1. ENVIRONMENTAL EFFECT ON CELLULAR DIFFERENTIATION REGULATION
Michael Ishak, Jacob Kott, Jose Ortega, Stephen Miller
Department of Biological Sciences, University of Maryland, Baltimore County
1000 Hilltop Circle, Baltimore, MD 21250
Abstract
The
objec)ve
of
this
study
is
to
inves)gate
the
effect
of
varying
growth
environments
on
cellular
differen)a)on
in
the
green
alga
Volvox
carteri
and
how
such
effects
are
executed
at
the
molecular
level.
V.
carteri
possesses
only
two
cell
types,
reproduc)ve
cells
(gonidia)
and
soma)c
cells.
In
the
wild
type
strain
EVE,
soma)c
cells
remain
differen)ated
because
they
express
the
regA
gene,
which
suppresses
growth
and
dedifferen)a)on.
In
a
mutant
strain
pReg,
some
soma)c
cells
dedifferen)ate
into
gonidia,
and
the
phenotype
becomes
stronger
as
cultures
become
more
crowded.
This
observa)on
led
to
the
hypothesis
that
soma)c
cell
differen)a)on
can
be
influenced
by
external
macronutrient
(sulfate,
phosphate,
or
nitrogen)
concentra)ons,
especially
when
regA
or
other
cell-­‐differen)a)on
genes
are
mutated.
To
test
this
idea,
EVE
and
pReg
were
cultured
in
media
limited
for
sulfate,
phosphate,
or
nitrogen,
and
it
was
found
that
depriva)on
for
each
of
these
macronutrients
led
to
increased
rates
of
dedifferen)a)on
in
mutant
pReg
individuals
but
not
in
EVE.
To
determine
whether
a
defect
in
the
regA
gene
might
be
responsible
for
the
pReg
phenotype,
we
are
cloning
and
sequencing
PCR
products
made
from
the
pReg
regA
gene,
with
results
to
be
reported.
Summary
• Mutation (insertion in intron 4) in the
sequenced region of the regA gene in the
pReg mutant.
• Somatic cell dedifferentiation
increases as nutrients decrease.
• Sulfate and phosphate starvation cause
largest phenotypic effects.
• Nitrogen starvation causes smaller
phenotypic effects.
Further study
• Quantitative analysis of somatic cell
dedifferentation using MetaMorph
imaging software
• Sequencing the remaining pReg regA
gene.
The V. carteri life cycle
Introduction
Molecular Work
• Primers were designed for introns 3, 4,
5, 7 and exons 4, 5, 6, 7, 8.
• PCR products (red) were cloned in
Pgen vector and sequenced.
pReg with 25% nitrogen pReg with 50% nitrogen pReg with 100% nitrogen
pReg with 0% phosphate pReg with 25% phosphate pReg with 100% phosphate
pReg with 0% sulfate pReg with 25% sulfate pReg with 100% sulfate
EVE with 0% sulfate EVE with 25% sulfate EVE with 100% sulfate
• V. carteri has on
average a two-day
life cycle.
• Two cell types:
gonidia and somatic
• Somatic cells
differentiate from
precursor cells
• Macronutrient cues (MN) compete against developmental cues
(D)
• MN cues and D cues are suspected to influence regA expression
and somatic cell fate
Model for macronutrient regulation of regA
Results