Primary Ciliary Signaling in the Skin—Contribution to Wound Healing and Scarring

We studied cell recruitment following optic tectum (OT) injury in zebrafish (Danio rerio), which has a remarkable ability to regenerate many of its organs, including the brain. The OT is the largest dorsal layered structure in the zebrafish brain. In juveniles, it is an ideal structure for imaging and dissection. We investigated the recruited cells within the juvenile OT during regeneration in a Pdgfrβ-Gal4:UAS-EGFP line in which pericytes, vascular, circulating, and meningeal cells are labeled, together with neurons and progenitors. We first performed high-resolution confocal microscopy and single-cell RNA-sequencing (scRNAseq) on EGFP-positive cells. We then tested three types of injury with very different outcomes (needle (mean depth in the OT of 200 µm); deep-laser (depth: 100 to 200 µm depth); surface-laser (depth: 0 to 100 µm)). Laser had the additional advantage of better mimicking of ischemic cerebral accidents. No massive recruitment of EGFP-positive cells was observed following laser injury deep in the OT. This type of injury does not perturb the meninx/brain–blood barrier (BBB). We also performed laser injuries at the surface of the OT, which in contrast create a breach in the meninges. Surprisingly, one day after such injury, we observed the migration to the injury site of various EGFP-positive cell types at the surface of the OT. The migrating cells included midline roof cells, which activated the PI3K-AKT pathway; fibroblast-like cells expressing numerous collagen genes and most prominently in 3D imaging; and a large number of arachnoid cells that probably migrate to the injury site through the activation of cilia motility genes, most likely being direct targets of the FOXJ1a gene. This study, combining high-content imaging and scRNAseq in physiological and pathological conditions, sheds light on meninges repair mechanisms in zebrafish that probably also operate in mammalian meninges.

Section: Ara Boosts the Ciliary Machinerysupporting

confidence: 67%

Molecular and Cellular Analysis of the Repair of Zebrafish Optic Tectum Meninges Following Laser Injury

Banerjee

Joly

Jouneau

et al. 2022

“…Upon ligand binding, the activated receptors are translocated from the ciliary tip toward the base of the cilium, where SMAD transcription factors are activated [ 10 ]. The primary cilium, therefore, plays a key role in the formation of myofibroblasts, first through the uptake of TGF-β1 receptors and second through the regulated process of cilium assembly and disassembly [ 1 , 11 ]. That is, myofibroblastic transformation is manifested by the initial growth of a primary cilium, which is crucial for the progression of trans-differentiation, followed by the complete loss of the primary cilium when the myofibroblastic phenotype is reached [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

Heterogeneity of the NIH3T3 Fibroblast Cell Line

Rahimi

Cai

Hoyer‐Fender

2022

The embryonic mouse fibroblast cell line NIH3T3 is widely used in life science research, including the study of cell cycle control and primary cilia. Fibroblasts are the most important cell type in connective tissue, as they produce components of the extracellular matrix and determine tissue architecture. However, they are very heterogeneous and consist of subtypes specific to their organ of residence, among others. The NIH3T3 cell line was derived from whole mouse embryos that developed to pre-birth and is therefore most likely composed of different fibroblast subtypes. Furthermore, prolonged proliferation may have influenced their cellular composition. A heterogeneous cell population is unsuitable for any sophisticated research project. We found that the proportion of ciliated cells in the total NIH3T3 cell population was highly variable and asked whether this was a consequence of cellular heterogeneity and what molecular signatures were associated with it. We have established sub-cell lines by clonal expansion of single cells and characterized them morphologically and molecularly. Eventually, a myofibroblast-like and a fibroblast-like cell line were generated that differ in ciliation and proliferation. These homogeneous cell lines are valuable for a more detailed study of their molecular signatures, not least to uncover further the molecular pathways that contribute to the formation of the primary cilium.

“…However, injury-stimulated migration of fibroblasts and their wound-healing capacity critically depend on a functional primary cilium [ 2 ]. In the initial phase of wound healing, activated platelets trigger the release of growth factors, such as platelet-derived growth factor (PDGF), that signal through the tyrosine kinase receptor PDGFRα located in the primary cilium membrane of fibroblasts causing re-entry into the cell cycle from quiescence and proliferation [ 3 ].…”

Section: Introductionmentioning

confidence: 99%

Expression of α-Tubulin Acetyltransferase 1 and Tubulin Acetylation as Selective Forces in Cell Competition

Rahimi

Cai

Kılıҫ

et al. 2021

The wound healing response of fibroblasts critically depends on the primary cilium, a sensory organelle protruding into the environment and comprising a stable axonemal structure. A characteristic marker for primary cilia is acetylation of axonemal tubulin. Although formation of primary cilia is under cell cycle control, the environmental cues affecting ciliation are not fully understood. Our purpose was, therefore, to study the impact of culture conditions on cilia formation in NIH3T3 fibroblasts. We quantified ciliation in different NIH3T3 sub-cell lines and culture conditions by immunodetection of primary cilia and counting. Quantitative Western blotting, qRT-PCR, and proliferation assays completed our investigation. We observed large differences between NIH3T3 sub-cell lines in their ability to generate acetylated primary cilia that correlated with cytoplasmic tubulin acetylation. We found no increased activity of the major tubulin deacetylase, HDAC6, but instead reduced expression of the α-tubulin acetyltransferase 1 (Atat1) as being causative. Our observations demonstrate that cells with reduced expression of Atat1 and tubulin acetylation proliferate faster, eventually displacing all other cells in the population. Expression of Atat1 and tubulin acetylation are therefore selective forces in cell competition.