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Molecular and Cellular Biology, February 1999, p. 1558-1568, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Localization of Distant Urogenital System-, Central Nervous
System-, and Endocardium-Specific Transcriptional Regulatory
Elements in the GATA-3 Locus
Ganesh
Lakshmanan,1
Ken H.
Lieuw,1
Kim-Chew
Lim,1
Yi
Gu,1
Frank
Grosveld,2
James Douglas
Engel,1,* and
Alar
Karis2,3
Department of Biochemistry, Molecular Biology
and Cell Biology, Northwestern University, Evanston, Illinois
602081;
Department of Cell Biology and
Genetics, Erasmus University School of Medicine, Rotterdam 3000, Holland2; and
Institute of Molecular
and Cell Biology, University of Tartu, Tartu EE2400,
Estonia3
Received 2 September 1998/Returned for modification 8 October
1998/Accepted 26 October 1998
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ABSTRACT |
We found previously that neither a 6-kbp promoter fragment nor even
a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3
gene was sufficient to recapitulate its full transcription pattern
during embryonic development in transgenic mice. In an attempt to
further identify tissue-specific regulatory elements modulating the
dynamic embryonic pattern of the GATA-3 gene, we have examined the
expression of two much larger (540- and 625-kbp) GATA-3 YACs in
transgenic animals. A lacZ reporter gene was first inserted
into both large GATA-3 YACs. The transgenic YAC patterns were then
compared to those of embryos bearing the identical lacZ
insertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ "knock-ins"). We found that most of the
YAC expression sites and tissues are directly reflective of the
endogenous pattern, and detailed examination of the integrated YAC
transgenes allowed the general localization of a number of very distant
transcriptional regulatory elements (putative central nervous system-,
endocardium-, and urogenital system-specific enhancers). Remarkably,
even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and
150 kbp of 5' and 3' flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus
and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.
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INTRODUCTION |
GATA-3 belongs to a family of
transcription factors that bind to the consensus sequence
(A/T)GATA(A/G) and share a steroid hormone receptor superfamily
C4 zinc finger DNA binding motif (14, 24, 27,
41) that is also evolutionarily conserved in lower eucaryotic
GATA factors. The GATA factor family is composed of six vertebrate
members (1, 19, 41), and from gene ablation studies,
GATA-1 through GATA-4 have been shown to be individually indispensable for embryonic development (26, 28, 32, 37, 38). GATA-1 is expressed in myeloerythroid lineage cells and Sertoli cells of the testis (13, 25, 33). GATA-2 is
expressed in multipotential hematopoietic progenitors, megakaryocytes,
mast cells, and endothelial cells as well as in an overlapping pattern with GATA-3 in the placenta and central nervous system (CNS)
(6, 15, 29, 30, 34). GATA-3 is, like GATA-2,
expressed more widely than GATA-1, and it is the only family member
expressed in T lymphocytes (41, 42). Based on comparisons of
cDNA sequences and intron/exon boundaries, the GATA-4, -5, and -6 factors constitute a distinct subfamily, principally implicated in
cardiac and ventral/dorsal patterning (28).
Previous in situ hybridization analysis showed that GATA-3
transcription is controlled both temporally and spatially during early
embryonic development (8, 31). GATA-3 mRNA is detected at high levels in the ectoplacental cone at 8.5 days postcoitus (dpc)
and persists over the course of the next several gestational days.
Within the embryo proper, GATA-3 is expressed first and most
abundantly in the CNS and peripheral nervous system (PNS), the kidney,
the adrenal gland, and the primitive thymus; these initial experiments
also suggested that there might be weak expression in the heart and the
skin (8).
Our initial transgenic experiments examining GATA-3 transcriptional
regulation using plasmid constructs identified a number of discrete
regulatory elements (namely, the genital tubercle and branchial arch
elements), but these promoter proximal sequences were clearly unable to
fully recapitulate the wild-type developmental expression pattern of
the GATA-3 locus (20). Therefore, we isolated and
characterized yeast artificial chromosomes (YACs) bearing the
GATA-3 gene, in anticipation that they would provide a means of
analyzing this locus as a single, large contiguous fragment of genomic
DNA (17). We identified two GATA-3 YACs that together delineate approximately 1 Mbp of gene flanking sequence, with the
GATA-3 structural gene located approximately in the center. We
found that a 120-kbp YAC lacZ reporter transgene (called
C4lacZ), which contains approximately 35 kbp of 5'
GATA-3 flanking sequence as well as 60 kbp of 3' GATA-3
flanking sequence, could direct the expression of the reporter gene at
new anatomical sites not identified previously in the smaller plasmid
expression constructs. However, even this 120-kbp YAC failed to direct
the expression of the reporter gene in several tissues that are known
to normally express GATA-3. In accord with this observation, the
120-kbp C4 YAC was not able to rescue embryonic lethality caused by the
original gene targeted mutation (17).
To localize the regulatory determinants of its expression during
embryogenesis, we have significantly extended the boundaries of the
murine GATA-3 locus under scrutiny. Here, we describe the expression profile of a lacZ reporter gene that was targeted
to the initiation codon of the chromosomal GATA-3 gene in ES cells, generating GATA-3/lacZ "knock-in" mice (11,
44) as the reference point. We then asked whether large
transgenic YACs encoding GATA-3 could reproduce this same pattern.
In independent transgenic lines bearing a 625-kbp GATA-3 YAC, the
lacZ transgene reflected the endogenous expression profile
in all tissues except the thymus and specific neural crest-derived
cells (i.e., the sympathetic chain and the adrenal gland), while the
smaller, 540-kbp YAC conferred a less complete pattern.
Surprisingly, these studies indicate that the complete
GATA-3 locus lies beyond the boundaries of even the largest YAC
(625 kbp) examined here. Nonetheless, detailed structural analysis of
these integrated transgenes led to the general localization of at least
three positive regulatory elements that direct the expression of
GATA-3 in distinct tissues. The element(s) required for GATA-3
expression in the endocardial cushions of the embryonic heart is
located quite far from the 3' end of the gene, between +105 and +145
kbp with respect to the GATA-3 transcription initiation site. At
least two other tissue-specific element(s) are located far 5' to the
gene: these elements regulate GATA-3 expression in the developing
CNS and the urogenital system, and reside between 
6 to 35 kbp
and 35 to 150 kbp, respectively. These studies also lead
to the conclusion that the patterning elements controlling GATA-3 expression in specific cell lineages derived from the neural crest (in the sympathetic chain and in the adrenal medulla) are located
beyond the boundaries of the 625-kbp YAC and therefore lie more than
450 kbp 5', or more than 150 kbp 3', to the GATA-3 structural gene.
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MATERIALS AND METHODS |
LacZ targeting of GATA-3 YACs.
The
Escherichia coli lacZ gene was targeted into the initiation
codon within the first coding exon of the GATA-3 gene in B124 and
B125 YACs by homologous recombination in yeast (4, 40), as
described previously (17). The resulting YACs therefore
precisely mimic the structure of the original term line targeted
mutation (32) as well as that of the lacZ gene in
the knock-ins (12).
Preparation and analysis of high-molecular-weight DNA.
Yeast
and mouse thymic DNA agarose plugs were prepared as described
previously (17). Pulsed-field gel electrophoresis (PFGE) was
performed by using 1% agarose gels in 0.5× Tris-borate-EDTA at
14°C. For resolution of DNA up to 2 Mbp in size, the electrophoresis conditions were 120 V with 10- to 200-s ramped switch time for 20 h. The gels were transferred onto nylon membranes (BioRad) and
hybridized at 65°C. The blots were then washed and finally exposed
for autoradiography. Probes were generated by random primer labeling
(7).
Isolation of YAC DNA for microinjection.
The protocol used
was essentially as described (17) except for the following
modifications. For the 540- and 625-kbp YACs, a 25- to 80-s ramped
switch time was used for 20 h. The YAC DNA was excised, rotated
90° (perpendicular to the electric field), and cast in a NuSieve 4%
agarose gel (FMC Corp.). The electrophoresis conditions used were 300-s
ramped switch time for 15 h. The concentrated YAC DNA band was
excised and equilibrated with injection buffer (10 mM Tris-HCl [pH
7.2], 0.1 mM EDTA, 70 mM spermine, and 30 mM spermidine) on ice for 2 to 24 h prior to digestion with 
-agarase (4 U/100 µl) for 2 to 4 h. Finally, the YAC DNA was dialyzed against injection buffer
by using a floating dialysis membrane (100-kDa exclusion limit) for 2 to 24 h. The integrity of the YAC DNA was verified by PFGE prior
to microinjection.
Transgenic mice.
Transgenic mice were generated using
standard protocols (20) and identified by PCR by using
lacZ and YAC left and right vector arm primers, and by
Southern blotting (17).
LacZ staining and tissue sectioning.
The morning that
vaginal plugs were detected was designated 0.5 dpc. Embryos were
isolated at gestation days from 10.5 to 18.0 and stained with X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) (39). They were then frozen in O.C.T. compound (Sakura
Finetek) and sectioned at 10-µm thickness prior to counterstaining
with nuclear fast red.
Genomic mapping.
The generation of probes used in mapping
the transgenic YAC lines has been described previously (17,
20). To illustrate the analysis that must be performed to
characterize the integrity of YAC transgenes in each line, the
deduction of the transgene structure of the most complicated multicopy
line described in the present study, B125Z89 (see Fig. 1E), is
discussed below.
Since the transgenes in line B125Z89 segregated together, we assumed
that they represented multiple copies integrated at a single genomic
site. The 5'-most probe, p138, is located at approximately 400 kbp
with respect to the GATA-3 transcription start site (see Fig. 1E
and also reference 17) and hybridized to one band of approximately 650 kbp in line B125Z89 (see Fig. 1A) as well as to an
endogenous 1-Mbp NotI fragment and a nonspecific band (see Fig. 1A through D). The 5' p143 probe lies at approximately 125 kbp
(17) and hybridized to two bands that were 650 and 900 kbp as well as to the same endogenous and nonspecific bands as the p138
probe (see Fig. 1B). These data indicated that line B125Z89 contained
one transgene copy (represented by the 650-kbp band) that was
contiguous between 400 and 125 kbp, and that a second copy (900 kbp) was fragmented somewhere between these two points. The 5' N probe
is located at 4.5 kbp (17, 20) and hybridized to the same
650- and 900-kbp fragments as well as to an additional 280-kbp band
(see Fig. 1C). This indicated that both larger bands (i.e., 650- and
900-kbp bands) were contiguous from 125 to 4.5 kbp, while the
280-kbp band represented a third YAC copy that was fragmented between
125 and 4.5 kbp.
The 3' p131 probe, which is located at approximately +80 kbp,
hybridized to a full-length
NotI fragment (175 kbp) that is
more intense than a single-copy band, as well as to three larger
fragments. Thus, the 3' p131 probe detected five transgene copies,
three of which were fragmented between +80 kbp and the right arm
of the
YAC, while the two remaining copies represent intact 175-kbp
3' end
fragments. Similar mapping studies using
SfiI restriction
endonuclease (
16) demonstrated that both the 5' N and p131
probes
(Fig.
1E) detect no aberrant bands, and thus all the transgenes
that contain the mGATA-3 gene in line Z89 are contiguous within
the
5' 120-kbp or the 3' 100-kbp
SfiI fragments. In summary, the
data indicate that none of the breakpoints in any of the transgene
copies in this line occurred between
110 and +80 kbp, including
the
entire 23-kbp GATA-3 structural gene (
8,
17,
20). Thus,
both the sets of data for
SfiI (
16) and
NotI restriction mapping
(see Fig.
1A through D) indicate
that the minimum contiguous mGATA-3
lacZ YAC present in
line Z89 must be >500 kbp but leave open the
equally likely
possibility that this line carries one intact transgene
copy.
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RESULTS |
Generation of GATA-3 YAC transgenic lines.
YAC B124
contains approximately 450 kbp of the 5' end genomic information, the
GATA-3 structural gene (approximately 25 kbp; see reference
8), and 65 kbp of 3' end genomic information, while
YAC B125 is identical to YAC B124 except for 85 kbp of additional sequence at the 3' end (8, 17). Both YACs were modified by insertion of the lacZ gene at the site of the GATA-3
initiation codon by sequential homologous integration and excision in
yeast (4, 17). Gel-purified B124lacZ and
B125lacZ YAC DNAs were injected into fertilized ova to
generate transgenic founders (4, 5).
Thirteen transgenic founders bearing the larger, B125
lacZ,
YAC were obtained from 133 pups. Of these, ten transmitted the
transgene through the germ line, and seven of them contained the
lacZ gene as well as both the left and right YAC vector arms
as
detected by the initial PCR screens. The copy numbers of the
B125
lacZ YAC lines ranged from one to five (Fig.
1, and data not shown).
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FIG. 1.
Structural integrity of the B125lacZ
transgenes. Agarose plugs containing thymus DNA from the transgenic
lines Z70, Z71, Z72, Z73, and Z89 were digested in situ with
NotI restriction endonuclease. After PFGE electrophoresis,
the DNA was transferred to a nylon membrane and independently
hybridized to either p138 probe (A) or p143 probe (B) or 5' N probe (C)
or p131 probe (D). (E) Summary diagram for five of the
B125lacZ YAC transgenic lines. The top line shows an
abbreviated map of the GATA-3 locus (S denotes SfiI sites;
data not shown) and the positions of several markers used as probes, as
well as the positions of the two NotI (N) sites in the YAC
at 4.5-kbp (20) in the locus and in the YAC right vector
arm. The shaded line represents mouse DNA flanking the integrated
transgene. The actual order of integration of multicopy transgenes
(lines Z72 and Z89) is arbitrary, since they have not been determined,
but all are integrated at a single site in the mouse genome. For those
two multicopy lines, it is not possible to determine which fragment
lying 5' to the genomic NotI site is physically contiguous
with which fragment lying 3' to the site, and thus the 5' and 3'
fragments in these two lines are depicted as separate and not
connected. Lines Z70, Z71, and Z73 contain fragmented, single-copy
transgenes. E, endogenous mGATA-3 NotI fragment; N,
nonspecific hybridization.
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Thymus nuclei recovered from each established line were digested with
NotI or
SfiI followed by PFGE. The DNA was
transferred
to nylon, and the blots were then probed with radiolabeled
DNA
fragments from throughout the locus (p138, p143, 5' N and p131;
Fig.
1), hence enabling us to identify the approximate positions
of
breakpoints in the integrated transgenes (
4,
17,
23).
These
experiments, for which a representative description is provided
in
Materials and Methods, showed that four of the B125
lacZ
lines
contained large internal segments of the GATA-3 locus,
including
unaltered 5' and 3'
SfiI fragments flanking the
unique
NotI site
at
4.5 kbp (Fig.
1E; references
8 and
20). A summary of
the
B125
lacZ YAC maps in the five lines characterized here is
diagrammed in Fig.
1E. Although only two of the five lines harbored
transgenes that contained large uninterrupted blocks of contiguous
B125
lacZ YAC DNA, comparison of the integrated transgene
structures
to the expression patterns of these YAC lines turned out to
be
uniquely informative (see below and
Discussion).
Six transgenic lines bearing the smaller, B124
lacZ, YAC were
obtained from 52 pups, and five were determined by PCR to contain
the
lacZ gene and both left and right YAC vector arms. Four of
the five lines transmitted the transgene through the germ line,
while
one line was mosaic (multiple sites of transgene integration)
and
therefore was not characterized further. When the four remaining
lines
were analyzed for copy number and integrated YAC structure,
two were
found to contain intact B124
lacZ transgenes, while the
other
two were badly fragmented and hence not examined further.
The copy
numbers of the four B124
lacZ YAC transgenic lines ranged
from one to three (
16).
GATA-3 expression from the chromosomal locus.
In order to
establish a reference point for endogenous GATA-3 expression, we
characterized the pattern for GATA-3LacZ/+
knock-in mice and compared this pattern to that detected by in situ
hybridization (8, 31). Generation of the
GATA-3LacZ knock-in allele, which results in
precisely the same lacZ insertion in the GATA-3 genomic
locus in ES cells as that in the YACs, has been described
(12). Although both the YAC and ES cell targeting events
placed the reporter gene at the GATA-3 initiation codon, one
difference was that the knock-in reporter gene incorporated a nuclear
localization signal, thus allowing us to clearly distinguish cell
morphology in expressing tissues.
Whole-mount staining for
-galactosidase of
GATA-3
LacZ/+ embryos showed that GATA-3
expression began in the ectoplacental cone around
8.5 dpc and was also
observed in the branchial arches and cloaca
or genital tubercle
(
16,
19). Strong staining was also noted
in the midbrain,
hindbrain, and spinal cord (abbreviated collectively
as the CNS below;
references
16 and
19-22). By
10.5 dpc, expression
became more intense in the CNS and in the
branchial arches and
was also prominent in the otic vesicle, the
developing eye, the
heart, the mesonephric duct, and the cloaca (Fig.
2A and below).
By 12.5 dpc, the pattern
was largely unchanged, with localized
expression in the CNS, in the
developing eye, organs of the inner
ear, the jaw, and the neck region
(elaborated from the developing
branchial arches) as well as in
proximal regions of the developing
limbs, the mesonephros, and the
mesonephric ducts (Fig.
2B). Expression
was also apparent in the
developing sympathetic trunk (see below)
and umbilical vessels. The
cloaca continued to strongly express
GATA-3 as it developed into
the urogenital sinus and rectum (see
below). All of these sites and
times of expression were previously
detected in the in situ
hybridization studies (
8).
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FIG. 2.
LacZ expression of
GATA-3LacZ/+ versus
GATA-3lacZ YAC transgenic embryos. The expression
patterns of GATA-3LacZ/+ and two GATA-3
B125 lacZ YAC transgenic lines are shown here at three
developmental stages (10.5, 12.5, and 13/14 dpc) for direct comparisons
of their coincidence. The embryos were cleaved roughly along the
lateral midline and then stained with X-Gal. The 10.5- and 12.5-dpc
embryos are displayed as the outside halves of the embryos, while the
13/14-dpc embryos are displayed as a view with the internal organs
presented en face. A, B, and C, GATA-3LacZ/+
embryos at 10.5, 12.5, and 13 dpc, respectively; D, E, and F, line
B125Z71 transgenic embryos at 10.5, 12.5, and 14 dpc, respectively; and
G, H, and I, line B125Z89 transgenic embryos at 10.5, 12.5, and 13 dpc,
respectively. Although the intensity of staining varies, consistent
patterns of expression are detected in all three cases in the CNS
(labeled mb, hb, and sc, for midbrain, hindbrain, and spinal cord,
respectively), the eye (e), the head mesenchyme (hm), the otic vesicle
(ov), the branchial arches/jaw (ba/j), the limb buds (lb), the heart
(h), the umbilical vessels (uv), the mesonephric duct (md), the
ureteric bud (ub), the urogenital sinus (us), the ureters (u), and the
kidneys (ki). The labeling in the lung of the single-copy Z71 embryo
(panel F) was not detected in the other B125lacZ lines nor
in the GATA-3LacZ/+ embryos and therefore
was rejected as ectopic staining due to the transgene integration
position.
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By 14 dpc,
-galactosidase continued to be most predominantly
expressed in the CNS (Fig.
2C) and in the sympathoadrenal system
(see
below). As the embryo matured further, expression diminished
in the
spinal cord but persisted in the midbrain. The eyes, the
semicircular
canals of the inner ear, the jaws (both mandibular
and maxillary
structures), the neck region, and the base of the
tongue also continued
to express
lacZ (Fig.
2C). In the circulatory
system,
expression was confined to the base of the heart, outflow
vessels, and
umbilical vessels (Fig.
2C), but by days 14 to 15,
when the development
of the fetal circulatory system was complete,
expression had vanished
from these
sites.
In the urogenital system, staining was strong in the mesonephros and
mesonephric duct (Fig.
2C). At later stages in embryogenesis,
expression persisted in structures derived from the mesonephros
(the
epididymis) and the mesonephric ducts (the van deferens;
see below).
Expression was quite prominent in the metanephric
duct and ureteric bud
and continued as the metanephric duct differentiated
into the renal
collecting tubules and the ureters (Fig.
2C and
below). The primitive
urinary bladder, derived from the ventral
aspect of the urogenital
sinus, strongly expressed the reporter
gene (Fig.
2C and below), and
this expression persisted until
bladder development was complete. The
epithelium lining the mesonephric
tubules and ducts also expressed
-galactosidase (data not shown).
The tubules derived from the
metanephric duct, which eventually
contribute to the adult kidney, also
expressed the
lacZ gene (e.g.,
Fig.
3A). The epithelium lining the bladder
was also stained (
16),
as were neural crest descendent cells
that contributed to the
adrenal medulla (Fig.
3A).
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FIG. 3.
Differential expression of 14.5-dpc
GATA-3LacZ/+ and B125lacZ
embryos. A and B, axial sagittal sections at the level of the
developing kidney and adrenal gland. The cells of the adrenal medulla
(ad) stain only in the GATA-3LacZ/+ embryos,
while the tubules of the kidney (ki) in both embryos express
-galactosidase. C and D, transverse sections at the axial level of
the cervical sympathetic trunk (st). The sympathetic trunk is negative
for -galactosidase expression in the YAC embryo (panel C), while the
GATA-3LacZ/+ allele is expressed strongly
(panel D). E and F, sagittal sections of developing hair follicles (hf)
in the skin, showing expression of -galactosidase in the dermal
papillae of both GATA-3LacZ/+ and
B125lacZ embryos. G and H, sagittal sections showing
expression in the developing mammary glands (mg) of both
GATA-3LacZ/+ and B125lacZ89
embryos.
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In the developing ear, expression was confined to the developing
semicircular canals, saccule, and cochlea (Fig.
4A).
-galactosidase
expression was
also detected in the ganglion of the vestibulocochlear
cranial nerve.
However, we found no evidence for expression in
the trigeminal and
facial ganglia, as reported previously for
our in situ hybridization
experiments (
8). Sectioning through
the developing eye
revealed that expression was confined to the
differentiating cuboidal
cells in the equatorial zone that form
secondary lens fibers (Fig.
4D).
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FIG. 4.
Expression in the ear, eye, nervous system, and heart of
GATA-3LacZ/+ and B124lacZ and
B125lacZ embryos. A through C, transverse sections at the
level of the developing ear showing expression of -galactosidase in
saccules of the semicircular canals (scc) in the inner ear of 12.5-dpc
embryos. D through F, sagittal sections at the level of the developing
eye of 12.5-dpc embryos showing expression of -galactosidase in the
posterior lens fibers (lf). G through I, transverse sections at the
level of the thoracic spinal cord. Neurons expressing -galactosidase
are located in the ventrolateral (vln) areas of the spinal cord in
these 13.5- to 14.5-dpc embryos. J through L, transverse sections at
the level of the developing heart. Note that the endocardial cushions
(ec) express -galactosidase in both the
GATA-3LacZ/+ and B125lacZ
13.5-dpc embryos, but not in the B124lacZ embryo at the same
stage.
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In the heart, where only weak expression was detected in our previous
in situ experiments (
8), sectioning of the
lacZ
gene-targeted
embryos showed that expression was confined to cells that
contribute
to the endocardial cushions at the atrioventricular junction
as
well as in the outflow track (Fig.
4J) and was temporally visualized
only between 10.5 and 14 dpc. Caudal cervical cross-sections revealed
that
-galactosidase was expressed in the developing thyroid gland
(data not shown), which was also not evident from in situ
hybridization.
Other sites of expression included the mammary gland
(Fig.
3G)
and the hair bulb and dermal papillae of hair follicles (Fig.
3E). Since expression in the mammary gland and hair follicles
was also
observed for a 6-kbp GATA-3 promoter reporter transgene
(
20), these data indicate that these two sites were simply
overlooked
in the earlier in situ hybridization analysis
(
8).
GATA-3 expression from 540- and 625-kbp YAC reporter
transgenes.
The expression patterns of the
GATA-3LacZ/+ mice revealed by whole-mount
staining (Fig. 2 and 5) and tissue
sectioning (Fig. 3 and 4) were compared to the patterns reflected in
two B125lacZ YAC transgenic lines as well as in two
B124lacZ transgenic lines at multiple stages during
embryogenesis. Only the most informative sections are reproduced here
to highlight specific tissue or temporal expression differences among
the three different kinds of mice examined
(GATA-3LacZ/+ knock-in mice and
B124LacZ or B125LacZ YAC transgenic embryos).
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FIG. 5.
Expression of YAC B125lacZ during urogenital
and renal development. (A) A 11.5-dpc B125Z71 embryo displaying
lacZ expression in the mesonephric duct and the ureteric bud
(ub), which gives rise to the adult kidney. The ureteric bud is
beginning to expand into the metanephric blastema (mb). The urogenital
sinus (us) also stains. (B) A 12.5-dpc embryo displaying expression in
the metanephric blastema (mb) that has differentiated from the ureteric
bud to form the primitive renal pelvis. This embryo also displays
transgene expression in remnants of the mesonephros (ms) and the
mesonephric duct (md). (C, D, and E) The same region, or the isolated
organs, from 14.5-, 15.5-, and 16.5-dpc embryos, respectively. The
mesonephros has regressed, while the metanephros (the definitive
kidney; ki) continues to differentiate, enlarge, and gradually ascend
rostrally from the us site of origin. The metanephric (collecting)
tubule continues to divide to form the collecting system and expresses
intense -galactosidase activity (D, E, and F). (F) A dissected
kidney attached to its ureter (u), and the testis (te), epididymis
(ep), and vas deferens (v) (the ep and v are derived from the
mesonephric duct) of an embryo at 17.5 dpc; strong -galactosidase
activity is detected throughout the collecting system and in the ep.
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For the B125
LacZ YAC studies, transgenic line Z71 was chosen
for further analysis because it bears only one transgene copy.
NotI and
SfiI mapping of this line showed not
only that the 5'
breakpoint in the Z71 transgene lies between 125 and
150 kbp from
the GATA-3 transcription start site (Fig.
1B and C)
but also that
the entire 3' end (175 kbp) is intact (Fig.
1D). Line Z89
was
also selected for further analysis because, in addition to
providing
independent confirmation for genuine sites of B125 YAC
expression,
it bears at least one substantially intact copy of the
entire
625-kbp YAC (Fig.
1E). Both the Z71 and Z89 transgenic YAC
expression
patterns did not significantly differ from one another
(other
than ectopic staining in the lungs of Z71 embryos; Table
1 and
Fig.
2D to I), leading us to
conclude that many of the regulatory
elements controlling GATA-3
transcription (see below) must lie
within an approximately 300-kbp
radius surrounding the gene described
by transgene B125Z71 (containing,
at most, 150 kbp of 5' end as
well as an intact 175-kbp 3' end). The
B124
LacZ YAC contains 85
kbp less 3'-end genomic information
than the B125
lacZ YAC, and
thus comparison of the two
expression patterns allowed us to further
delimit the position of 3'
GATA-3 regulatory sequences (Table
1 and Fig.
3 and
4).
To precisely define sites of
-galactosidase expression at
the cellular level, we sectioned
GATA-3
LacZ/+, B124
lacZ, and
B125
lacZ transgenic embryos at 12.5 and 14.0 dpc.
These
observations showed that the CNS patterns of the YAC transgenic
animals
were coincident with that in the germ-line mutant
lacZ-targeted
animals (Fig.
2). At 12.5 dpc, expression in
the brain was confined
to the mantle layers of the diencephalon, the
mesencephalon, and
the pontine region of the metencephalon. Expression
was also found
in the myelencephalon, which forms the medulla oblongata
(Fig.
2, and data not shown), and in ventrolateral neurons within the
spinal cord (Fig.
4G). However, in the cervical region of the
spinal
cord, neither YAC was expressed in neurons of the sympathetic
trunk
(e.g., Fig.
3D).
Comparisons among the GATA-3
LacZ/+
knock-in and B124
lacZ and B125
lacZ YAC transgenic
embryos demonstrated that most of the sites
of expression are
coincident (Table
1 and Fig.
2 to
4). The tissues
in which neither YAC
transgene is expressed are the thymus (see
the Discussion) and specific
differentiated lineages contributing
to the sympathetic nervous system
(e.g., in the adrenal gland
[Fig.
3B] and the sympathetic trunk
[Fig.
3D]). Therefore, regulatory
elements controlling expression in
those cell types are not present
in either
YAC.
In summary, analysis of the expression patterns of the
lacZ
transgenes and the GATA-3
LacZ/+ knock-in
mice allows further refinement of the position of
cis-regulatory
elements directing GATA-3 expression.
When we compared the profile
to that previously detected by in situ
hybridization (
8), a
promoter GATA-3 transgene
(
20), or a smaller, 120-kbp YAC reporter
transgene
(C4
lacZ; reference
17), we found that a
number of
previously unresolved
cis-regulatory elements for
GATA-3 could
be roughly positioned within the locus (Table
1 and
Fig.
6).
Taken together, detailed
comparisons to earlier data reveal that
the elements controlling the
expression of GATA-3 in the ectoplacental
cone, the CNS, the eye,
and the thyroid gland all lie within the
smallest (120 kbp) C4 YAC
(
17) but outside the boundaries described
by the GATA-3
promoter transgene, which is expressed in the branchial
arches, genital
tubercle/cloaca, mammary glands, and whisker follicles
(
20).
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 6.
Summary of localization of GATA-3 regulatory
elements. The top of the diagram depicts the genomic GATA-3 locus
(bold line), while the bottom shows the positions of several regionally
defined regulatory elements in the locus. The 5'-most element
identified here contains the urogenital element(s) and was defined by
the 5' breakpoint in the B125Z71 transgene at approximately 150 kbp
and at its 3' boundary by the 5' end of YAC C4Z (17) at 35
kbp. The CNS element(s) is defined by the 5' end of the C4Z transgene
at 35 kbp and the NotI site at 4.6 kbp (17,
20). The branchial arch (BA) and genital tubercle (GT) elements
were defined previously (20). The element conferring
GATA-3 expression in the endocardial cushions was originally
defined by the differences in the 3' boundaries of the B124 (+85 kbp)
and B125 (+175 kbp) lacZ transgenes (above), and more recent
studies have localized the element to within +105 and +145 kbp 3' to
the gene (see Discussion and reference 10).
|
|
Both B125
lacZ and B124
lacZ YAC transgenes
reproducibly express
-galactosidase in the umbilical vessels
(data not shown) and
in the developing urogenital system,
particularly in the mesonephric
duct as well as in the
metanephric duct and ureteric bud (Fig.
5). Therefore, the
elements controlling GATA-3 expression in the
umbilical vessels and
in the mesonephric and the metanephric ducts
lie beyond the boundaries
of the 120-kbp C4 YAC but inside the
boundaries described by both the
B124
lacZ and B125
lacZ YACs (Fig.
1E). Since the
C4 and B124 YACs share the same 3' genomic boundary,
this deductively
narrows the positions of these elements to between
35 and
450 kbp
5' to the
gene.
Neither of the B124
lacZ transgenic lines expresses
-galactosidase in the endocardial cushion tissues in the heart (Fig.
4K),
while GATA-3
LacZ/+ and both
B125
lacZ lines express
-galactosidase there (Fig.
4J
and
L), demonstrating that the element(s) directing GATA-3 expression
in the endocardial cushion lie in very distant 3' flanking sequences.
These results delimit the position of a positive endocardium-specific
regulatory element for the GATA-3 gene to between +85 and +175
kbp
3' to the GATA-3 gene (also see the Discussion). Finally,
these
experiments demonstrate deductively that the
cis-regulatory
elements controlling GATA-3 in a specific subset of PNS derivative
lineages (the sympathetic trunk and the adrenal medulla) lie beyond
either 450 kbp 5' to or 150 kbp 3' to the GATA-3 structural
gene.
| |
DISCUSSION |
During the last decade, numerous technical advances have allowed
us to dissect and decipher the effects of specific genetic perturbations introduced into the mouse genome by homologous gene targeting. Homozygous loss of GATA-3 represents one typical
category of germ line-targeted mutants: embryos missing GATA-3
activity survive until midgestation but suffer multiple phenotypic
abnormalities at the time of death, including partially penetrant or
incompletely expressive malformation of the spinal cord, brain, and jaw
(32). The homozygous null mutant embryos also display other
fully penetrant defects (e.g., in either blood vessel formation or
vascular connection to internal organs) at the time of demise in utero.
Additionally, from other studies we know that defects in GATA-3
function profoundly affect the differentiation of cell lineages which
mature later in development (e.g., during T-cell differentiation;
reference 43), and these same defects would likely
be manifested in vivo were the mutant embryos to survive long enough to
initiate thymic organogenesis.
While gene ablation studies can sometimes provide a definitive answer
to the question of the functional significance of a particular gene of
interest, early embryonic lethal mutations can also serve as a
significant barrier to further analysis of the gene in lineages that
develop after the time of death. In order to circumvent this, various
strategies, such as conditional knockouts, have been developed
recently. Other alternate avenues include tissue- or lineage-specific
gene ablation and the analysis of hypomorphic alleles.
An alternative strategy distinct from those mentioned above would be to
define the entire genetic locus by isolation of completely complementing transgenes, thereby delimiting the boundaries for all
chromatin and transcriptional elements necessary to specify the
complete expression pattern of the gene of interest. With these genomic
sequences in hand, one could then methodically refine the positions of
individual tissue-specific regulatory regions and then examine the in
vivo consequences of deleting specific control elements (and therefore
ablation of specific cell lineages) from the fully complementing
transgene in a null mutant background. Since the developmental
expression profile of GATA-3 is dynamic in both space and time, and
since previous attempts to define the boundaries of the locus by using
either plasmid expression or small YAC constructs have failed, we have
examined even larger GATA-3 YACs in this study. The experiments
presented here underscore a major concern with this strategy of gene
rescue: even the largest (625 kbp) GATA-3 transgene lacks the
patterning element(s) that are critical for its expression in certain
sympathetic ganglia and the adrenal gland.
The studies presented here provide direct evidence that an upper limit
for the mouse GATA-3 locus has not yet been defined. However, we
explicitly note that there are several caveats to the overall
conclusions, since analysis of transgenes of this size presents several
unique challenges. First, it is clearly impossible to exhaustively
characterize every line with respect to the detailed internal structure
of the integrated transgenes. Thus, a small deletion or inversion (note
that even 1% of the B124 or B125 YAC transgenes would comprise 5 or 6 kbp) would probably be undetected in the PFGE assays. Second, and along
the same lines as the first complication, regardless of the detail
applied to both conventional and PFGE mapping, we cannot ever
completely eliminate the possibility that the original YACs differ
slightly in structure from the genomic locus. While one can surmount
objections regarding possible position-of-integration effects by
examining multiple transgenic lines, as we have done here, a third
caveat regarding the general utility of this strategy is that one can position regulatory elements by mapping breakpoints in the transgenes only if a breakpoint happens to occur at an informative position. In
other words, the breakpoint mapping strategy for positioning of
regulatory elements within the locus depends on fortuitous opportunity
rather than on intentional, directed mutagenesis.
Despite these multiple complications, this strategy has allowed us to
position several regulatory elements that confer proper patterning and
temporal expression to a target gene without reference to surrogate
methods, and indeed the overall strategy appears well suited for
defining very distant constituents of a genetic locus. For example, we
demonstrated previously that a 120-kbp YAC transgene (17)
confers expression in the CNS, whereas a 6-kbp mGATA-3 promoter
construct contained regulatory elements that were able to confer
expression in other sites where the gene is normally detected
(20).
Given that the 120-kbp C4 YAC did not confer expression at a number of
sites where GATA-3 is known to be transcribed (17), we
examined the expression patterns of two much larger YACs
and compared them with the pattern obtained from targeting
lacZ into the endogenous GATA-3 gene locus
(GATA-3LacZ/+). The
GATA-3LacZ/+ knock-in embryos showed
expression in all of the tissues where GATA-3 is synthesized,
including those where previous studies had indicated either ambiguous
or weak expression (Table 1), with the exception of the thymus. Neither
the heterozygous germ line mutant nor the YAC transgenic animals
display expression in the thymus, one of the most prominent sites of
midembryonic GATA-3 expression (41-43), indicating a
general failure of lacZ expression in T lymphocytes. While
lacZ could not be detected in thymocytes of any of these
animals by normal X-Gal staining protocols (Materials and Methods), its
detection was made possible by using a far more sensitive reagent,
FACS-Gal (12). We do not know, at the present time, why
detection of lacZ expression in the thymus is fraught with
such complications.
Transgenic animals bearing the 540-kbp B124lacZ and 625-kbp
B125lacZ YACs showed several sites of normal GATA-3
embryonic expression in addition to those found in mice bearing smaller transgenes; these sites include the heart, umbilical vessels, and
mesonephric and metanephric ducts (Table 1). While the position(s) of
regulatory elements within the B125 YAC that are required for the
generation of this expression pattern are not yet finely localized, they must lie beyond the limits of C4 YAC but within the boundaries described by B125lacZ transgene Z71, which is broken at the
5' end. The evidence indicates that the elements directing the
expression of GATA-3 in the umbilical vessels, the inner ear, and
the mesonephros and metanephros reside between 35 kbp (the 5' end of
the C series of YACs, including the C4lacZ; reference
17) and 150 kbp (the approximate 5' breakpoint in
B125lacZ line 71) 5' to the GATA-3 structural gene (Fig.
1E and Fig. 6). Expression in the endocardial cushions of the heart is
regulated by an element within the 85-kbp sequences that differ between
the B124 and B125 YAC 3' ends. More recent studies have refined the
position of this element to an approximately 45-kbp sequence within
this interval (Fig. 6; reference 10), and similarly,
the CNS element has now been refined to a single 1-kbp fragment lying
within the 6 to 35 kbp interval (22). Continued analysis
should allow us to resolve the precise position of the urogenital and
heart element(s) as well as the identity of putative upstream
developmental effectors (10, 22). In this manner, we hope to
determine the epistatic relationships in the regulatory cascades that
lead to GATA-3 function in specific organs.
Recapitulating a complex embryonic gene expression pattern that
parallels normal GATA-3 expression can be achieved by simply extending the limits of DNA surrounding the gene. This observation conceals several implications. First, the data suggest that expression of the transgene in these tissues is principally controlled by positive
transcriptional regulatory elements, since extending the sequences
under scrutiny from those lying very close to the gene (20)
to ones lying substantially further away adds to the pattern and
consistency of expression established by smaller constructs. Second,
the positive regulatory elements appear to be discrete, since addition
of new segments of DNA to those analyzed previously confers
reproducible -galactosidase accumulation at sites where GATA-3
is normally expressed.
To place the present analysis in perspective, it might be instructive
to compare these data to other better characterized genetic regulatory
models. The present data indicate that the full extent of the
GATA-3 locus includes at least 150 kbp of 3' and 150 kbp of 5'
flanking sequence information (Fig. 6). If we assume that the
regulatory elements controlling the (presently unaccounted for)
expression in the sympathetic chain and adrenal medulla lie immediately
outside of the B125 YAC, the locus must be minimally 325 kbp in size,
including the 25-kbp structural gene (8). Thus, the
GATA-3 locus is at least four times the size of the human
-globin gene locus (35), at least three times larger than
the known extent of any of the murine hox gene clusters (e.g., see
reference 9), and even somewhat larger than the entire mating type locus complex on yeast chromosome III (e.g., see
reference 36). Although other genes have been
inferred to have even more distant regulatory sequences (from mutant
mapping, breakpoint inversion, and similar genomic mapping
studies [references 2 and 3 and references
therein]), the mouse GATA-3 locus at present defines the most
distant regulatory elements contributing to a single gene locus that
has been characterized by direct physical isolation.
Many investigations have not shown that transcriptional control
elements linked to reporter genes can confer the wild-type expression
pattern to at least a subset of the appropriate tissues where and when
that gene is normally expressed. Nonetheless, despite a geometric
increase during the last decade in documenting regulatory elements that
are required for control of metazoan transcription, phenotypic rescue
of recessive loss-of-function mutants in the mouse has been achieved in
only surprisingly few cases. The studies presented here show that the
boundaries of mammalian loci which display complex embryonic expression
patterns may extend much further than has previously been assumed and
thus may represent one rather daunting hurdle that could be encountered
in attempts to rescue other developmental regulatory genes.
| |
ACKNOWLEDGMENTS |
Ganesh Lakshmanan and Ken Lieuw contributed equally to this work,
and both should be considered first authors.
We thank Jie Fan for outstanding technical assistance, members of the
Engel lab, in particular Jorg Bungert, Ko Onodera, and Yinghui Zhou,
for insightful discussions and help, and Gauri Tilak for assistance
with transgenic analysis. We also thank Rick Gaber, Rob Nakamura, and
Hong Liang for advice about yeast and for reagents.
This work was supported by an MSTP grant to Northwestern University
(NIH T32 GM08152; to K.H.L.), a Scanlon fellowship from Evanston
Hospital (to G. L.), research grants from the NWO (The Netherlands; to F.G. and A.K.) and the National Institutes of Health
(GM28896; to J.D.E.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Molecular Biology and Cell Biology, Northwestern
University, 2153 N. Campus Dr., Evanston, IL 60208-3500. Phone: (847)
491-5139. Fax: (847) 467-2152. E-mail: d-engel{at}nwu.edu.
| |
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Molecular and Cellular Biology, February 1999, p. 1558-1568, Vol. 19, No. 2
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Abstract
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