Phenotypic Analysis of Autism-associated mutations in C. elegans

Author: Judy Kim

Mentor: Dr. Paul Sternberg

Co-Mentor: Sandy Wong

Editors: Sherry Wang and Jonathan Chan

Introduction

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with a large genetic component. Among these rare variants that are related to ASD, missense mutations (a type of point mutation where a single nucleotide change results in a codon that codes for a different amino acid) make up nearly 30% of the rare variants.1 Because missense mutations are so abundant, they are usually difficult to study in most organisms; nearly five decades ago, however, biologist Sydney Brenner developed the nematode Caenorhabditis elegans as a genetic model for understanding developmental biology and neurobiology.2 C. elegans is a model organism to study mutations because it is easy to genetically manipulate, has a life cycle of three days, is transparetnt, a well-annotated genome, etc.4 Properly understanding missense mutations in C. elegans might help us better understand Autism’s mechanism and effects in humans.       

Patients with syndromes affected by BRAF mutations were often found to exhibit autistic features, such as social impairment and internalizing and externalizing problems, as measured by commonly used ASD-related diagnostic tools.5,6

Similarly, TRIO has been found that when expressed in neurons, these mutations produce a wide range of alternations in glutamatergic synapse function that is similar to those observed in current animals models of ASD. Among the human genome, there are two genes, BRAF and TRIO, which are well-known oncogenes (genes that can transfer into tumors). BRAF and TRIO were chosen because these are the human orthologs of lin-45 and unc-73, respectively.  BRAF codes for the B-raf protein, which is involved in sending signals inside the cells that are key in cell growth, was shown to be mutated in some human cancers.7 TRIO, coding for a guanidine nucleotide exchange factor, was frequently amplified and abundantly expressed in soft tissue sarcomas.8 Extensive research has focused on the cancer-inducing effects of these genes, but little is known about the genes’ neuronal functions. Molecular genetics research has been done with BRAF and TRIO on other animals such as rabbits,9 dogs,10 and mice.11 A previous study conducted on C. elegans indicated that there is interaction between lin-45 and unc-73 after using a RNA interference method in C. elegans to test approximately 65,000 pairs of genes for their ability to interact genetically.12

Chemosensory cues can lead to chemotaxis, rapid avoidance, and changes in movement; these behaviors are mainly regulated by chemosensory organs, which contain eleven pairs of chemosensory neurons.13 AWA and AWC are type types of olfactory neurons that sense volatile odors and is tested through chemotaxis to volatile chemoattractants in C. elegans. The lin-45 mutant is known to be phenotypically defective for these neurons during chemotaxis, meaning the worms cannot sense the odor.14 Olfactory signaling is initiated by interactions between odorants and olfactory receptors.

odr-10 was used as a positive control used because these mutants have a specific defect in chemotaxis to diacetyl, of several odorants detected by AWA olfactory neurons.14 Since there is little known about these genes and their impact on neuronal function, we wanted to investigate this function in C. elegans, specifically by comparing the established wild-type strain to the newly generated double mutant worms by genotyping and setting up multiple crosses.

These studies inform the various types of neuronal functions that preexist in these genes. A study that investigated the neuronal functions of lin-45 and unc-73 demonstrated that although the nervous and immune systems influence each other, the complex nature of these systems in mammals makes it difficult to determine how neuronal signaling influences the immune response.15 Another study also found that serotonergic chemosensory neurons modify the C. elegans immune response by regulating G-protein signaling in epithelial cells.15  Finally, we also know that unc-73, signals through nucleotide exchange factors such as RhoGEF-2 to regulate pharynx and vulva musculature and to modulate synaptic neurotransmission.16

In this study, we investigate whether diacetyl is an attractant to N2 wild-type and PS double mutant strains that were generated to ensure that the chemotaxis assay was working properly. The purpose of the chemotaxis assay is to investigate whether the worms are attracted to diacetyl. The odr-10(ky225) gene impair C. elegans so these mutants served as a chemotaxis to low concentrations of the odorant diacetyl.13 Our null hypothesis held that the odr-10(ky225) mean of the Region of Interest value is equal that of N2. After confirming the assay was working, we investigate whether diacetyl was an attractant to the double mutant strains like it was for N2 wild-type. The null hypothesis of the question was that the mean of the wild-type, mean of unc-73(sy898);lin-45 (sy875), mean of unc-73(sy896);lin-45 (sy875), and mean of unc-73(sy892);lin-45 (sy875) were all equal to each other. If our null hypothesis is void, then we know that there is statistical significance among the means.

Methods

The C. elegans used in this project were all synchronized at the L4 stage. After genotyping the F1(first filial) and F2 (second filial) crosses and finding new double mutant strains by identifying mutants that were homozygous for both unc-73 and lin-45, chemotaxis was performed to examine the worms’ attraction towards diacetyl and ethanol (Figure 1). Chemotaxis is the movement of an organism in a direction corresponding to a gradient of increasing or decreasing concentration of a particular substance such as diacetyl, an attractant that has been used consistently in previously studied literature.

Figure 1. Cross Overview. This diagram highlights the ideal F1 and F2 after each cross to ultimately generate a new double mutant strain.

Genotyping

The cross overview highlights the F1 and F2 crosses that were performed to find the new double mutant strains. In the first cross, we placed +/+; +/+ males in to a dish of +/+; lin-45/lin-45 hermaphrodites. From this cross, males were picked to use in the next cross. In the second cross, +/+; lin-45/+ males were crossed to unc-73/unc-73; +/+ hermaphrodites. After about three days, worms from the F1 progeny were then genotyped. For the F2 progeny, we repeated a similar process to find homozygous mutants in both lin-45 and unc-73 to generate new double mutant strains.

To find the double mutant strains, the F1 and F2 were genotyped as following:

Worms were lysed adding 25uL of proteinase K from stock (10mg/mL) to 100uL of lysis buffer for use in PCR. In worm lysis, the lysis buffer solution was first prepared. Lysed worms were placed in wells such that one well in each 8-strip tube was used for the negative template control and another well was used for the N2 wild-type worms. Afterwards, the worms were centrifugated, then frozen for 15 minutes at -80°C. Finally, lysis was performed on the thermocycler for a period of 1 hour at -80°C.

PCR MasterMix was performed to amplify the lysis product. Once the lysis reaction was completed, DNA was also added. Two sets of PCR for lin-45 and unc-73 were performed. After the PCR was completed, lin-45 and unc-73 restriction enzyme in buffer to DI water and 5uL of DNA from the PCR were added to cut the DNA in the expected regions after incubating at 37°C for 1 hour.

Gel electrophoresis (1 hr, 130 V) was run to locate the wells that had the homozygous mutants for lin-45 and unc-73. Then, we distinguished the wells that were common in both strains to generate new double mutant strains. If, in the F2 stage, we could not find wells that were common in both strains, we had to separate the F2 and run another set of genotyping.

Chemotaxis assay

Approximately 20 L4 stages worms of these 3 double mutant strains, with approximately 5 worms per strain, in addition to N2 wild-type strains, and a mutant of odr-10, with five worms per plate were prepared. Four acrylic plates of each strain with a total of 12 plates were prepared for chemotaxis. After a few days, the worms were then used in chemotaxis. First, diacetyl (1:10,000 in ethanol) was prepared and placed on a pre-drawn template (Figure 2).

Figure 2. Chemotaxis diagram with indicated regions of ethanol and diacetyl.

1uL of 1M sodium azide was added on each spot to paralyze the worms with its toxicity and arrest worms within a centimeter of where it is placed. For each plate, 1uL of ethanol was pipetted on the spots labeled as “X” and 1uL of diacetyl on “O”. The worms were then washed multiple times using S-basal buffer. After the final supernatant removal, about 100 worms were transferred to the center of the chemotaxis plate under the microscope. The worms were chemotaxed for 1 hour, then burned.

Results

Generation of unc-73 and lin-45 double mutants     

After genotyping the F2 progeny, three double mutant strains, unc-73(sy898);lin-45 (sy875), unc-73(sy896);lin-45 (sy875), and unc-73(sy892);lin-45 (sy875), were generated. An example of a F2 gel image is shown in Figure 3, which highlights the homozygous, heterozygous, and wild-type wells. The double mutant strains are those that are homozygous for both unc-73 and lin-45.

Figure 3. Gel Image example of F2 #115/80. In unc-73, there was aband length of 436 for wild-type and a band length of 187, 249 for the mutant.

Verification of chemotaxis assays

As seen in previous literature, the odr-10 mutant had a higher chemotaxis ROI index than did N2, confirming that more worms were concentrated in one region and more attracted to the diacetyl than they were to the ethanol. The final image of the worms after the worms were burned on the Bunsen burner is shown in Figure 4.

The plates were captured and the worms were counted using ImageJ.The different regions on the chemotaxis plates are illustrated in Figure 2. The chemotaxis ROI index was calculated as the following:

For statistical testing, a statistic software (SPSS) was used to perform a one-way ANOVA with post-hoc Tukey HSD. A one-way ANOVA is used to determine if there are any statistically significant differences between the means of two or more independent groups. When there was a statistically significant difference in group means, a post-hoc Tukey HSD tested whether significant differences occurred between groups.

Figure 4. N2 Chemotaxis image taken. After 1 hour of chemotaxis, the worms were flamed using a Bunsen Burner.  
Figure 5. Chemotaxis ROI result for N2 and CX 3410. Bar graph shows the ROI index for both the wild-type strain and the positive control strain. CX3410 is the strain that has the odr-10(ky225) gene with the diacetyl.

Examination of unc-73 and lin-45 double mutants in chemotaxis assay

After verifying our chemotaxis assay, we used it to test the diacetyl chemosensory functions in the double mutants of unc-73 and lin-45. Region of Interest (ROI) value, standard deviation, and standard error of the double mutant strains are shown in Table 1.

Table 1.  Chemotaxis results of each strain.

Figure 5 demonstrates our chemotaxis assay was consistent with the results from previous studies that observed a higher ROI value for N2 than that of CX3410. This means that more wild-type worms were attracted towards diacetyl than they were towards ethanol. With an α- level of .05 and a t-test, odr-10 mutant shows statistically lower values in chemotaxis score compared to N2.; t(1), p < .0001. Thus, we rejected the null hypothesis that the mean of the wild-type is not equal to the mean of odr-10(ky225) and therefore expect an equal distribution in both regions 1 and 5, as shown in the plate template.


Figure 6. Chemotaxis ROI results for N2 vs. PS7876, PS774, and PS7878. Bar graph shows the ROI index for both the wild-type strain and the double mutant strains.

Figure 6 demonstrates that mutants are differently attracted to diacetyl. With an α- level of .05 and a one-way ANOVA test, there was a statistically significant difference between the means of the double mutants N2, unc-73(sy898);lin-45 (sy875), unc-73(sy896);lin-45 (sy875), unc-73(sy892);lin-45 (sy875); p < .05.However, the post-hoc Tukey test results show that there is no statistical significance among the means of the double mutant strains to the wild-type strain (p < .01).

Table 2. Chemotaxis strain information with the human amino acid change.

Discussion

Our result showed that N2 was significantly different from the diacetyl defective odr-10 mutant, proving the validity of our assay. However, the chemosensory index of double mutants did not differ from wild-type, indicating the unc-73 and lin-45 double mutants can sense diacetyl as well as wild-type.

Our results did not support the hypothesis that unc-73 and lin-45 double mutants exhibit diacetyl sensory defects. This may due to the huge variations in some double mutant strains such as PS7876, PS7878, and PS777. It may also be due to the missense mutants on these two genes located in domains that do not interact with each other.12, 13, 14  C. elegans only has 32 chemosensory neurons, but chemosensation is its most complex sensory measure and the main mechanism by which the animal makes judgments of its environment. Since there is a chemical specificity of odr-10 and the small number of sensory neurons, each cell might need to express many receptors for the animal to recognize the full spectrum of salient odorants.12

Future studies could explore a larger sample size and better control maturity and synchronicity of worms. Even though there was no statistical significance among the comparison of each double mutant to the mean of the wild-type ROI, we hope to eventually use this method in the human gene alignment to characterize new missense alleles for ASD patients (Table 2). In future studies, with a longer period of time and a larger sample size, more double mutant strains will be able to generated, chemotaxed and studied. This information will ultimately contribute to implications of medical practices and genetic studies. 

Conclusion

To investigate the genetic interaction of BRAF and TRIO, we use C. elegans equivalents of human missense mutations genes, unc-73 and lin-45. In this study, we generated unc-73 and lin-45 double mutants and examined the diacetyl chemosensory functions in these mutants. First we determined if diacetyl is an attractant to N2 wild-type and PS double mutant strains that were generated to make sure that the chemotaxis assay was consistent with previous literature. After confirming the assay was working, we investigate whether diacetyl was an attractant to the double mutant strains like it was for N2 wild-type and found that there was a statistically significant difference among the means.

Acknowledgements

I would like to thank my mentors, Sandy Wong and Professor Paul Sternberg for their guidance and support throughout the duration of this study. I would also like to thank the rest of the Sternberg Lab for their support. Lastly, I would like to thank the Caltech Student-Faculty Programs for giving me the opportunity to participate in research and the Simons Foundation Autism Research Initiative (SFARI) for awarding me as a SURF Fellow recipient with funding towards this project.

References

1. Simons Foundation Autism Research Initiative. (n.d.). Retrieved from https://www.sfari.org/

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12. Lehner, C. Crombie, J. Tischler, A. Fortunato, A. G. Fraser, Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways. Nat. Genet. 38, 896 (2006). 

13. Hirotsu, T., Saeki, S., Yamamoto, M., & Iino, Y. (2004). Corrigendum: The Ras–MAPK pathway is important for olfaction in Caenorhabditis elegansNature, 432(7017), 653-653.

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15. Anderson, A., Laurenson-Schafer, H., Partridge, F. A., Hodgkin, J., & McMullan, R. (2013). Serotonergic chemosensory neurons modify the C. elegans immune response by regulating G-protein signaling in epithelial cells. PLoS pathogens9(12), e1003787.

16. Steven, R., Zhang, L., Culotti, J., & Pawson, T. (2005). The UNC-73/Trio RhoGEF-2 domain is required in separate isoforms for the regulation of pharynx pumping and normal neurotransmission in C. elegans. Genes & development19(17), 2016-29.

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