Biology subjects

Zolessi, F. R.

Publications and source records attributed to Zolessi, F. R..

6 recordsLinked to original sources

bioRxiv2025

Mechanical conditions preventing live cell extrusion during primary neurulation in amniotes

During primary neurulation in amniote embryos, the neural plate gives rise to the neural tube in a process requiring the coordination of forces at different scales throughout a geometrically complex tissue. The ways in which this process fails inform us of the complex mechanical conditions required for its correct completion. Previous results showed that the functional disruption of MARCKS, a protein which simultaneously interacts with the plasma membrane and actin filaments, resulted in neural tube closure defects with apical cell extrusion. Here, we demonstrate that this is an example of "live cell extrusion", wherein extruded cells are not undergoing apoptosis. This suggests that extrusion in this case might be due to a mechanical instability in the neural plate. Using an expanded energy-based vertex model of pseudostratified epithelia we then show that extrusion may be elicited by a reduction in the relative surface tension of apical and basal interfaces with respect to cell-cell interfaces. Finally, by considering a continuum description of a simplified epithelium we derive an approximate quantitative threshold for single-layered epithelial stability in the form of a power law relating cell density to the relative value of interfacial surface tensions. Our work serves to explain an example of how alterations in polarization and forces at the single-cell level can produce tissue-scale instabilities which not only greatly alter its morphology but can also ultimately lead to severe developmental defects. HighlightsO_LIPMA treatment induces live apical cell extrusion in the chick embryo neural plate C_LIO_LIOur energy-based vertex model mechanically simulates a pseudostratified epithelium C_LIO_LIHigher apical/basal compared to lateral surface tension leads to epithelial stability C_LIO_LILower apical/basal surface tension leads to epithelial instability and cell extrusion C_LIO_LIA power law between cell density and surface tension defines the stability threshold C_LI

developmental biology↗

bioRxiv2023

Development and characteristics of photoreceptor actin-based apical processes in zebrafish (Danio rerio)

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). While CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the processes remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, while heat shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically-oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Muller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development, and surrounding environment of photoreceptor microvilli in the zebrafish retina.

cell biology↗

bioRxiv2023

Spectral phasor plot for hyperspectral imaging of acridine orange photophysics within the cell

Acridine Orange (AO) exhibits complex photophysical properties that have been utilized to study its interactions with RNA and DNA. Traditionally, AO has been used to stain cells, distinguishing nucleus and cytoplasm based on differences in DNA and RNA composition. Its labeling behavior varies depending on factors such as concentration and pH. AO fluorescence is rich and complex; therefore, acquiring the full spectrum or the fluorescence lifetime is beneficial for understanding its intricate photophysics. Nevertheless, handling 4D datasets can be challenging and often relies on sophisticated or highly parameterized models for their analysis. Model-free approaches, such as spectral phasor (SP) analysis for hyperspectral imaging (HSI), are well suited to navigating these analytical challenges. Using SPs principles of linear combination and reciprocity, we identified spectral fingerprints of RNA and DNA in in vitro experiments that remain consistent in both live and fixed cells. Interestingly, SP analysis of AO fluorescence in live cells revealed a third spectral component at 640 nm. The linear properties of SP analysis enabled the quantification of AO bound to RNA, DNA, and the third component. Co-labeling with AO and LysoTracker suggested that some of these subcellular structures are associated with acidic compartments. Moreover, SP analysis distinguished AO self-interactions from its RNA binding in cytoplasmic compartments. This study establishes SP analysis as a robust tool for hyperspectral imaging of AO, thereby enhancing our understanding of its intricate photophysics and unlocking new opportunities for advanced in vivo cell imaging applications.

biophysics↗

bioRxiv2022

Zebrafish Slit2 and Slit3 act together to regulate retinal axon crossing at the midline

Slit-Robo signaling regulates midline crossing of commissural axons in different systems. In the zebrafish, all retinofugal axons cross at the optic chiasm to innervate the contralateral tectum. Here, the mutant for the Robo2 receptor presents severe axon guidance defects, which were not completely reproduced in a Slit2 ligand null mutant. Since slit3 is also expressed around this area at the stage of axon crossing, we decided to analyze the possibility that it collaborates with Slit2 in this process. We found that the disruption of slit3 expression by sgRNA-Cas9 injection caused similar, albeit slightly milder, defects than those of the slit2 mutant, while the same treatment in the slit2-/- background caused much more severe defects, comparable to those observed in robo2 mutants. Tracking analysis of in vivo time-lapse experiments indicated differential but complementary functions of these secreted factors in the correction of axon turn errors around the optic chiasm. Interestingly, RT-qPCR analysis showed a mild increase in slit2 expression in slit3 deficient embryos, but not the opposite. Our observations support the previously proposed "repulsive channel" model for Slit-Robo action at the optic chiasm, with both Slits acting in different manners, most probably relating to their different spatial expression patterns.

developmental biology↗

bioRxiv2020

Slit2 is essential for correct retinal ganglion cell axon fasciculation and midline crossing in the zebrafish

Slit-Robo signaling has been implicated in regulating several steps of retinal ganglion cell axon guidance, with a central role assigned to Slit2. We report here the phenotypical characterization of a CRISPR-Cas9-generated zebrafish null mutant for this gene, along with a detailed analysis of its expression pattern by WM-FISH. All evident defects in the optic axons in slit2-/- mutants were detected outside the retina, coincident with the major sites of expression at the ventral forebrain, around the developing optic nerve and anterior to the optic chiasm/proximal tract. Anterograde axon tracing experiments in zygotic and maternal-zygotic mutants, as well as morphants, showed the occurrence of axon sorting defects, which appeared mild at the optic nerve level, but more severe in the optic chiasm and the proximal tract. A remarkable sorting defect was the usual splitting of one of the optic nerves in two branches that surrounded the contralateral nerve at the chiasm. Although all axons eventually crossed the midline, the retinotopic order appeared lost at the proximal optic tract, to eventually correct distally. Time-lapse analysis demonstrated the sporadic occurrence of axon misrouting at the chiasm level, which could be responsible for the sorting errors. Our results support previous evidence of a channeling role for Slit molecules in retinal ganglion cell axons at the optic nerve, in addition to a function in the segregation of axons coming from each nerve and from different retinal regions at the medio-ventral area of the forebrain.

developmental biology↗

bioRxiv2020

Photoreceptor progenitor dynamics in the zebrafish embryo retina and its modulation by primary cilia and N-cadherin

BackgroundPhotoreceptors of the vertebrate neural retina are originated from the neuroepithelium, and like other neurons, must undergo cell body translocation and polarity transitions to acquire their final functional morphology, which includes features of neuronal and epithelial cells. MethodsWe analyzed this process in detail on zebrafish embryos using in vivo confocal microscopy and electron microscopy. Photoreceptor progenitors were labeled by the transgenic expression of EGFP under the regulation of the photoreceptor-specific promoter crx, and genes of interest were knocked-down using morpholino oligomers. ResultsPhotoreceptor progenitors detached from the basal retina at pre-mitotic stages, rapidly retracting a short basal process as the cell body translocated apically. They remained at an apical position indefinitely to form the outer nuclear layer (ONL), initially extending and retracting highly dynamic neurite-like processes, tangential to the apical surface. Many photoreceptor progenitors presented a short apical primary cilium. The number and length of these cilia was gradually reduced until nearly disappearing around 60 hpf. Their disruption by knocking-down IFT88 and Elipsa caused a notorious defect on basal process retraction. Time-lapse analysis of N-cadherin knock-down, a treatment known to cause a severe disruption of the ONL, showed that the ectopic photoreceptor progenitors initially migrated in an apparent random manner, profusely extending cell processes, until they encountered other cells to establish cell rosettes in which they stayed acquiring the photoreceptor-like polarity. ConclusionAltogether, our observations indicate a complex regulation of photoreceptor progenitor dynamics to form the retinal ONL, previous to the post-mitotic maturation stages.

developmental biology↗