Download Lin-42 - York College of Pennsylvania

Temporal Regulation of Cell Development: The Heterochronic Gene
Pathway in Caenorhabditis elegans
David Cooper, Department of Biology, York College of Pennsylvania
http://lc-molecular.wikispaces.com/
http://neurodojo.blogspot.com
Introduction
In Caenorhabditis elegans, a
complex set of genes have
been discovered, collectively
known as the heterochronic
gene pathway.
These genes control the timing
of cell development, ensuring
that structures develop at the
proper time during maturation.
Some genes are understood,
while others have remained
difficult to characterize.
Methods (1)
Two separate experiments were conducted:
• Two crosses to examine the impact on
Seam Cells and expression of the hbl-1
gene of a lin-42;lin-14 double mutant
• An RNAi experiment to look for altered
phenotypes when lin-42 and lin-14 mutants
are grown under other mutant backgrounds
What is RNAi?
Crosses were started with a control N2
strain which, by definition, has no mutations.
• Utilizes a viral defense mechanism
against single-stranded RNA
• Tricks organism into shutting off its own
gene
E. coli induced to separately produce four
different types of RNAi:
• Vector, lin-42, lin-41, hbl-1
Worm Strain
RNAi
Hbl-1 (BVB3)
Lin-41 (I4J11)
1) Determine if a Lin-42;Lin-14
double mutant alters the seam
cell count or the expression of
the hbl-1 gene
2) Compare and contrast four
different strains grown under
four different mutant
backgrounds
Wild Type
Alae: L4
a: 16.75
SCN
Vector (BVB8)
(n=20)
Results (1)
Hbl-1::GFP; Lin-42; Lin-14:
• Cross appeared unsuccessful
• Rolling phenotype observed in males
• Rolling males did not seem to be able
to successfully mate
SCM::GFP; Lin-42; Lin-14:
• Cross was successful.
• Hermaphrodites produced that had:
Seam cell marker, blipped
• Able to produce offspring with same
phenotype
• Average Seam Cell Number: 11.4
(n=5)
Average = 11.4
Adults should have 16
Previous reports: no symmetric
double division
Lin-42; lin-14 hbl-1 expression
•
•
Phenotype does not match
expected. Results inconclusive.
lin-42 has been shown in the past
to suppress hbl-1 phenotype
RNAi
Results (2)
Lin-14 produces a nuclear
protein believed to be critical to
early larval development.
The specific objectives of this
study were:
•
•
•
Four different strains plated onto each RNAi.
• Wild type, lin-42, lin-42; lin-14, lin-28
• lin-28 mutants have the same
phenotype as lin-42
The protein lin-42 is known to
be related to the family of
proteins responsible for
circadian rhythms in mammals.
Objectives
Lin-42; lin-14 Seam Cells
• Can be done fairly efficiently and quickly
The heterochronic genes Lin42 and Lin-14 were the focus of
this study.
These affect development of
Seam Cells as well as the
expression of the hbl-1 gene
Results (Summary):
Methods (2)
Lin-42
(pkk220*)
Alae: L4
SCN: 10.91
(n=22)
Alae: L4
SCN: 15.39
(n=18)
Alae: L4
SCN: 15.85
(n=20)
Lin-42
Lin-42;Lin-14
Lin-28
Alae: L3
BLP
Alae: L3
SCN: 11.15
(n=20)
Alae: L3
DPYb
Alae: L3
STRL
Alae: L3
SCN: 9.7
(n=20)
DPY
STRLc
Alae: L3
DPY
BLP
Alae: L3
SCN: 10.75
(n=24)
Alae: L3
Normal
Shape
No apparent
effects
Alae: L3
BLP
Alae: L3
SCN: 10.68
(n=25)
Table 1: Results of the RNAi experiments. Each strain was plated onto each of the four
RNAi types.
a Seam Cell Number
b Dumpy Phenotype
c Unconfirmed Sterility
Results:
• Lin-42 on Lin-41 RNAi: Animals appeared
sterile and were highly deformed
• Lin-28 on Hbl-1 RNAi: Average Seam Cell
number was highly unusual
•
lin-42 animals on lin-41 RNAi have
a very severe phenotype
• lin-28 animals on hbl-1 RNAi have
an unusual seam cell count
• Corresponds with previously
established reports on hbl-1
RNAi
Conclusions
• Analysis of the results imply that
lin-14 is epistatic to lin-42.
• Further investigation into the two
unusual RNAi strain phenotypes
may help reveal more about the
different genes relationships to one
another.
Literature Cited
AMBROS, V., 1989, A hierarchy of regulatory genes controls a
larva-to-adult developmental switch in C. elegans. Cell
57: 49-57.
AMBROS, V., HORVITZ, H.R., 1984, Heterochronic mutants of
the nematode Caenorhabditis elegans. Science 226:
409-416.
MOSS, E.G., 2007, Heterochronic genes and the nature of
developmental times. Current Biology 17: R425R434.
Acknowledgements
I would like to thank the University of
Medicine and Dentistry of New Jersey –
Graduate School of Biomedical Science for
hosting the SURE Program.
I would also like to thank Dr. Eric Moss, Dr.
Bhaskar Vadla, Jennifer Alaimo, and Kevin
Kemper for helping me to conduct this
research and interpret my data.
Lastly I would like to thank Dr. Kaltreider for
being my mentor and helping me refine my
written report and poster.