Modelling the Fluid Mechanics of Cilia and Flagella in Reproduction and Development

Cilia and flagella are actively bending slender organelles, performing functions such as motility, feeding and embryonic symmetry breaking. We review the mechanics of viscous-dominated microscale flow, including time-reversal symmetry, drag anisotropy of slender bodies, and wall effects. We focus on the fundamental force singularity, higher order multipoles, and the method of images, providing physical insight and forming a basis for computational approaches. Two biological problems are then considered in more detail: (1) left-right symmetry breaking flow in the node, a microscopic structure in developing vertebrate embryos, and (2) motility of microswimmers through non-Newtonian fluids. Our model of the embryonic node reveals how particle transport associated with morphogenesis is modulated by the gradual emergence of cilium posterior tilt. Our model of swimming makes use of force distributions within a body-conforming finite element framework, allowing the solution of nonlinear inertialess Carreau flow. We find that a three-sphere model swimmer and a model sperm are similarly affected by shear-thinning; in both cases swimming due to a prescribed beat is enhanced by shear-thinning, with optimal Deborah number around 0.8. The sperm exhibits an almost perfect linear relationship between velocity and the logarithm of the ratio of zero to infinite shear viscosity, with shear-thickening hindering cell progress.Comment: 20 pages, 24 figure

Mechanical Stretch and PI3K Signaling Link Cell Migration and Proliferation to Coordinate Epithelial Tubule Morphogenesis in the Zebrafish Pronephros

Organ development leads to the emergence of organ function, which in turn can impact developmental processes. Here we show that fluid flow-induced collective epithelial migration during kidney nephron morphogenesis induces cell stretch that in turn signals epithelial proliferation. Increased cell proliferation was dependent on PI3K signaling. Inhibiting epithelial proliferation by blocking PI3K or CDK4/Cyclin D1 activity arrested cell migration prematurely and caused a marked overstretching of the distal nephron tubule. Computational modeling of the involved cell processes predicted major morphological and kinetic outcomes observed experimentally under a variety of conditions. Overall, our findings suggest that kidney development is a recursive process where emerging organ function “feeds back” to the developmental program to influence fundamental cellular events such as cell migration and proliferation, thus defining final organ morphology

Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis

Live Imaging of Innate Immune Cell Sensing of Transformed Cells in Zebrafish Larvae: Parallels between Tumor Initiation and Wound Inflammation

Live imaging and genetic studies of the initial interactions between leukocytes and transformed cells in zebrafish larvae indicate an attractant role for H2O2 and suggest that blocking these early interactions reduces expansion of transformed cell clones

Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development.

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development

The role of chemokine receptor trafficking in regulating neutrophil migration to inflammatory sites

Neutrophils are the first immune cells to be recruited to sites of tissue injury or infection. Upon detection of an inflammatory stimulus, neutrophils exit the vasculature and migrate directionally through the interstitial tissue towards the target site. Once at the target site, neutrophils may either focalise and form clusters or may exhibit a more dispersive and exploratory behaviour. Focalisation acts to concentrate local neutrophil effector responses but excess clustering can prove detrimental, resulting in undesirable tissue damage. Neutrophil dispersal promotes the encounter of alternative signals and therefore drives resolution of the response. A fine balance between focalisation and exploration must exist to ensure that the inflammatory response is effective but also transient and self-resolving.  Neutrophils are recruited to target sites by gradients of attractant molecules, a major class of which is chemokines. Chemokines bind to G-protein coupled receptors (GPCRs) and initiate complex intracellular signalling cascades which ultimately result in directional neutrophil migration. Upon ligand binding, GPCRs can undergo multiple trafficking fates which may in turn influence sensitivity to the gradient. However, the functional significance of receptor trafficking during neutrophil responses in vivo remains unknown. Here, I address this question using zebrafish Cxcl8a (a homologue of human CXCL8) which signals through two G-protein coupled receptors, Cxcr1 and Cxcr2. Through new in vivo biosensors, I show that Cxcr1 and Cxcr2 exhibit differential trafficking in response to endogenous gradients. Cxcr1 is extensively internalised whilst Cxcr2 is sustained on the cell membrane. Live-imaging of receptor knockout neutrophils revealed that Cxcr1 promotes neutrophil clustering at wounds, whilst Cxcr2 drives dispersal. Through receptor mutagenesis I show that neutrophil dispersal relies on Cxcr1 internalisation and membrane sustenance of Cxcr2. Thus, I show that differential trafficking of two receptors balances the rise and fall of neutrophil inflammatory responses. To my knowledge, this is the first study to functionally link receptor dynamics to neutrophil migration behaviour in vivo.MRC 3-year studentshi