Transcript Symposium Poster - uospur

Investigation of Embryonic Lethal Cell Division Defects In Temperature Sensitive
Caenorhabditis elegans Mutants
Karra Rutherford1, Aleesa Schlientz2, and Bruce Bowerman2
1Saint Leo University, 2University of Oregon Institute of Molecular Biology
Abstract
Methods
Cell division is a highly specialized process that must be meticulously
regulated to ensure fidelity of the distribution of genetic material, cellular
function, and ultimately organism survival. Improper cell division can result in
aneuploidy, cell death, or abnormal growth such as cancer. Errors in early cell
division, the focus of this investigation, can be particularly detrimental to
organism development and viability; potentially leading to infertility,
spontaneous abortion, or birth defects. In order to study essential gene
requirements in early embryonic division, we screened temperature-sensitive
C. elegans mutants for highly penetrant embryonic lethal mutations. Using
Nomarski differential interference contrast (DIC) microscopy we were able to
view the first few rounds of cell division of live embryos in real-time, allowing
for phenotype analysis of mutations of interest.
Complementation Schemes
A
Score for
Penetrance
DIC Imaging
P0
Genetic
Complementation
Tests
Fluorescent
Confocal Imaging
B
P0
Results
Wild type
or1200
~60-80% of
protein
sequences have
human
homologous
genes
-222s
-186s
0s
192s
2318s
1764s
478s
1428s
926s
626s
2872s
1894s
B
or854
Wild type
15℃ Embryonic
Lethality
28.6% (128/447)
26℃ Embryonic
Lethality
96.9% (565/583)
Dominance Test
6.5% (17/261)
A. DIC still of mutant or854 in comparison to wild type N2. White triangle indicates a polar body. B. Testing for lethality at both the
permissive and restrictive temperature, with high viability at 15℃ and high lethality at 26℃, confirms that we are working with a TS
conditional allele. Dominance testing indicates the mutation is recessive and exhibits aberrant function when up shifted to the
restrictive temperature.
Expression of
Wild Type
Phenotype
SNP Mapping
Mutagenized strain
(N2)
C
Polymorphic strain
(Hw)
Allow to
self-fertilize
Induction of
Mutant
Phenotype
Select ~15 F2
homozygotes
Temperature sensitive (TS) mutants are a powerful genetic tools when studying essential gene requirements. These conditional
alleles are heat sensitive; when grown at a permissive temperature worms express the wild type phenotype, and when upshifted
to a restrictive temperature the mutant phenotype is induced due to inactivation of gene product. In studying embryonic lethal
mutations, this allows for bypassing earlier essential requirements during larval and adult germline development.3
Acknowledgements
University of Oregon Summer Program for Undergraduate Research:
Peter O’Day
Marilyn Drennan
Cohorts
B
SNP locus:
Hw/Total
:
1
0.5
2
0
3
0
4
0.5
A. A genetic cross between the TS mutant of interest and the polymorphic HW strain.
B. SNP reads/Total reads indicate location of mutation. C. Interpretation of the
sequencing data shows possible chromosomal location of the mutation.
Ratio of Hw SNP reads/Total read depth
A
Bowerman Lab:
Bruce Bowerman
Aleesa Schlientz
Chien-Hui Chuang
Molly Jud
Kenji Sugioka
3288s
Meiotic Cell Division Mutant Phenotypes
A. Cellular differentiation in C. elegans. B. Series of
asymmetric divisions in C. elegans that result in organism
orientation and development of the founder cells. This
investigation focused on mutations that caused defects
within the first three mitotic divisions 2
𝑜𝑟854 +
50%
𝑏𝑎𝑙
viable
2246s
or1326
0s
456s
A
Many genes are conserved between humans and C. elegans.
Genes that are evolutionarily conserved between distantly
related species are very likely to be essential for organism
survival. C. elegans are therefore a relevant model when
studying certain human health issues.1
1478s
DIC time-lapse images comparing mutants or1200 and or1326 to wildtype N2. Time points are relative to pronuclear meeting. Black
dots indicate spindle poles, white arrows indicate spindle orientation during anaphase, white circle indicates membrane contractility,
and white triangle indicates large polar body.
7,390 remaining
sequences may
lead to discovery
of novel genes
𝑜𝑟854
+
50%
+
𝑧𝑦𝑔−11
𝑋
𝑧𝑦𝑔−11
𝑏𝑎𝑙
Two possible outcomes of the genetic complementation test between the TS mutant of interest or854 and zyg-11
1160s
408s
398s
𝑜𝑟854
𝑜𝑟854
or854 Complementation Results
-134s
7,954 sequences
match known
human
transcripts
0s
𝑜𝑟854
50%
𝑏𝑎𝑙
viable
viable
Mitotic Cell Division Mutant Phenotypes:
C. elegans as a Model for Essential Gene Function:
𝑜𝑟854
50%
𝑧𝑦𝑔−11
nonviable
or
F1
Background
𝑋
𝑧𝑦𝑔−11
𝑏𝑎𝑙
Outcrossing
F1
SNP Mapping
𝑜𝑟854
𝑜𝑟854
zyg-11
Plate
26℃ Embryonic Lethality
Plate
26℃ Embryonic Lethality
D6
6.6% (5/76)
E12
94.3% (50/53)
D7
5.4% (2/37)
E13
100% (79/79)
D8
100% (74/74)
G4
91.5% (108/118)
D9
4.6% (3/65)
G5
100% (27/27)
D10
97.1% (67/69)
G6
0% (0/63)
E5
22.2% (8/36)
G7
94.4% (51/54)
E6
6.7% (2/30)
G8
2.1% (2/97)
E7
96.8% (61/63)
H2
100% (84/84)
E8
100% (58/58)
H3
0% (0/60)
E9
93.1% (27/29)
H4
3.5% (3/86)
E10
95.5% (64/67)
H5
100% (102/102)
E11
5.4% (3/55)
56.5% (13/23) of F1 progeny tested produced a high rate of inviable embryos. This supports complementation outcome A, that if the
mutations do not complement, it would be expected that 50% of the F1 progeny would produce nonviable embryos.
Future Directions
Both or1200 and or1326 exhibit phenotypes of interest to the lab, they are
currently being outcrossed to wild type N2’s to confirm these phenotypes are
due to a single mutation rather than a combination of mutations. Pending the
results of these tests, they will go on to be genetically mapped and
outcrossed for complementation.
Chromosomal position (Mbp)
References
Funding:
R01GM049869
NSF DBI/BIO 1460735
1. Lai, Chun-Hung, Chang-Yuan Chou, Lan-Yang Ch'ang, Chung-Shyan Liu, and Wen-chang Lin. "Identification of Novel Human Genes Evolutionarily Conserved in Caenorhabditis Elegans by Comparative
Proteomics." Genome Research 10.5 (2000): 703-13.
2. Rose, Lesilee, and Pierre Gonczy. "Polarity Establishment, Asymmetric Division and Segregation of Fate Determinants in Early C. Elegans Embryos." WormBook (2014): 1-43.
3. O'rourke, Sean M., Clayton Carter, Luke Carter, Sara N. Christensen, Minh P. Jones, Bruce Nash, Meredith H. Price, Douglas W. Turnbull, Aleena R. Garner, Danielle R. Hamill, Valerie R. Osterberg, Rebecca
Lyczak, Erin E. Madison, Michael H. Nguyen, Nathan A. Sandberg, Noushin Sedghi, John H. Willis, John Yochem, Eric A. Johnson, and Bruce Bowerman. "A Survey of New Temperature-Sensitive,
Embryonic-Lethal Mutations in C. Elegans: 24 Alleles of Thirteen Genes."PLoS ONE 6.3 (2011): n. pag. Web.