Quantifying spatiotemporal gradient formation in early Caenorhabditis elegans embryos with lightsheet microscopy

Abstract

Major steps in embryonic development, e.g. body axes formation or asymmetric cell divisions, rely on symmetry-breaking events and gradient formation. Using three-dimensional time-resolved lightsheet microscopy, we have studied a prototypical example for self-organized gradient formation in the model organism Caenorhabditis elegans. In particular, we have monitored in detail the formation of a concentration and mobility gradient of PIE-1 proteins as well as the partitioning behavior of vital organelles prior to the first, asymmetric cell division. Our data confirm the emergence of a concentration gradient of PIE-1 along the embryo’s anterior–posterior (AP) axis but they also reveal a previously unseen depletion zone near to the cell cortex that is not present for MEX-5 proteins. Time-resolved spatial diffusion maps, acquired with SPIM-FCS, highlight the successive emergence of a mobility gradient of PIE-1 along the AP axis and suggest an almost linear decrease of fast diffusing PIE-1 proteins along the AP axis. Quantifying the subordinated dissemination of vital organelles prior to the first cell division, i.e. gradient formation on larger length scales, we find a significant asymmetry in the partitioning of the endoplasmic reticulum and mitochondria to the anterior and posterior part of the cell, respectively. Altogether, our spatiotemporally resolved data indicate that current one-dimensional model descriptions for gradient formation during C. elegans embryogenesis, i.e. a mere projection to the AP axis, might need an extension to a full three-dimensional description. Our data also advocate the use of lightsheet microscopy techniques to capture the actual three-dimensional nature of embryonic self-organization events.

Extracting, quantifying, and comparing dynamical and biomechanical properties of living matter through single particle tracking

“…Subsequent patterns (e.g. concentration and mobility gradients for MEX-5 and PIE-1 proteins 93,95,204 ) are triggered by this initial patterning, including even the condensation of membraneless organelles into so-called p-granules in the posterior daughter cell. 205,206 Taken together, it appears fair to say that vital selforganisation phenomena in living matter rely crucially on transport processes: unambiguously revealing and naming them is of the utmost importance to obtain a quantitative understanding of living matter.…”

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults

“…Similarly, the C. elegans embryo facilitated showing the effectiveness of fourdimensional (4D) live imaging with the Dual Inverted Selective Plane Illumination Microscope (diSPIM) system (Kumar et al, 2014). LSFM has also advanced our understanding C. elegans embryogenesis (Chardès et al, 2014;Duncan et al, 2019), such as helping to reveal how the rigid egg shell contributes to asymmetrical cell divisions (Fickentscher and Weiss 2017), how circuit structures are organized within the nerve ring (the C. elegans brain) (Moyle et al, 2021), and how the zinc finger protein PIE-1 concentration gradient is established and maintained in the zygote (Benelli et al, 2020).…”

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults

“…Similarly, the C. elegans embryo facilitated showing the effectiveness of four-dimensional (4D) live imaging with the Dual Inverted Selective Plane Illumination Microscope (diSPIM) system (Kumar et al 2014). LSFM has also advanced our understanding C. elegans embryogenesis (Chardès et al 2014; Duncan et al 2019), such as helping to reveal how the rigid egg shell contributes to asymmetrical cell divisions (Fickentscher and Weiss 2017), how circuit structures are organized within the nerve ring (the C. elegans brain) (Moyle et al 2021), and how the zinc finger protein PIE-1 concentration gradient is established and maintained in the zygote (Benelli et al 2020).…”