Biology subjects

Whitfield, T. T.

Publications and source records attributed to Whitfield, T. T..

7 recordsLinked to original sources

bioRxiv2026

A dynein-driven nucleokinesis program enables neural crest migration through confined tissues in vivo

Cell migration through confined tissue environments requires precise coordination between the nucleus and the cytoskeleton. Translocation of the nucleus through small pores is a limiting step in confined cell migration. Work in immune and cancer cells has illuminated a key role for actin polymerization and Myosin-II contractility in facilitating nucleus translocation through confinement. However, the microtubule cytoskeleton can also adaptively stabilise under confinement. Microtubule-dependent nucleokinesis constitutes an evolutionarily conserved cellular program ensuring directional nucleus translocation by deployment of cytoplasmic dynein motors. However, whether microtubule-driven nucleokinesis can operate during cell migration through confinement remained so far unexplored. The zebrafish neural crest (NC) provides a unique in vivo system to address this question. NC cells are a highly migratory, multipotent precursors of the vertebrate peripheral nervous system, which traverse diverse environments along the anterior-posterior axis of the embryo. In the head, cranial neural crest (cNC) cells migrate through loosely organised tissues, whereas trunk neural crest (tNC) cells navigate narrowly confined tissue spaces. Here, we show that tNC cells engage a microtubule motor-driven program of nuclear deformation and translocation during confined migration in vivo. Under confinement, tNC cells reorganize their microtubules from a perinuclear meshwork into a polarized, centrosome-associated bundle positioned ahead of the nucleus. Laser ablation reveals that this microtubule array actively pulls and deforms the nucleus. Using pharmacological and genetic approaches, we discover that dynein-dependent pulling forces enable nucleus translocation through confinement. Strikingly, this process occurs independently of Rho/ROCK/myosin II-mediated contractility, revealing a novel neuronal-like mode of nucleokinesis operating in confined tissue environments. Together, our findings uncover a conserved nuclear translocation program deployed during neural crest migration and suggest that microtubule-based nucleokinesis represents a fundamental strategy for navigating tissue confinement in vivo during both central and peripheral nervous system development.

cell biology↗

bioRxiv2024

Paired and solitary ionocytes in the zebrafish olfactory epithelium

The sense of smell is generated by electrical currents that are influenced by the concentration of ions in olfactory sensory neurons and mucus. In contrast to the extensive morphological and molecular characterization of sensory neurons, there has been little description of the cells that control ion concentrations in the zebrafish olfactory system. Here, we report the molecular and ultrastructural characterization of zebrafish olfactory ionocytes. Transcriptome analysis suggests that the zebrafish olfactory epithelium contains at least three different ionocyte types, which resemble Na+/K+-ATPase-rich (NaR), H+-ATPase-rich (HR), and Na+/Cl- cotransporter (NCC) cells, responsible for calcium, pH, and chloride regulation, respectively, in the zebrafish skin. In the olfactory epithelium, NaR-like and HR-like ionocytes are usually adjacent to one another, whereas NCC-like cells are usually solitary. The distinct subtypes are differentially distributed: NaR-like/HR-like cell pairs are found broadly within the olfactory epithelium, whereas NCC-like cells reside within the peripheral non-sensory multiciliated cell zone. Comparison of gene expression and serial-section electron microscopy analysis indicates that the NaR-like cells wrap around the HR-like cells and are connected to them by shallow tight junctions. The development of olfactory ionocyte subtypes is also differentially regulated, as pharmacological Notch inhibition leads to a loss of NaR-like and HR-like cells, but does not affect NCC-like ionocyte number. These results provide a molecular and anatomical characterization of olfactory ionocytes in a stenohaline freshwater teleost. The paired ionocytes suggest that both transcellular and paracellular transport regulate ion concentrations in the olfactory epithelium, while the solitary ionocytes may enable independent regulation of ciliary beating.

neuroscience↗

bioRxiv2021

Bmper is required for morphogenesis of the anterior and posterior semicircular canal ducts in the developing zebrafish inner ear

BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator of BMP signalling with both pro- and anti-BMP roles in different developmental contexts. The inner ears of bmper mutant embryos develop with truncations of the anterior and posterior semicircular canal ducts. To image the developing ear in live embryos, we have exploited a new transgenic line, Tg(smad6b:EGFP), which exhibits strong GFP expression in the otic epithelium. Morphometric analysis indicates defects in the bmper mutant ear from early stages of semicircular canal formation, correlating with a specific reduction in BMP signalling activity and specific loss of dlx5a expression in dorsal otic epithelium. Subsequent changes to cell shape occur at the truncation site and the dorsolateral septum. The bmper mutations that we describe are adult viable; truncation of the anterior and posterior semicircular canal ducts persists into adulthood. Our results argue against a major role for Bmper in specification of the pre-placodal region, induction of the otic placode, or development of the neural crest, processes in which Bmper function has previously been implicated. Instead, we conclude that a key requirement for Bmper function in the zebrafish is to promote BMP signalling during patterning and morphogenesis of the semicircular canal system.

developmental biology↗

bioRxiv2021

Origami: Single-cell oriented 3D shape dynamics of folding epithelia from fluorescence microscopy images

A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets aided by spatio-temporal cell shape changes at the cellular-level. Studying cell shape dynamics and polarised processes that underpin them, requires orienting cells within the epithelial sheet. In epithelia with highly curved surfaces, assigning cell orientation can be difficult to automate in silico. We present Origami, a MATLAB-based image analysis pipeline to compute oriented cell shape-features. Our automated method accurately computed cell orientation in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia.

developmental biology↗

bioRxiv2020

Olfactory rod cells: a rare cell type in the larval zebrafish olfactory epithelium with an actin-rich apical projection

We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending about 10 m from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. The cells also differ from secondary sensory cells such as hair cells of the inner ear or lateral line, or sensory cells in the taste bud, as they are not associated with established synaptic terminals. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium.

developmental biology↗

bioRxiv2019

Identification of compounds that rescue otic and myelination defects in the zebrafish adgrg6 (gpr126) mutant

Adgrg6 (Gpr126) is an adhesion class G protein-coupled receptor with a conserved role in myelination of the peripheral nervous system. In the zebrafish, mutation of adgrg6 also results in defects in the inner ear: otic tissue fails to down-regulate versican gene expression and morphogenesis is disrupted. We have designed a whole-animal screen that tests for rescue of both up- and down-regulated gene expression in mutant embryos, together with analysis of weak and strong alleles. From a screen of 3120 structurally diverse compounds, we have identified 68 that reduce versican b expression in the adgrg6 mutant ear, 41 of which also restore myelin basic protein gene expression in Schwann cells of mutant embryos. Nineteen compounds unable to rescue a strong adgrg6 allele provide candidates for molecules that interact directly with the Adgrg6 receptor. Our pipeline provides a powerful approach for identifying compounds that modulate GPCR activity, with potential impact for future drug design.

developmental biology↗

bioRxiv2018

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, but neither loss nor gain of hmx3a function was sufficient to generate ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

developmental biology↗