Egg-laying behavior of the Caenorhabditis elegans has been extensively used in several research studies, and it has been served as an effective behavioral assay for a number of fundamental processes in phenotypic identification, neuronal cell biology and signal transduction1, etc. of the. Although laying an egg is the simplest mechanism that all the nematodes are capable of, specific motor functions associated with the egg-laying are among the first C. elegans behaviors to be subjected to extensive genetic analysis12,13,14,15. C.elegans are hermaphrodites and self-fertile in nature. In our research, diverse egg-laying patterns associated with different mutant strains are used to identify the distinct phenotypic trait associated with its fecundity defects1. Fecundity is the number of viable offspring produced by an individual. In nematode worms, this is a useful metric for measuring the effects of genetic variants or various treatments.
Why do we choose lmn-1?
lmn-1 (nuclear LaMiN) refers to human LMNA ortholog strain (laminA/C) which is helpful in the binding activity of both histones and proteins. Mutations in the human LMNA A/C gene are responsible for several diseases, such as Emery-Dreifuss muscular dystrophy, familial partial lipodystrophy, limb-girdle muscular dystrophy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria syndrome7,2. Several mutant residues of human LMNA A/C gene are also conserved in the lamin genes of the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster8, and the sequence conservation between Ce-lamin and human lamin A leads to several respective laminopathy diseases10, allowing testing the disease phenotypes in C. elegans9. Several transgenic C. elegans expressing disease-linked lamin mutations were generated8 at InVivo Biosystems to better understand the correlation of mutations and their associated human diseases.
Purpose of this study
There are several phenotypes associated with the lmn-1 gene, such as embryonic lethal, larval lethal, reduced brood size, shortened life span, sterile progeny, locomotion variant, and several others as mentioned in wormbase.org. We developed an assay, in which we could use the viable homozygous worm variants employing its reproductive patterns to find the associated phenotype. Moreover, this egg-viability assay upon different humanized lmn-1 worm variants is based on the hypothesis that the benign and malignant strains have a fecundity defect in terms of the total eggs laid, unhatched eggs, viable progeny, and 2-day brood size to the N2 control strains. The main purpose of this assay is to find the potential humanized LMNA worm variants that have a fecundity defect. For uniformity, we are going to start this assay with the synchronized L4 hermaphrodites. In order to do that, we are going to follow a simple bleaching and synchronization protocol which we will discuss as follows.
Synchronizing worms by bleaching
We are employing this bleaching procedure to isolate eggs from the bodies of gravid hermaphrodites. The main reason why we employ this technique is to get all the eggs hatched at the same time across different worm variants and made to synchronize them at L4 stages which could then be useful in minimizing the errors in data recordings and also providing uniformity throughout the experiment. We start this procedure with the well-fed L4 adult worms with OP50 which are later subjected to bleaching treatment. Bleaching the worms involves several wash steps, vortexing it, plating the unhatched eggs in a cell culture plate (figure 1), and subjected to overnight incubation which can later be plated onto a fresh plate (figure 2). As a result, we can achieve synchronized worms which is a crucial step in performing this assay.
Figure 1: The worm strains set up for the NEST 12 cell culture plate incubator upon the shaker at 20°C. The yellow tape holding the coverings open is required for the constant airflow while shaking at overnight incubation.
Figure 2: Above images show the hatching pattern of the eggs before and after overnight incubation. A) Left image shows the unhatched eggs which are waiting for its overnight incubation period at 20°C. B) Right image shows the L1s generated from the eggs after overnight incubation and ready for plating into the OP50 plate.
Initial Project Design - Version 1.0
To calculate the egg-laying and brood size, we designed the initial research workflow by gathering information across published research articles mentioning the egg - viability assay procedures11,1. As an initial test run, we used one of our humanized pathogenic worm variants, and used N2 as a control strain.
Firstly, we synchronized both the mutant strains and wild-type worms as per the synchronization bleaching protocol. We did five replicates per condition and scored the dead embryos and larvae in the interval of 24 hours and 48 hours respectively. Data collection was performed until the parent worm in all the replicates were dead and the results were mentioned below in graphs 1 and 2. Based on these results, we noticed a significant difference in the total progeny and total-eggs laid between the two conditions. The results gave solid data mentioning lower egg-viability and brood size relatively respectively compared to the wild type worms. The overall process is pretty laborious and time-consuming to perform this experiment across multiple worm strains. As an end result, we designed a new project design that will shorten the time frame and increase data efficiency. The general workflow graphic is shown below in figure 3.
Figure 3: The overall assay process that was used in calculating the total number of larvae and dead embryos across different stages.
Graph 1 (left): The data plots reveal the total progeny between wildtype and mutant variants proving the humanized worm strains have a reduced progeny compared to wild types. Graph 2 (right): The data plots reveal the total eggs laid across different days were significantly lesser in number in the mutant ones than compared to wild type ones.
Egg viability assay - Version 2.0
To better assess the egg viability patterns, we redesigned this assay in order to achieve better results in an efficient manner. The purpose of this specific assay is to calculate the total number of eggs laid, unhatched eggs, and viable progeny. As a standard, we used 35mm plates seeded with OP50. For this assay, we transferred 10 synchronized worms onto three plates per condition (For example, N2, LMNA humanized worm variants, etc.). For maximizing the assay efficiency, we did three replicates for each condition particular to the different worm strain. We used N2 as a control strain. For data collection, we recorded the number of the total eggs laid, unhatched eggs, and viable progeny at the end of 24 hours by counting the number of unhatched eggs and L1 stage worms. Counting the eggs and L1s is an error-prone process. To minimize errors, you could use an alternative technique to count the number of eggs and L1s, such as using the WormLab imaginary to capture the magnified plate image.
Figure 4. Egg viability assay graphic mentioned the steps involved were generated by Lucidchart diagrams
2-day brood size assay version 2.0
To assess the 2-day brood size for different worm variants. For this condition, we did five replicates for all the worm variants. In this 2-day brood size assay, we also used N2 strains as a control strain. We transferred one synchronized L4 hermaphrodite for each benign and malignant variant into a 35mm plate seeded with OP50. After incubation for 48 hours at 20°C, we counted the worms at stages from L2 - L4 stages which were scored for uniformity across the experiment. For more precise counting, we counted twice in the interval of 24 hours each, one after 24 hours and the other after 48 hours.
Figure 5. 2-day brood assay graphic mentioned the steps involved were generated by Lucidchart diagrams
In our experiment, we used six different lmna-1 worm strains, three were benign and three were pathogenic. All different worm strains were subjected to the same conditions as mentioned above. In the egg-viability assay, two among three pathogenic worm strains show a significantly higher number in the total number of eggs laid, unhatched eggs, and viable progeny than compared to a significantly lower number in the benign strains. In the 2-day brood size assay, all three benign worm strains were shown in increased brood size numbers compared to the pathogenic strains. The data reveals a clear distinction in the data plots between benign and pathogenic humanized worm strains.
In our lab experiment, We collected the data for N2 wild type worms for a 2-day brood size assay. For more precise counting, we counted twice in the interval of 24 hours each, one after 24 hours and the other after 48 hours. The results are tabulated below:
Figure 6. Total number of N2 from five replicates at the end of Day 1 (24 hrs) - 243. Total number of N2 from five replicates at the end of Day 2 (48 hrs) - 687
As a result of this egg-viability analysis, we can conclude that benign and pathogenic lmna-1 worm strains show distinct variability between the number of eggs laid, unhatched eggs, viable progeny, and 2-day brood size. Benign variants show significantly reduced numbers of eggs laying, and unhatched eggs compared to the pathogenic variants. Moreover, benign strains show increased brood size numbers across all three different variants compared to the pathogenic variants. By using this assay, we are able to identify the specific phenotype associated with the nematodes by relatively evaluating its results.
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