SUMMARY Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.

The “cancerized field” concept posits that cells in a given tissue share an oncogenic mutation or insult and are thus cancer-prone, yet only discreet clones within the field initiate tumors. Nearly all benign nevi carry oncogenic BRAFV600E mutations, but they only rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCP’s) and is specifically re-expressed in melanoma. We show by live imaging of transgenic zebrafish crestin reporters that, within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model. We show the crestin element is regulated by NCP transcription factors, including sox10. Forced sox10 overexpression in melanocytes accelerated melanoma formation, consistent with activation of a NCP gene signature and super-enhancers leading to melanoma. Our work highlights the importance of NCP state reemergence as a key event in melanoma initiation.

Author Contributions G.M. designed and performed the experiments, analyzed and interpreted the data, and wrote the manuscript. M.A.M. designed and supervised the research, analyzed and interpreted the data, and wrote the manuscript. C.V.C. co-designed and assisted with high spatiotemporal resolution microscopy experiments. E.J.H. and J.R.P. co-designed and assisted with zebrafish experiments. N.M. and T.P. co-designed and performed the BBB-on-a-chip experiments. C.C.D. assisted with some of the experiments. L.I.Z. and D.E.I. provided zebrafish and BBB-on-a-chip models, respectively.

Despite profound importance in development and cancer, the extracellular cues that target cell invasions through basement membrane barriers remain poorly understood 1. A central obstacle has been the difficulty of studying the interactions between invading cells and basement membranes in vivo 2,3. Using the genetically and visually tractable model of C. elegans anchor cell (AC) invasion, we show that unc-6 (netrin) signaling, a pathway not previously implicated in controlling cell invasion in vivo, is a key regulator of this process. Site of action studies reveal that prior to invasion localized UNC-6 secretion directs its receptor, UNC-40, to the AC’s plasma membrane in contact with the basement membrane. There, UNC-40 polarizes a specialized invasive membrane domain through the enrichment of actin regulators, F-actin and phosphatidylinositol 4,5-bisphosphate. Cell ablation experiments indicate that UNC-6 promotes the formation of invasive protrusions from the AC that break down the basement membrane in response to a subsequent vulval cue. Together, these results characterize an invasive membrane domain in vivo, and reveal a novel role for netrin in polarizing this domain towards its basement membrane target.

Localized activation of netrin signaling induces focused F-actin formation and the protrusive force necessary for physical displacement of basement membrane during cell transmigration.

Summary Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, we have recently shown that netrin signaling orients a specialized invasive cell membrane domain towards the basement membrane. Here, we demonstrate that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part, by targeting the netrin receptor UNC-40 (DCC) to the AC’s plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2 and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain.

The basement membrane is a dense, highly cross-linked, sheet-like extracellular matrix that underlies all epithelia and endothelia in multicellular animals. During development, leukocyte trafficking, and metastatic disease, cells cross the basement membrane to disperse and enter new tissues. Based largely on in vitro studies, cells have been thought to use proteases to dissolve and traverse this formidable obstacle. Surprisingly, recent in vivo studies have uncovered a remarkably diverse range of cellular- and tissue-level strategies beyond proteolysis that cells use to navigate through the basement membrane. These fascinating and unexpected mechanisms have increased our understanding of how cells cross this matrix barrier in physiological and disease settings.

Large gaps in basement membrane (BM) occur at sites of cell invasion and tissue remodelling in development and cancer. Though never followed directly in vivo, BM dissolution or reduced synthesis have been postulated to create these gaps. Using landmark photobleaching and optical highlighting of laminin and type IV collagen, we find that a new mechanism, BM sliding, underlies BM gap enlargement during uterine-vulval attachment in C. elegans. Laser ablation and mutant analysis reveal that the invaginating vulval cells promote BM movement. Further, an RNA interference and expression screen identify the integrin INA-1/PAT-3 and VAB-19, homolog of the tumour suppressor Kank, as regulators of BM opening. Both concentrate within vulval cells at the BM gap boundary and halt expansion of the shifting BM. BM sliding followed by targeted adhesion represents a new mechanism for creating precise BM breaches that can be used by cells to break down compartment boundaries.