Faculty Bibliography

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.

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.

Cell-extracellular matrix interactions have been studied extensively using cells cultured in vitro. These studies indicate that focal adhesion (FA)-based cell-extracellular matrix interactions are essential for cell anchoring and cell migration. Whether FAs play a similarly important role in vivo is less clear. Here, we summarize the formation and function of FAs in cultured cells and review how FAs transmit and sense force in vitro. Using examples from animal studies, we also describe the role of FAs in cell anchoring during morphogenetic movements and cell migration in vivo. Finally, we conclude by discussing similarities and differences in how FAs function in vitro and in vivo.

Cytoskeletal networks play an important role in regulating nuclear morphology and ciliogenesis. However, the role of microtubule (MT) post-translational modifications in nuclear shape regulation and cilium disassembly has not been explored. Here we identified a novel regulator of the tubulin polyglutamylase complex (TPGC), C11ORF49/CSTPP1, that regulates cytoskeletal organization, nuclear shape, and cilium disassembly. Mechanistically, loss of C11ORF49/CSTPP1 impacts the assembly and stability of the TPGC, which modulates long-chain polyglutamylation levels on microtubules (MTs) and thereby balances the binding of MT-associated proteins and actin nucleators. As a result, loss of TPGC leads to aberrant, enhanced assembly of MTs that penetrate the nucleus, which in turn leads to defects in nuclear shape, and disorganization of cytoplasmic actin that disrupts the YAP/TAZ pathway and cilium disassembly. Further, we showed that C11ORF49/CSTPP1-TPGC plays mechanistically distinct roles in the regulation of nuclear shape and cilium disassembly. Remarkably, disruption of C11ORF49/CSTPP1-TPGC also leads to developmental defects in vivo. Our findings point to an unanticipated nexus that links tubulin polyglutamylation with nuclear shape and ciliogenesis.

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- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals.

Zebrafish provide an excellent model for in vivo cell biology studies because of their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers because of inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N or C termini with fluorescent proteins by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes that are crucial for epithelial biology and organ development, including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, the apical polarity protein aPKC and the ECM receptor Integrin β1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

Animal embryogenesis requires a precise coordination between morphogenesis and cell fate specification. During mesoderm induction, mesodermal fate acquisition is tightly coordinated with the morphogenetic process of epithelial-to-mesenchymal transition (EMT). In zebrafish, cells exist transiently in a partial EMT state during mesoderm induction. Here, we show that cells expressing the transcription factor Sox2 are held in the partial EMT state, stopping them from completing the EMT and joining the mesoderm. This is critical for preventing the formation of ectopic neural tissue. The mechanism involves synergy between Sox2 and the mesoderm-inducing canonical Wnt signaling pathway. When Wnt signaling is inhibited in Sox2-expressing cells trapped in the partial EMT, cells exit into the mesodermal territory but form an ectopic spinal cord instead of mesoderm. Our work identifies a critical developmental checkpoint that ensures that morphogenetic movements establishing the mesodermal germ layer are accompanied by robust mesodermal cell fate acquisition.

Animal development entails the organization of specific cell types in space and time, and spatial patterns must form in a robust manner. In the zebrafish spinal cord, neural progenitors form stereotypic patterns despite noisy morphogen signaling and large-scale cellular rearrangements during morphogenesis and growth. By directly measuring adhesion forces and preferences for three types of endogenous neural progenitors, we provide evidence for the differential adhesion model in which differences in intercellular adhesion mediate cell sorting. Cell type-specific combinatorial expression of different classes of cadherins (N-cadherin, cadherin 11, and protocadherin 19) results in homotypic preference ex vivo and patterning robustness in vivo. Furthermore, the differential adhesion code is regulated by the sonic hedgehog morphogen gradient. We propose that robust patterning during tissue morphogenesis results from interplay between adhesion-based self-organization and morphogen-directed patterning.

Chemoattractant gradients frequently guide migrating cells. To achieve the most directional signal, such gradients should be maintained with concentrations around the dissociation constant (K_d)^1-6 of the chemoreceptor. Whether this actually occurs in animals is unknown. Here we investigate whether a moving tissue, the zebrafish posterior lateral line primordium, buffers its attractant in this concentration range to achieve robust migration. We find that the Cxcl12 (also known as Sdf1) attractant gradient ranges from 0 to 12 nM, values similar to the 3.4 nM K_d of its receptor Cxcr4. When we increase the K_d of Cxcl12 for Cxcr4, primordium migration is less directional. Furthermore, a negative-feedback loop between Cxcl12 and its clearance receptor Ackr3 (also known as Cxcr7) regulates the Cxcl12 concentrations. Breaking this negative feedback by blocking the phosphorylation of the cytoplasmic tail of Ackr3 also results in less directional primordium migration. Thus, directed migration of the primordium is dependent on a close match between the Cxcl12 concentration and the K_d of Cxcl12 for Cxcr4, which is maintained by buffering of the chemokine levels. Quantitative modelling confirms the plausibility of this mechanism. We anticipate that buffering of attractant concentration is a general mechanism for ensuring robust cell migration.

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 the attractant and adhere to each other to coordinate their movements and migrate with robust directionality.