From the
NGFI-A (also known as EGR-1, zif/268, and Krox-24) is a zinc
finger transcription factor induced in many cell types by a variety of
growth and differentiation stimuli. To determine if NGFI-A plays a
requisite role in these processes, we used homologous recombination to
mutate both alleles of NGFI-A in embryonic stem (ES) cells and examined
its effect on growth and differentiation. We find that ES cells lacking
NGFI-A exhibit similar growth rates and serum-induced gene expression
profiles compared to wild-type parental cells. They are capable of
differentiating into neurons, cardiac myocytes, chondrocytes, and
squamous epithelium. Chimeric mice were generated from targeted ES
cells, and their progeny were crossed to produce homozygous mutant
mice. Growth and histological analyses of mice lacking NGFI-A confirm
the finding in ES cells that NGFI-A is not required for many of the
processes associated with its expression and suggest that the function
of NGFI-A is either more subtle in vivo or masked by redundant
expression provided by other gene family members such as NGFI-C,
Krox-20, or EGR3.
The cellular immediate early genes (IEGs)
The NGFI-A gene (also termed EGR-1, Krox-24,
and zif/268)
(5, 6, 7, 8) was
originally identified by virtue of its induction by nerve growth factor
in the rat pheochromocytoma cell line PC12 and by mitogenic stimuli in
fibroblasts and in lymphocytes
(5, 6, 7, 8, 9) . The NGFI-A
gene encodes a Cys
To assess the
importance of NGFI-A in the variety of cellular processes associated
with its induction, we used homologous recombination followed by
culture in elevated G418 to inactivate both copies of NGFI-A in
embryonic stem (ES) cells. In this report, we demonstrate that NGFI-A
is not required for the growth and differentiation of ES cells. We
confirm and extend these findings by examining homozygous mutant mice
derived from the targeted ES cells and find that mice lacking NGFI-A
exhibit no overt defects in growth or differentiation.
AB1 and D3 ES cells were passaged
every 2-3 days, maintained with daily changes of ES medium (DMEM
containing 15% fetal calf serum (HyClone or Sigma), 10 µM
2-mercaptoethanol, 10-20 µg of LIF (Life Technologies, Inc.)
antibiotics), and cultured on
DNA for PCR was prepared
from cells by lysis in 50 µL of 1
For
serum response analysis, ES cells deprived of serum for 12-24 h
in DMEM containing 0.5% fetal calf serum were induced with fetal calf
serum at a final concentration of 20% and harvested at various times
after induction. Total RNA was prepared by the guanidinium
isothiocyanate method
(26) . 15 µg of total RNA were
electrophoresed in 2.2 M formaldehyde gels, transferred onto
Sureblot nylon membranes (Oncor), and probed with either a 400-bp
BglII/ XbaI fragment of the 3`-UTR of NGFI-A,
full-length c- fos cDNA (ATCC), a 410-bp BglII
fragment of the 3`-UTR of NGFI-B, or full-length cyclophilin cDNA, and
exposed for 12-24 h to PhosphorImager screens. Immunoblots were
prepared
(27) from 5
For lymphocyte proliferation assays, single cell suspensions of
spleens from adult mice were prepared by mincing between frosted
slides. Cells were washed three times in Hanks' balanced salt
solution and plated into flat bottom 96-well plates at 5
Teratomas were produced by
intraperitoneal or subcutaneous injection of 10
For histological analysis,
mice were perfused transcardially, and their tissues fixed overnight in
4% paraformaldehyde in PBS. Bones were decalcified following fixation
by overnight incubation in 10% formic acid in PBS. Tissues were
processed for paraffin embedding, sectioned into 6-7-µm
sections, and stained as described in figure legends.
We have eliminated NGFI-A from ES cells by homologous
recombination and have examined its effect on proliferative and
differentiative processes associated with its expression. Our finding
that ES cells proliferate normally without NGFI-A suggests either that
NGFI-A has no role in ES cell proliferation, or that redundant
mechanisms can compensate for NGFI-A function. We did not observe a
compensatory increase, however, in the expression of other gene family
members in the mutant cells. Similar observations have been reported
for the targeted disruption of the immediate early genes c -fos and c -jun in which the lack of either gene did not
significantly impair ES cell growth
(35, 36) .
Several reports have described the induction of NGFI-A during
differentiation into a variety of cell types, including neurons,
myocytes, and osteoblasts. We have shown by in vitro differentiation and analysis of teratomas that NGFI-A is not
essential for the differentiation of embryonic stem cells into multiple
lineages. Additionally, NGFI-A is induced in cardiac tissue in response
to hypertrophic signals and has been reported to regulate transcription
of the
As indicated by the weight analysis, growth in general of mice
lacking NGFI-A appears to be normal. It is also surprising to find an
intact proliferative response to mitogenic signals in
NGFI-A
We thank Dr. Allan Bradley of Baylor University for
the PGKneopA vector and the AB1 ES cells, Dr. Steven Potter of the
University of Cincinnati for the D3 ES cells and advice on their
culture, Dr. Andy McMahon of Harvard University for the
MC1neopA
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
are thought to mediate processes of cell growth and
differentiation
(1, 2) . Upon stimulation by growth
factors, cytokines, or membrane depolarization, these basally quiescent
genes become rapidly expressed without the requirement for de novo protein synthesis. Many of these genes encode transcription
factors such as c -fos and c -jun, as well as NGFI-A
and NGFI-B (also known as nur/77) which are thought to regulate genetic
programs that ultimately result in long term phenotypic changes
(3, 4) .
-His
zinc finger protein
which shares strong sequence homology to a family of genes that
includes NGFI-C
(10) , Krox-20
(11) , EGR3
(12) ,
and more distantly, the Wilms' tumor suppressor gene WT1
(13, 14, 15) . The expression of NGFI-A has been
detected in a variety of differentiation paradigms such as embryonal
carcinoma cells into myocytes or neurons, pre-osteoblasts into
osteoblasts, and myelomonocytic precursors into macrophages
(16, 17, 18, 19) . While most of the
evidence for the involvement of NGFI-A in these processes has been
correlative, antisense strategies directed against NGFI-A have shown
that NGFI-A is necessary for macrophage differentiation as well as for
T lymphocyte proliferation
(20, 21) .
NGFI-A Gene Targeting
To construct the targeting
vectors, a 4.5-kb EcoRI/ XbaI fragment encompassing
1.0 kb relative to the transcription start site to the 3`-UTR of
the NGFI-A gene was isolated from a
Dash II Balb/c mouse genomic
library (Stratagene) and cloned into the corresponding sites in
pGEM-7z(+) (Promega). The neomycin resistance gene cassette
MC1neopA
Act
(22) , PGKneo, or PGKneopA
(23) was
cloned into a unique NdeI site located upstream of the DNA
binding domain using XhoI linkers. The thymidine kinase gene
cassette MC1TK was cloned 3` of the short arm of homology as an
XhoI/ SalI fragment into a SalI site carried
over from the phage polylinker to create either pA(MC1neopA)TK,
pA(PGKneopA)TK, or pA(PGKneo)TK.
-irradiated neomycin-resistant
primary murine embryonic fibroblasts (MEFs). ES cells were shown in
preliminary experiments to contribute extensively to chimeric mice
(30-75% as determined by coat color) when injected into day 3.5
C57Bl/6 host blastocysts
(24) . NsiI-linearized
targeting vector (25 µg) was electroporated into approximately
10
trypsinized ES cells suspended in 0.9 ml of
HEPES-buffered saline (pH 7.2) using a BTX electroporator at 260 V, 500
µF. After plating onto 10-cm MEF feeder plates for 24 h, drug
selection was applied using G418 (330 µg/ml) (Life Technologies,
Inc.) with or without 2 µM ganciclovir (Syntex).
Approximately 10
colonies survived selection in G418 alone
with 2-5-fold reduction in numbers with ganciclovir, while no
reduction was seen in cells transfected with a vector lacking the TK
expression cassette. Colony halves were picked and pooled in groups of
five for PCR analysis, while the corresponding half was plated onto
individual 96 wells containing MEF feeders.
PCR buffer, digested for
3 h to overnight in 10 µg/ml proteinase K, which was then
heat-inactivated at 95-100 °C for 15 min
(25) . Half
of the lysate was used for PCR analysis using primers specific for the
neomycin resistance gene and the 3`-untranslated region of NGFI-A
external to the targeting vector, with conditions previously determined
from cells transfected with a positive control template. Individual
clones were identified from positive pools and expanded for Southern
analysis. Blots were exposed to x-ray film (Amersham Corp.) or to
PhosphorImager screens (Molecular Dynamics).
Serum Response and Proliferation Assays
ES cells
conditioned to grow in the absence of MEF feeders were plated in
triplicate onto gelatinized six-well plates to yield approximately 1
10
colonies. At various times, cells were
trypsinized, and cell counts were determined in duplicate for each
well. Error bars represent standard deviation from the mean.
10
uninduced or
serum-stimulated ES cells or NGF-induced PC12 cells and probed by ECL
chemiluminescence (Amersham Corp.) using the anti-NGFI-A mAb 6H10.
10
cells/well in 200 µl of RPMI 1640 containing 10%
fetal calf serum, 2 mM glutamine, 50 µg/ml gentamycin, 10
mM HEPES, and 2
10
M
2-mercaptoethanol. Following exposure to either concanavalin A (Sigma)
or anti-CD3 antibody, cells were incubated at 37 °C at 5% CO
for 72 h, followed by an additional incubation for 20 h in the
presence of added [
H]thymidine (0.4
µCi/well). For stimulation by anti-CD3 antibody, cells were plated
onto anti-CD3 antibody-coated plates which were prepared by incubating
2C11 hybridoma cell culture supernatant at various dilutions for 12 h
at 37 °C, after which the medium was aspirated and the plates were
allowed to dry.
In Vitro Differentiation of ES Cells and Teratoma
Production
For differentiation assays, ES cells were lightly
trypsinized, aggregated to form simple embryoid bodies
(24) ,
and plated 4-5 days later onto gelatinized plates. Neurons,
beating cardiac myocytes, and other cell types were distinguishable
morphologically between 4 and 12 days after plating. Indirect
immunofluorescence staining of cardiac myosin was performed on cells
fixed in 1% paraformaldehyde in PBS by incubating mAb 3A6.8 made
against cardiac myosin(
)
followed with
rhodamine-coupled goat anti-mouse secondary antibody and visualized on
a Zeiss Axiophot fluorescence microscope.
ES cells
into SCID mice. Tumors were evident by 3 weeks after injection, at
which time the mice were sacrificed. Tumors were fixed in 4%
paraformaldehyde in PBS overnight, incubated in 30% sucrose in PBS,
embedded in OCT (TissueTek), cut into 7-µm sections, and stained
with hematoxylin and eosin.
Production and Analysis of Mice
Blastocyst
injection
(24) of two D3 ES cell-derived clones A4 and Z,
singly targeted by the vectors pA(MC1neopA)TK and pA(PGKneo)TK,
respectively, resulted in chimeras which transmitted the agouti allele as well as the targeted allele to their offspring. The
resulting heterozygotes were mated to produce homozygous mutants
lacking the wild type allele. All subsequent analyses were performed on
mice derived from either of these clones.
Production of Homozygous Mutant NGFI-A ES
Cells
To disrupt NGFI-A, we used a 4.5-kb genomic fragment of
the NGFI-A gene to construct a replacement-type targeting vector
(28) , which upon homologous recombination, introduces several
in-frame stop codons that truncate the NGFI-A coding sequence upstream
of the DNA binding domain. The negative selectable marker thymidine
kinase (MC1TK) was attached at the end of the genomic fragment to
select against nonhomologous recombination (Fig. 1 a)
(29) . Linearized targeting vector was electroporated into AB1
or D3 ES cells and transfectants were selected by culture in G418 and
ganciclovir. From 580 doubly resistant colonies, four had incorporated
the targeting vector by homologous recombination as assessed by PCR and
verified by Southern blot (Fig. 1 b). No other
integration sites were detected when blots were probed specifically for
the neomycin cassette (data not shown). From these heterozygous clones,
homozygous mutant ES cells were generated by culturing in elevated
concentrations of G418 (3-4 mg/ml)
(30) . Surviving
colonies were screened by Southern blot to identify clones homozygous
for the targeted allele. (Fig. 1 c).
Figure 1:
Targeted inactivation
of both alleles of the NGFI-A gene. a, schematic
representation of the targeting vector pA(MC1neopA)TK, wild type, and
mutated NGFI-A gene, indicating predicted restriction fragment lengths
when probed with probe A3`. Arrowheads denote the location of
primers used for PCR screens. Similar results were obtained with the
targeting vectors pA(PGKneopA)TK and pA(PGKneo)TK (data not shown).
b, Southern blot of PstI-digested DNA from two
positive clones ( lanes 1 and 2) and a non-targeted
clone ( lane 3) demonstrating the presence of the 1.9-kb band
corresponding to the mutated NGFI-A allele. c, Southern blot
of PstI-digested DNA from 12 colonies derived from the parent
clone A1 ( lane C) surviving selection in high G418 (3 mg/ml)
demonstrating the loss of the 2.5-kb band corresponding to the wild
type allele ( lanes 1-4, 6, 10, and
11).
The Loss of NGFI-A Does Not Affect ES Cell
Growth
Because NGFI-A is rapidly induced in quiescent
fibroblasts upon treatment with serum
(6, 8, 31) , we examined the possibility that
serum also induced NGFI-A in quiescent ES cells. After treatment of ES
cells with serum for 1 h, NGFI-A mRNA and protein were detected in wild
type cells, but were absent in the homozygous mutant cells, confirming
that the targeting vector disrupted the NGFI-A gene (Fig. 2,
a and b). We next examined the effect of this
mutation on ES cells in 1) their growth rate and 2) their serum
induction profiles of other immediate-early genes. To compare growth
rates, equivalent numbers of wild type and mutant cells were plated and
cell counts were taken at various times afterward. No significant
differences were observed in cell number during the exponential growth
phase (Fig. 2 c), with cell doubling times ranging from
13-15 h in both wild type and mutant cells. The expression
profiles of c- fos and NGFI-B mRNAs demonstrated characteristic
serum induction patterns, as evidenced by an increase in mRNA levels
within 1 h, but no differences were observed between wild type
( WT) and mutant ( KO) cells (Fig. 2 b).
We also examined whether the loss of NGFI-A affected the expression of
the closely related genes Krox-20, EGR-3, and NGFI-C, but no
differences were observed (data not shown). Thus, NGFI-A is not
essential for ES cell proliferation or the response to serum.
Figure 2:
Analysis of RNA, protein, and growth rate
of NGFI-A ES cells. a, homozygous
mutant ES cells lack NGFI-A protein. 5
10
wild type
( WT) and mutant ( KO) ES cells were serum starved for
24 h before stimulating 1 h with 20% fetal calf serum (+
serum) or DMEM alone (
serum). The cells were
lysed in Laemmli buffer and protein blot analysis was performed using
the NGFI-A-specific mouse mAb 6H10 (27). PC12 cells stimulated with NGF
for 1 h were used for comparison ( lane 5). Molecular size
markers (in kilodaltons) are indicated on the right.
b, Northern analysis comparing the serum induction profiles of
NGFI-A, c -fos and NGFI-B mRNA in wild type ( WT) and
homozygous mutant ( KO) ES cells. Cells were serum stimulated
as in a and ES cells harvested for RNA at the indicated time
points ( h). 15 µg of total RNA were blotted and probed for
NGFI-A (3.5 kb), NGFI-B (2.4 kb), c -fos (1.3 kb), and
cyclophilin (0.8 kb). Blots were exposed to PhosphorImager screens for
24 h except for NGFI-B, which was exposed for 48 h. c, growth
rates of wild type D3 (+/+) and homozygous mutant
(
/
) ES cells. Approximately 1
10
ES
cells/well were plated onto gelatin-coated 6-well plates, and cell
counts determined at indicated times. Each point represents mean
(± S.D.) of triplicate determinations. Similar results were
obtained with AB1 ES cells (not shown).
The Loss of NGFI-A Does Not Affect ES Cell
Differentiation
NGFI-A is induced during the differentiation of
a variety of cell types
(5, 17, 18) .
Additionally, NGFI-A has been implicated in the regulation of
-cardiac myosin heavy chain gene expression
(32) . To
determine whether the loss of NGFI-A affected these processes, we
assessed the ability of ES cells to differentiate into neurons and
cardiac myocytes
(24) . ES cells were aggregated to form
embryoid bodies which were then allowed to attach and undergo
differentiation. After several days, both wild type and
NGFI-A
cells formed neurons with extensive
neurite outgrowth (Fig. 3 a). Rhythmically contracting
myocytes were also observed, and these stained positive with a
monoclonal antibody which recognizes cardiac myosin
(Fig. 3 b).
Figure 3:
Analysis of differentiation of ES cells
in vitro. Embryoid bodies formed by aggregation of
NGFI-AES cells were plated and cultured for
4-12 days. Various cell types were observed including
( a) neurons sending out extensive neurites (phase contrast,
200
) or ( b) beating cardiac myocytes, stained here with
monoclonal antibody made against cardiac myosin followed with rhodamine
conjugated goat anti-mouse antibody. Fluorescence specifically
localizes to regions of contractile myocytes (
350). Similar
results were obtained with +/
and +/+ ES cells
(not shown).
To examine the role of NGFI-A in the
formation of a greater variety of tissues, we examined the ability of
NGFI-A deficient ES cells to form teratomas after injection into SCID
mice
(24) . Three weeks after injection, tumors from these mice
were analyzed histologically. Hematoxylin and eosin-stained tumor
sections revealed the presence of a variety of tissue types including
cartilage, with their chondrocytes arranged in characteristic groups or
cell ``nests'' (Fig. 4 a, arrow);
squamous epithelium, forming a stratified layer of flattened cells
(Fig. 4 b, arrowheads); and striated muscle,
arranged in longitudinal myofibrils containing cross-striations
(Fig. 4 c, arrows). The presence of these
tissues indicate that, although NGFI-A is expessed in these tissues in
embryonic and adult animals, it is not required for their formation
(33) .(
)
Taken together, the in vitro differentiation and histological analysis of teratomas demonstrate
that the absence of NGFI-A does not abolish the ability of ES cells to
differentiate into a variety of cell types.
Figure 4:
Histological analysis of teratomas derived
from NGFI-A ES cells. Three weeks after
injection of NGFI-A
ES cells into SCID mice,
the resulting teratomas were analyzed by histology and were shown to
contain various tissues including: a, cartilage
( arrow); b, squamous epithelium
( arrowheads); and c, striated muscle tissue
( arrows). Magnification,
420; hematoxylin and
eosin.
Mice Lacking NGFI-A Exhibit a Normal Growth Rate and Lack
Any Obvious Defects in Cellular Differentiation
Injection of
targeted ES cells into blastocysts resulted in chimeras which
transmitted the agouti as well as the targeted allele to their
offspring. Homozygous mutant mice were produced by intercross matings
of the resulting heterozygotes. The NGFI-A mutant mice were born at
expected Mendelian frequencies, appeared healthy, and were virtually
indistinguishable from their littermates. The NGFI-A mutant mice were
monitored for growth by weighing at various ages. In accord with the
findings obtained from ES cells in vitro, NGFI-A does not
appear to be required for growth in general as both mutant and wild
type animals gained weight at the same rate (Fig. 5).
Figure 5:
Growth
assessment of NGFI-A mice. Weight comparison
of NGFI-A
mice and their wild type or
heterozygous littermates at various ages. Values represent the mean of
at least four animals/group ± S.D.
NGFI-A
is highly expressed in the thymus
(34) and in T-lymphocytes
(6) which have been previously shown to require NGFI-A for
proliferation
(21) . Therefore, the T cell compartment of
NGFI-Amice was further investigated.
Histological analysis of the thymus indicated a normal architecture in
NGFI-A
mice, as evidenced by a well
demarcated cortex ( C) and medulla ( M) (see
Fig. 7
, c and d). To examine the proliferative
response of peripheral lymphocytes, [
H]thymidine
incorporation was measured following activation by concanavalin A or
anti-CD3 antibody. Surprisingly, both control and
NGFI-A
lymphocytes demonstrated an equivalent
response (Fig. 6, A and B), indicating that
NGFI-A is not required for T-lymphocyte proliferation in response to
these stimuli.
Figure 7:
Histological survey of
NGFI-A and control mice. Histological
analysis comparing littermate controls ( a, c,
e, g) and NGFI-A
mice
( b, d, f, h). a and
b, coronal brain sections stained for neurons in the dentate
gyrus ( dg) and pyramidal cell layer ( py) of the
hippocampus (cresyl violet). c and d, sections of
thymus showing the cortex ( c) and medulla ( m)
(hematoxylin and eosin). e and f, longitudinal
section of the humerus with epiphyseal plate (Alcian blue
counterstained with hematoxylin and eosin). g and h,
higher magnification of e and f depicting
proliferative ( p), hypertrophic ( h), and calcifying
( c) zones of the epiphyseal plate. Magnification,
a-f,
52; g and h,
420.
Figure 6:
Proliferation assays of
NGFI-A lymphocytes. Proliferative responses
of peripheral lymphocytes to ( A) concanavalin A or
( B) anti-CD3 antibody as determined by incorporation of
[
H]thymidine. Values represent the mean of
triplicate wells ± S.D.
To explore the possibility that NGFI-A is required
for the formation of a variety of tissues and cell types, a
histological survey was performed. Examination of coronal brain
sections stained with cresyl violet revealed a normal neuronal
organization and cellularity in the hippocampus (Fig. 7, a and b) and cortex (not shown), indicating that, despite
the extensive expression of NGFI-A in the developing brain
(34) , NGFI-A does not appear to be required for its basic
organization. In situ hybridization analysis has also
demonstrated coordinate regulation of NGFI-A and c- fos in the
developing long ends of bones where they may regulate bone or cartilage
formation
(33) .Longitudinal sections of the
humerus taken from mice at 9 months of age showed a bone marrow cavity
with normal cellularity, contained within a spongy bone shaft and
epiphysis (Fig. 7, e and f). Higher
magnification of the epiphyseal plate demonstrated zones of
proliferation ( p), hypertrophy ( h), and calcification
( c) of normal thickness compared to wild type samples
(Fig. 7, g and h). Thus, NGFI-A does not appear
to be required for bone or cartilage formation. These results confirm
the findings in ES cells that differentiation into a variety of cell
types remains intact despite the absence of NGFI-A.
-cardiac myosin heavy chain gene (
CMHC)
(32, 37) . Our observation that
NGFI-A
ES cells can differentiate into
beating embryoid bodies, which occurs only when both
- and
-cardiac myosin heavy chain genes are expressed
(38) ,
suggests that NGFI-A is not essential for the expression of this gene.
lymphocytes, which is in direct
contrast to previous studies using antisense oligonucleotides directed
against NGFI-A
(21) . Furthermore, histological analysis of
NGFI-A
mice indicate a variety of cell types
including neurons, thymocytes, chondrocytes, and osteocytes. These
results reinforce our findings in ES cells that NGFI-A is not required
for many of the processes associated with its expression and suggest
either that the expression of NGFI-A is superfluous to these processes,
or that other factors or gene products provide functional redundancy.
It is notable that NGFI-A is a member of a gene family that includes
NGFI-C, EGR3, and Krox-20. Because these genes are often coordinately
regulated, a likely explanation may be that these genes provide
adequate functional compensation for the absence of NGFI-A without
exhibiting a compensatory increase in expression. Significant overlap
in function and expression has been observed in other gene families
involved in signal transduction, most notably the src family
of tyrosine kinases. Targeted mutation of these genes results in
minimal or undetectable phenotypes despite their widespread pattern of
expression. It may therefore be necessary to target other members of
the NGFI-A gene family before the full range of function of these genes
can be fully recognized.
act vector, Dr. Simon Halegua of the University of
Pennsylvania for the cyclophilin probe, and Dr. Stacy Smith of
Washington University for the gift of the 3A6.8 mAb. We especially
thank Kathy Frederick and Dave Donermeyer of Dr. Paul Allen's
laboratory at Washington University for help with the SCID mouse
injections and for performing the lymphocyte proliferation assays,
respectively. We also thank members of the Milbrandt laboratory for
their suggestions and comments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.