover, cdx4 mutants show disruptions to the segmental pattern of trunk NC cell migration due to loss of normal leader/follower cell dynamics. Finally, using cell transplantation to generate chimeric specimens, we show that Cdx4 does not function in the paraxial mesoderm-the environment adjacent to which crest migrates-to influence migratory behaviors. We conclude that cdx4 plays a critical, and likely tissue autonomous, role in the establishment of trunk NC migratory behaviors. Together, our results indicate that cdx4 functions as an early NC specifier gene in the posterior body of zebrafish embryos.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

The neural crest is regionalized along the anteroposterior axis, as demonstrated by foundational lineage-tracing experiments that showed the restricted developmental potential of neural crest cells originating in the head. Here, we explore how recent studies of experimental embryology, genetic circuits and stem cell differentiation have shaped our understanding of the mechanisms that establish axial-specific populations of neural crest cells. Additionally, we evaluate how comparative, anatomical and genomic approaches have informed our current understanding of the evolution of the neural crest and its contribution to the vertebrate body.

The neural crest—a key innovation of the vertebrates—gives rise to diverse cell types including melanocytes, neurons and glia of the peripheral nervous system, and chondrocytes of the jaw and skull. Proper development of the cephalic region is dependent on the tightly-regulated specification and migration of cranial neural crest cells (NCCs). The core PCP proteins Frizzled and Disheveled have previously been implicated in NCC migration. Here we investigate the functions of the core PCP proteins Prickle1a and Prickle1b in zebrafish cranial NCC development. Using analysis of pk1a and pk1b mutant embryos, we uncover similar roles for both genes in facilitating cranial NCC migration. Disruption of either gene causes pre-migratory NCCs to cluster together at the dorsal aspect of the neural tube, where they adopt aberrant polarity and movement. Critically, in investigating Pk1-deficient cells that fail to migrate ventrolaterally, we have also uncovered roles for pk1a and pk1b in the epithelial-to-mesenchymal transition (EMT) of pre-migratory NCCs that precedes their collective migration to the periphery. Normally, during EMT, pre-migratory NCCs transition from a neuroepithelial to a bleb-based and subsequently, mesenchymal morphology capable of directed migration. When either Pk1a or Pk1b is disrupted, NCCs continue to perform blebbing behaviors characteristic of pre-migratory cells over extended time periods, indicating a block in a key transition during EMT. Although some Pk1-deficient NCCs transition successfully to mesenchymal, migratory morphologies, they fail to separate from neighboring NCCs. Additionally, Pk1b-deficient NCCs show elevated levels of E-Cadherin and reduced levels of N-Cadherin, suggesting that Prickle1 molecules regulate Cadherin levels to ensure the completion of EMT and the commencement of cranial NCC migration. We conclude that Pk1 plays crucial roles in cranial NCCs both during EMT and migration. These roles are dependent on the regulation of E-Cad and N-Cad.

Cranial neural crest cells act as precursors to a variety of tissues in the craniofacial region, including: pigment cells, bone, and the peripheral nervous system. One interesting feature of neural crest cells is the migration they must make from the neuroepithelium to various sites within the developing embryo before they differentiate. Filopodia and lamellipodia are thought to play a critical role in this migration; however, much remains to be elucidated about the characteristics of this migration and the specific roles these protrusions play. We are constructing a light-sheet microscope to study the F-actin dynamics of filopodia and lamellipodia in migrating neural crest cells of zebrafish (Danio rerio) embryos. F-actin can be targeted to neural crest cells by injecting embryos with a DNA construct that uses the neural crest specific sox10 gene regulatory sequences to mosaically drive expression of LifeAct-GFP reporter. LifeAct-GFP will be used to visualize the formation of protrusions in the neural crest cells, as well as the migration of these cells throughout the embryo. Light-sheet microscopy allows for rapid visualization of the developing embryos with high three-dimensional resolution and a reduced risk of photobleaching. Our custom-built light-sheet microscope is based on the open source platform OpenSPIM. It includes a second illumination objective to prevent shadowing of our images and a stage that allows for 360 degree rotation of the sample. This modified design will allow for precise imaging of migrating neural crest cells with high z-dimension resolution at a speed that is conducive to capturing the rapid dynamics of pseudopodia generation.

Our understanding of the neural crest, a key vertebrate innovation, is built upon studies of multiple model organisms. Early research on neural crest cells (NCCs) was dominated by analyses of accessible amphibian and avian embryos, with mouse genetics providing complementary insights in more recent years. The zebrafish model is a relative newcomer to the field, yet it offers unparalleled advantages for the study of NCCs. Specifically, zebrafish provide powerful genetic and transgenic tools, coupled with rapidly developing transparent embryos that are ideal for high-resolution real-time imaging of the dynamic process of neural crest development. While the broad principles of neural crest development are largely conserved across vertebrate species, there are critical differences in anatomy, morphogenesis, and genetics that must be considered before information from one model is extrapolated to another. Here, our goal is to provide the reader with a helpful primer specific to neural crest development in the zebrafish model. We focus largely on the earliest events-specification, delamination, and migration-discussing what is known about zebrafish NCC development and how it differs from NCC development in non-teleost species, as well as highlighting current gaps in knowledge.

1590 Background: Genetic counseling is recommended for all women diagnosed with epithelial ovarian cancer, independent of family history. Despite the potential benefits to the patient and her family, referral rates remain low. We sought to assess referral rates at our institution and implement two strategies to improve both the rate of referral and the completion of counseling and testing – (1) discussion of referral for all patients reviewed at multidisciplinary treatment planning conference and (2) option of telephone counseling. Methods: Patients with a diagnosis of epithelial ovarian, fallopian tube or primary peritoneal cancer since 10/1/14 were identified through pathology reports and chart review was performed to obtain demographic data and cancer details including histology, grade and stage. Personal and family history of cancer, date of genetic counseling referral, method of genetic counseling (in-person vs. telephone), date of genetic testing and the result of genetic testing were also abstracted. Results: The rate of genetic counseling referral was 63.5% (214/337 patients). Of those referred, 61% (131/214) underwent counseling, with 77% (165/214) in person and the 23% (49/214) via telephone after initiation of the telephone counseling program in September 2017. Overall, 90% of patients who received genetic counseling underwent testing, including 92.7% of the in-person counseling cohort and 67.9% of the telephone counseling cohort to date. In total, 24.1% of patients harbored a pathogenic gene mutation, with BRCA1 mutations most common (16.5% of patients). Variants of undetermined significance were identified in 11.3% of patients. Conclusions: Telephone genetic counseling and mandatory discussion of referral at the time of treatment planning conference both appear to facilitate genetic counseling and ascertainment of actionable germline mutations. Overall, referral rates are high at our institution. The implementation of telephone-based genetic counseling programs has the potential to improve both counseling and testing rates, particularly when in-person counseling is not available or is delayed.

The zebrafish pancreas shares its basic organization and cell types with the mammalian pancreas. In addition, the developmental pathways that lead to the establishment of the pancreatic islets of Langherhans are generally conserved from fish to mammals. Zebrafish provides a powerful tool to probe the mechanisms controlling establishment of the pancreatic endocrine cell types from early embryonic progenitor cells, as well as the regeneration of endocrine cells after damage. This knowledge is, in turn, applicable to refining protocols to generate renewable sources of human pancreatic islet cells that are critical for regulation of blood sugar levels. Here, we review how previous and ongoing studies in zebrafish and beyond are influencing the understanding of molecular mechanisms underlying various forms of diabetes and efforts to develop cell-based approaches to cure this increasingly widespread disease.