STAR Protocols︱A protocol for fluorescence in vivo imaging of postembryonic development of C. elegans

Written by ︱ Li Tingting and editor in charge ︱ Wang Sizhen Caenorhabditis elegans (Caenorhabditis elegans) is a non-parasitic nematode that feeds on bacteria
.

In the 1960s, Sydney Brenner conducted research on developmental biology and neuroscience in C.
elegans, established a system for studying the development of multicellular organisms using C.
elegans as a model, and created a new era of C.
elegans model organism research
.

So far, researchers have made outstanding contributions to various aspects of research using C.
elegans, such as nervous system development, autophagy and apoptosis, human gene function and disease, and aging
.

 Postembryonic development is an inevitable process for multicellular organisms
.

Real-time imaging of dynamic events during nematode development can provide detailed information about the developmental process of organisms
.

For example, to analyze whether the loss of a cell is caused by cell division or apoptosis regulated by signaling pathways, to analyze which defects in the growth, pruning or anchoring of neuronal processes are caused by the reduction of neuronal processes, and to observe through live imaging in vivo Developmental processes are differentiated, thereby pointing the way for further genetic analysis
.

 On April 19, 2022, Zou Yan's research group from the School of Life Science and Technology, ShanghaiTech University published a research proposal paper titled "Live imaging of postembryonic developmental processes in C.
elegans" in STAR Protocols, a magazine under Cell.
Fluorescence real-time microscopy imaging of neuronal migration and dendritic growth during postembryonic development in C.
elegans
.

Zou Yan's research team used this technology to study in C.
elegans and discovered the important factor P5A ATPase that regulates neuron migration and dendritic growth.
The research work has been published in the journal Cell Reports[1,2]
.

The protocol first synchronizes the nematodes to obtain larvae of a specific developmental stage, then the larvae are anesthetized and fixed on agar slides, and the gaps in the coverslip and slide are sealed with a mixture of paraffin and petrolatum (Figure 1)
.

The agar slides containing nematodes were placed under a confocal microscope for long-term photography, and the acquired image data were processed and analyzed with the software ImageJ
.

Figure 1 Preparation of agar sheets and fixation of nematodes (Source: Li T, et al.
, STAR Protoc, 2022) The results of this study demonstrate asymmetric cell division, cell migration, and apoptosis in Q cell development
.

The Q cell divides to obtain daughter cells Qa and Qp, and then undergoes one and two cell divisions, respectively, and finally obtains three neurons (Q.
ap, Q.
paa, and Q.
pap) and two apoptotic cells (Q.
aa) and Q.
pp)
.

The backward migration of QL cells and their progeny eventually produces neurons: PQR, SDQL, and PVM, while the forward migration of QR cells and their progeny eventually produces neurons: AQR, SDQR, and AVM (Fig.
2)
.

Figure 2 Schematic diagram of the Q cell lineage and the migration process of its progeny cells (Image source: Li T, et al.
, STAR Protoc, 2022) This protocol also studies the growth and development of neuronal dendrites
.

PVD neurons are born at the L2 larval stage, the cell body is located on the posterior side of the body, and grows 1° dendrites along the anterior-posterior axis of the body, 2° perpendicular to the 1° dendrite, and 3° along the 2° horizontal direction.
There are gaps between adjacent dendrites and 4° dendrites grow in the vertical direction of the 3° dendrites, and the dendrite development of PVD neurons is completed at the late L4 stage (Fig.
3)
.

Figure 3.
Dendritic development of nematode PVD neurons (Credit: Li T, et al.
, STAR Protoc, 2022) Figure 4 Schematic diagram of the fluorescence intravital imaging scheme of postembryonic neuron development in nematodes (Credit: Li T, et al.
al.
, STAR Protoc, 2022) Article Conclusion and Discussion, Inspiration and Prospect This experimental protocol describes the complete process from preparation of nematodes to fluorescence real-time imaging (Fig.
4), and proposes the details and problems that need attention in the experiment and solutions so that researchers can refer to and quickly master the method
.

The researchers used this method to study cell division, migration, and apoptosis in C.
elegans development, as well as neuronal process growth, guidance, localization, and regeneration after injury, providing detailed information on cellular dynamic events in post-embryonic development of C.
elegans.

.

In addition, the authors also pointed out the limitations of this protocol: 1.
This protocol is not suitable for real-time imaging in the late embryonic development, because the nematodes cannot be fixed with anesthesia under the protection of the eggshell, and the nematodes are in the eggshell in the late embryonic development.
Twisting affects imaging
.

2.
The physiological function of C.
elegans is affected after the real-time imaging time exceeds 4 hours.
Some researchers report that the cell division and migration speed of C.
elegans are slowed down after imaging for more than 4 hours [3]
.

Link to the original text: https://doi.
org/10.
1016/j.
xpro.
2022.
101336 Li Tingting (first from the right in the front row) and Zou Yan (third from the left in the front row)
.

(Photo courtesy of Zou Yan's Laboratory, ShanghaiTech University) Li Tingting, a doctoral student at the School of Life Science and Technology, ShanghaiTech University, is the first author of the paper, and Researcher Zou Yan is the corresponding author
.

This work was supported by the National Natural Science Foundation of China (No.
32170974) and the Shanghai Natural Science Foundation (No.
22ZR1442400), and thanks to the Molecular Imaging Platform of the School of Life Sciences, ShanghaiTech University for their strong support
.

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Li, T.
, et al.
, P5A ATPase controls ER translocation of Wnt in neuronal migration.
Cell Reports, 2021.
37(4): p.
109901.
2.
Feng, Z.
, et al.
, CATP-8/P5A ATPase Regulates ER Processing of the DMA-1 Receptor for Dendritic Branching.
Cell Reports, 2020.
32(10):p.
108101.
3.
Chai, Y.
, et al.
, Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development.
Nat Protoc, 2012.
7(12): p.
2090-102.