Posterior Lateral Line Primordium Research Articles

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

Pulses of RhoA signaling stimulate actin polymerization and flow in protrusions to drive collective cell migration.

In animals, cells often move as collectives to shape organs, close wounds, or-in the case of disease-metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network. Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin-II-dependent actin flow and protrusion retraction at the base of the protrusions and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other contexts.

RhoA GEF Mcf2lb regulates rosette integrity during collective cell migration.

Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of ∼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.

MARVEL domain containing CMTM4 affects CXCR4 trafficking.

The MARVEL proteins CMTM4 and CMTM6 control PD-L1, thereby influencing tumor immunity. We found that defective zebrafish cmtm4 slowed the development of the posterior lateral line (pLL) by altering the Cxcr4b gradient across the pLL primordium (pLLP). Analysis in mammalian cells uncovered that CMTM4 interacted with CXCR4, altering its glycosylation pattern, but did not affect internalization or degradation of CXCR4 in the absence of its ligand CXCL12. Synchronized release of CXCR4 from the endoplasmic reticulum revealed that CMTM4 slowed CXCR4 trafficking from the endoplasmic reticulum to the plasma membrane without affecting overall cell surface expression. Altered CXCR4 trafficking reduced ligand-induced CXCR4 degradation and affected AKT but not ERK1/2 activation. CMTM4 expression, in contrast to that of CXCR4, correlated with the survival of patients with renal cell cancer in the TCGA cohort. Furthermore, we observed that cmtm4 depletion promotes the separation of cells from the pLLP cell cluster in zebrafish embryos. Collectively, our findings indicate that CMTM4 exerts general roles in the biosynthetic pathway of cell surface molecules and seems to affect CXCR4-dependent cell migration.

Rear traction forces drive adherent tissue migration in vivo.

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate, and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin/integrin-mediated coupling is required for efficient migration, and its loss is partly compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the primordium’s rear exerts higher stresses than the front, suggesting that this tissue pushes itself forward with its back. This unexpected strategy likely also underlies the motion of other tissues in animals.

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration.

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

Spatio-temporal analysis of collective migration in vivo by particle image velocimetry

Collective cell migration drives the formation of complex organ systems as well as certain tumour invasions and wound healing processes. A characteristic feature of many migrating collectives is tissue-scale polarity, whereby ‘leader’ cells at the tissue edge guide ‘followers’ cells that become assembled into polarized epithelial tissues. In this study, we employed particle image velocimetry (PIV) as a tool to quantitate local dynamics underlying the migration of the posterior lateral line primordium (pLLP) in zebrafish at a short time scale. Epithelial cadherin-EGFP was the fluorescent tracer in time-lapse images for PIV analysis. At the tissue level, global speed and directionality of the primordium were extracted from spatially averaged velocity fields. Interestingly, fluctuating velocity patterns evolve at the mesoscale level, which distinguishes the pseudo-mesenchymal leading front from the epithelialized trailing edge, and superimpose to the global deceleration of the whole primordium during the separation of a protoneuromast. Local velocity fields obtained by PIV proved sensitive to estimate the migration speed and directionality of the pLLP in zebrafish, predicting protoneuromast separation at short time scales. Finally, the PIV approach may be suitable for analysing the dynamics of other in vivo models of collective migration.

The H3K27 demethylase controls the lateral line embryogenesis of zebrafish.

Kdm6b, a specific histone 3 lysine 27 (H3K27) demethylase, has been reported to be implicated in a variety of developmental processes including cell differentiation and cell fate determination and multiple organogenesis. Here, we regulated the transcript level of kdm6bb to study the potential role in controlling the hearing organ development of zebrafish. A morpholino antisense oligonucleotide (MO) strategy was used to induce Kdm6b deficiency; immunohistochemical staining and in situ hybridization analysis were conducted to figure out the morphologic alterations and embryonic mechanisms. Kdm6bb is expressed in the primordium and neuromasts at the early stage of zebrafish embryogenesis, suggesting a potential function of Kdm6b in the development of mechanosensory organs. Knockdown of kdm6bb severely influences the cell migration and proliferation in posterior lateral line primordium, abates the number of neuromasts along the trunk, and mRNA-mediated rescue test can partially renew the neuromasts. Loss of kdm6bb might be related to aberrant expressions of chemokine genes encompassing cxcl12a and cxcr4b/cxcr7b in the migrating primordium. Moreover, inhibition of kdm6bb reduces the expression of genes in Fgf signaling pathway, while it increases the axin2 and lef1 expression level of Wnt/β-catenin signaling during the migrating stage. Collectively, our results revealed that Kdm6b plays an essential role in guiding the migration of primordium and in regulating the deposition of zebrafish neuromasts by mediating the gene expression of chemokines and Wnt and Fgf signaling pathway. Since histone methylation and demethylation are reversible, targeting Kdm6b may present as a novel therapeutic regimen for hearing disorders.

Expression of two uncharacterized protein coding genes in zebrafish lateral line system.

The lateral line system is a mechanosensory organ of fish and amphibians that detects changes in water flow and is formed by the coordinated action of many signalling pathways. These signalling pathways can easily be targeted in zebrafish using pharmacological inhibitors to decipher their role in lateral line system development at cellular and molecular level. We have identified two uncharacterized proteins, whose mRNA are expressed in the lateral line system of zebrafish. One of these proteins, uncharacterized protein LOC564095 precursor, is conserved across vertebrates and its mRNA is expressed in posterior lateral line primordium (pLLP). The other uncharacterized protein, LOC100536887, is present only in the teleost fishes and its mRNA is expressed in neuromasts. We show that inhibition of retinoic acid (RA) signalling reduces the expression of both of these uncharacterized genes. It is reported that inhibition of RA signalling during gastrulation starting at 7 hours post fertilization (hpf) abrogates pLLP formation, and inhibition of RA signalling at 10 hpf delays the initiation of pLLP migration. Here, we show that inhibition of RA signalling before and during segmentation (9-16 hpf) results in delayed initiation and reduced speed of pLLP migration, as well as inhibition of posterior neuromasts formation.

Zebrafish Posterior Lateral Line primordium migration requires interactions between a superficial sheath of motile cells and the skin.

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

Lamellipodia-like protrusions and focal adhesions contribute to collective cell migration in zebrafish

Collective cell migration is a process where cohorts of cells exhibit coordinated migratory behavior. During individual and collective cellular migration, cells must extend protrusions to interact with the extracellular environment, sense chemotactic cues, and act as points of attachment. The mechanisms and regulators of protrusive behavior have been widely studied in individually migrating cells; however, how this behavior is regulated throughout collectives is not well understood. To address this, we used the zebrafish posterior lateral line primordium (pLLP) as a model. The pLLP is a cluster of ~150 ​cells that migrates along the zebrafish trunk, depositing groups of cells that will become sensory organs. To define protrusive behavior, we performed mosaic analysis to sparsely label pLLP cells with a transgene marking filamentous actin. This approach revealed an abundance of brush-like protrusions throughout the pLLP that orient in the direction of migration. Formation of these protrusions depends on the Arp2/3 complex, a regulator of dendritic actin. This argues that these brush-like protrusions are an in vivo example of lamellipodia. Mosaic analysis demonstrated that these lamellipodia-like protrusions are located in a close proximity to the overlying skin. Immunostaining revealed an abundance of focal adhesion complexes surrounding the pLLP. Disruption of these complexes specifically in pLLP cells led to impaired pLLP migration. Finally, we show that Erk signaling, a known regulator of focal adhesions, is required for proper formation of lamellipodia-like protrusions and pLLP migration. Altogether, our results suggest a model where the coordinated dynamics of lamellipodia-like protrusions, making contact with either the overlying skin or the extracellular matrix through focal adhesions, promotes migration of pLLP cells.

Relationship between surrounding tissue morphology and directional collective migration of the posterior lateral line primordium in zebrafish.

Collective cell migration, in which cells assemble and move together, is an essential process in embryonic development, wound healing and cancer metastasis. Chemokine signaling guides cell assemblies to their destinations. In zebrafish posterior lateral line primordium (PLLP), a model system for collective cell migration, it has been proposed that the chemokine ligand Cxcl12a secreted from muscle pioneer cells (MPs) and muscle fast fibers (MFFs), which are distributed along with the horizontal midline, binds to the receptor Cxcr4b in PLLP and that Cxcl12a-Cxcr4b signaling guides the anterior-to-posterior migration of PLLP along the horizontal midline. However, how the surrounding tissues affect PLLP migration remains to be elucidated. Here, we investigated the relationship between the PLLP and the surrounding tissues and found that a furrow between the dorsal and ventral myotomes is generated by Sonic hedgehog (Shh) signaling-dependent MP and MFF differentiation and that the PLLP migrates in this furrow. When transient inhibition of Shh signaling impaired both the furrow formation and differentiation of cxcl12a-expressing MPs/MFFs, directional PLLP migration was severely perturbed. Furthermore, when differentiated MPs and MFFs were ablated by femtosecond laser irradiations, the furrow remained and PLLP migration was relatively unaffected. These results suggest that the furrow formation between the dorsal and ventral myotomes is associated with the migratory behavior of PLLP.

An image-based data-driven analysis of cellular architecture in a developing tissue.

Quantitative microscopy is becoming increasingly crucial in efforts to disentangle the complexity of organogenesis, yet adoption of the potent new toolbox provided by modern data science has been slow, primarily because it is often not directly applicable to developmental imaging data. We tackle this issue with a newly developed algorithm that uses point cloud-based morphometry to unpack the rich information encoded in 3D image data into a straightforward numerical representation. This enabled us to employ data science tools, including machine learning, to analyze and integrate cell morphology, intracellular organization, gene expression and annotated contextual knowledge. We apply these techniques to construct and explore a quantitative atlas of cellular architecture for the zebrafish posterior lateral line primordium, an experimentally tractable model of complex self-organized organogenesis. In doing so, we are able to retrieve both previously established and novel biologically relevant patterns, demonstrating the potential of our data-driven approach.

A negative-feedback loop maintains optimal chemokine concentrations for directional cell migration.

Chemoattractant gradients often guide migrating cells. To achieve the greatest directional signal over noise, such gradients should be maintained with concentrations around the chemoreceptor’s dissociation constant (Kd) 1–6. Whether this is true in animals is unknown. Here, we investigate whether a moving tissue, the zebrafish posterior lateral line primordium, buffers its attractant in this concentration range for robust migration. We find that the Cxcl12/Sdf1 attractant gradient ranges from 0 to 12 nM and thus borders around the 3.4 nM Kd of its receptor Cxcr4. When we increase the Cxcl12-Cxcr4 Kd, primordium migration is less directional. Furthermore, a negative feedback loop between Cxcl12 and its clearance receptor Ackr3/Cxcr7 regulates the Cxcl12 concentrations. Breaking this negative feedback by blocking the phosphorylation of Ackr3b’s cytoplasmic tail also results in less directional primordium migration. Thus, the primordium relies on a close match between the Cxcl12 concentration and the Cxcl12-Cxcr4 Kd for directed migration which it maintains by buffering the chemokine levels. Quantitative modeling confirms the plausibility of this mechanism. We anticipate that attractant concentration buffering is a general mechanism to ensure robust cell migration.

Shootins mediate collective cell migration and organogenesis of the zebrafish posterior lateral line system

The zebrafish sensory posterior lateral line is an excellent model system to study collective cell migration and organogenesis. Shootin1 is a cytoplasmic protein involved in neuronal polarization and axon guidance. Previous studies have shown that shootin1 couples actin filament retrograde flow with extracellular adhesive substrates at the leading edge of axonal growth cones, thereby producing mechanical force for the migration and guidance of axonal growth cones. However, the functions of shootin in peripheral cells remain unknown. Here we identified two novel shootin family members, shootin2 and shootin3. In zebrafish, shootin1 and shootin3 are expressed in the posterior lateral line primordium (PLLP) and neuromasts during embryonic development. A shootin1 mutant displayed a reduced speed of PLLP migration, while shootin1;shootin3 double mutation inhibited cell proliferation in the PLLP. Furthermore, our results suggest that shootin1 and shootin3 positively regulate the number of neuromasts and the number of cells in deposited neuromasts. Our study demonstrates that shootins mediate collective cell migration of the posterior lateral line primordium and formation of neuromasts in zebrafish.

Cadherin-Mediated Cell Coupling Coordinates Chemokine Sensing across Collectively Migrating Cells

The directed migration of cells sculpts the embryo, contributes to homeostasis in the adult, and, when dysregulated, underlies many diseases [1, 2]. During these processes, cells move singly or as a collective. In both cases, they follow guidance cues, which direct them to their destination [3-6]. In contrast to single cells, collectively migrating cells need to coordinate with their neighbors to move together in the same direction. Recent studies suggest that leader cells in the front sense the guidance cue, relay the directional information to the follower cells in the back, and can pull the follower cells along [7-19]. In this manner, leader cells steer the collective and set the collective's overall speed. However, whether follower cells also participate in steering and speed setting of the collective is largely unclear. Using chimeras, we analyzed the role of leader and follower cells in the collectively migrating zebrafish posterior lateral line primordium. This tissue expresses the chemokine receptor Cxcr4 and is guided by the chemokine Cxcl12a [20-23]. We find that leader and follower cells need to sense the attractant Cxcl12a for efficient migration, are coupled to each other through cadherins, and require coupling to pull Cxcl12a-insensitive cells along. Analysis of cell dynamics in chimeric and protein-depleted primordia shows that Cxcl12a-sensing and cadherin-mediated adhesion contribute jointly to direct migration at both single-cell and tissue levels. These results suggest that all cells in the primordium need to sense theattractant and adhere to each other to coordinate their movements and migrate with robust directionality.

Using Zebrafish to Study Collective Cell Migration in Development and Disease.

Cellular migration is necessary for proper embryonic development as well as maintenance of adult health. Cells can migrate individually or in groups in a process known as collective cell migration. Collectively migrating cohorts maintain cell-cell contacts, group polarization, and exhibit coordinated behavior. This mode of migration is important during numerous developmental processes including tracheal branching, blood vessel sprouting, neural crest cell migration and others. In the adult, collective cell migration is important for proper wound healing and is often misappropriated during cancer cell invasion. A variety of genetic model systems are used to examine and define the cellular and molecular mechanisms behind collective cell migration including border cell migration and tracheal branching in Drosophila melanogaster, neural crest cell migration in chick and Xenopus embryos, and posterior lateral line primordium (pLLP) migration in zebrafish. The pLLP is a group of about 100 cells that begins migrating around 22 hours post-fertilization along the lateral aspect of the trunk of the developing embryo. During migration, clusters of cells are deposited from the trailing end of the pLLP; these ultimately differentiate into mechanosensory organs of the lateral line system. As zebrafish embryos are transparent during early development and the pLLP migrates close to the surface of the skin, this system can be easily visualized and manipulated in vivo. These advantages together with the amenity to advance genetic methods make the zebrafish pLLP one of the premier model systems for studying collective cell migration. This review will describe the cellular behaviors and signaling mechanisms of the pLLP and compare the pLLP to collective cell migration in other popular model systems. In addition, we will examine how this type of migration is hijacked by collectively invading cancer cells.

Anosmin1 Shuttles Fgf to Facilitate Its Diffusion, Increase Its Local Concentration, and Induce Sensory Organs

Growth factors induce and pattern sensory organs, but how their distribution is regulated by the extracellular matrix (ECM) is largely unclear. To address this question, we analyzed the diffusion behavior of Fgf10 molecules during sensory organ formation in the zebrafish posterior lateral line primordium. In this tissue, secreted Fgf10 induces organ formation at a distance from its source. We find that most Fgf10 molecules are highly diffusive and move rapidly through the ECM. We identify Anosmin1, which when mutated in humans causes Kallmann Syndrome, as an ECM protein that binds to Fgf10 and facilitates its diffusivity by increasing the pool of fast-moving Fgf10 molecules. In the absence of Anosmin1, Fgf10 levels are reduced and organ formation is impaired. Global overexpression of Anosmin1 slows the fast-moving Fgf10 molecules and results in Fgf10 dispersal. These results suggest that Anosmin1 liberates ECM-bound Fgf10 and shuttles it to increase its signaling range.

Prpf4 is essential for cell survival and posterior lateral line primordium migration in zebrafish

Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we characterized a zebrafish prpf4t243 mutant harboring a Tol2 transposon-based gene trap cassette in the third intron of the prpf4 gene. Cells in the brain and spinal cord gradually undergo p53-dependent apoptosis after 28 hpf in prpf4t243 mutants, suggesting that a widespread function of prpf4 in neural cell survival. In addition, prpf4 is essential for survival of posterior lateral line primordial (pLLP) cells. prpf4 deficiency perturbs Fgf, Wnt/β-catenin and chemokine signaling pathways and impairs pLLP migration. RNA-Seq analysis suggests that prpf4 deficiency may impair spliceosome assembly, leading to compensatory upregulation of core spliceosomal genes and alteration of pre-mRNA splicing. Taken together, our studies uncover an essential role of prpf4 in pre-mRNA splicing, cell survival and pLLP migration.

Cxcl12a induces snail1b expression to initiate collective migration and sequential Fgf-dependent neuromast formation in the zebrafish posterior lateral line primordium.

The zebrafish posterior lateral line primordium migrates along a path defined by the chemokine Cxcl12a, periodically depositing neuromasts, to pioneer formation of the zebrafish posterior lateral line system. snail1b, known for its role in promoting cell migration, is expressed in leading cells of the primordium in response to Cxcl12a, whereas its expression in trailing cells is inhibited by Fgf signaling. snail1b knockdown delays initiation of primordium migration. This delay is associated with aberrant expansion of epithelial cell adhesion molecule (epcam) and reduction of cadherin 2 expression in the leading part of the primordium. Co-injection of snail1b morpholino with snail1b mRNA prevents the initial delay in migration and restores normal expression of epcam and cadherin 2 The delay in initiating primordium migration in snail1b morphants is accompanied by a delay in sequential formation of trailing Fgf signaling centers and associated protoneuromasts. This delay is not specifically associated with knockdown of snail1b but also with other manipulations that delay migration of the primordium. These observations reveal an unexpected link between the initiation of collective migration and sequential formation of protoneuromasts in the primordium.