发育生物学


现刊
往期刊物
0 Q&A 1054 Views Mar 20, 2022

Analysis of DNA double strand breaks (DSBs) is important for understanding dyshomeostasis within the nucleus, impaired DNA repair mechanisms, and cell death. In the C. elegans germline, DSBs are important indicators of all three above-mentioned conditions. Although multiple methods exist to assess apoptosis in the germline of C. elegans, direct assessment of DSBs without the need for a reporter allele or protein-specific antibody is useful. As such, unbiased immunofluorescent approaches can be favorable. This protocol details a method for using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to assess DNA DSBs in dissected C. elegans germlines. Germlines are co-labeled with DAPI to allow for easy assessment of DNA DSBs. This approach allows for qualitative or quantitative measures of DNA DSBs.


Graphic abstract:



Schematic for TUNEL labeling of C. elegans germlines.


0 Q&A 1253 Views Oct 20, 2021

Calcium ions trigger many cellular events, including the release of neurotransmitters at the synaptic terminal and excitotoxic cell death. Recently, we have discovered that a transient increase in the level of cytoplasmic Ca2+ triggers the exposure of phosphatidylserine (PS) on the surfaces of necrotic cells in the nematode Caenorhabditis elegans. PS serves as an “eat me” signal that attracts engulfing cells to engulf and degrade necrotic cells. During the above study, we developed a microscopic imaging protocol for real-time monitoring the levels of cytoplasmic Ca2+ and cell surface PS in Caenorhabditis elegans touch neurons. Previously, Ca2+ dynamics was monitored in neurons in Caenorhabditis elegans larvae in time periods ranging from milliseconds to seconds. Methods for monitoring Ca2+ dynamics for a relatively long period of time during embryonic development were not available, let alone for simultaneous monitoring Ca2+ and PS dynamics. The protocol reported here utilizes a deconvolution imaging system with an optimized experimental setting that reduces photo-damage and allows the proper development of embryos during the real-time imaging process. This protocol enables the simultaneous measurement of cytosolic Ca2+ and cell surface PS levels in necrotic touch neurons during embryonic development in a period longer than six hours. Our method provides an easy and sensitive approach to perform long-time Ca2+ and PS recording in living animals, simultaneously or individually. This protocol can be applied to study various cellular and developmental events that involve the dynamic regulation of Ca2+ and/or PS.

0 Q&A 3489 Views Feb 20, 2021

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency.


RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatio-temporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.

0 Q&A 8282 Views Mar 20, 2018
Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for measuring noncanonical Wnt signaling activity. We recently described the Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a post-transcriptional regulatory event in noncanonical Wnt signaling. In this protocol, we describe the generation of the stable GFP-Kif26b reporter cell line and a quantitative reporter assay for detecting and measuring Wnt5a signaling activities in live cells via flow cytometry.
1 Q&A 10944 Views May 5, 2013
During the development of the C. elegans hermaphrodite, 131 of the 1090 somatic cells generated undergo programmed cell death, among which 113 die during embryogenesis starting from 200-cell stage. The apoptotic cells (also called “cell corpses”) appear as highly refractile button-like objects and are easily identified using differential interference contrast (DIC) optics (Robertson et al., 1982).