en%2E2014-1815

Adrenal Development in Mice Requires GATA4 and
GATA6 Transcription Factors
Sergei G. Tevosian, Elizabeth Jiménez, Heather M. Hatch, Tianyu Jiang,
Deborah A. Morse, Shawna C. Fox, and Maria B. Padua
Department of Physiological Sciences (S.G.T., E.J., H.M.H., T.J., S.C.F., M.B.P.), College of Veterinary
Medicine, University of Florida, Gainesville, Florida 32610-0144; and Department of Applied Physiology
and Kinesiology (D.A.M.), College of Health and Human Performance, University of Florida, Gainesville,
Florida 32611-8200
The adrenal glands consist of an outer cortex and an inner medulla, and their primary purposes
include hormone synthesis and secretion. The adrenal cortex produces a complex array of steroid
hormones, whereas the medulla is part of the sympathetic nervous system and produces the
catecholamines epinephrine and norepinephrine. In the mouse, GATA binding protein (GATA) 4
and GATA6 transcription factors are coexpressed in several embryonic tissues, including the ad-
renal cortex. To explore the roles of GATA4 and GATA6 in mouse adrenal development, we con-
ditionally deleted these genes in adrenocortical cells using the Sf1Cre strain of animals. We report
here that mice with Sf1Cre-mediated double deletion of Gata4 and Gata6 genes lack identifiable
adrenal glands, steroidogenic factor 1-positive cortical cells and steroidogenic gene expression in
the adrenal location. The inactivation of the Gata6 gene alone (Sf1Cre;Gata6flox/flox
) drastically
reduced the adrenal size and corticosterone production in the adult animals. Adrenocortical apla-
sia is expected to result in the demise of the animal within 2 weeks after birth unless glucocorticoids
are provided. In accordance, Sf1Cre;Gata4flox/flox
Gata6flox/flox
females depend on steroid supple-
mentation to survive after weaning. Surprisingly, Sf1Cre;Gata4flox/flox
Gata6flox/flox
males appear to
live normal lifespans as vital steroidogenic synthesis shifts to their testes. Our results reveal a
requirement for GATA factors in adrenal development and provide a novel tool to characterize the
transcriptional network controlling adrenocortical cell fates. (Endocrinology 156: 2503–2517,
2015)
The mature adrenal (also suprarenal) glands are paired
endocrine organs located anteriomedially to the re-
spective kidneys (Latin r៮en៮es). Each adrenal is composed
of 2 interdependent parts, the outer cortex and the internal
medulla, enclosed in a fibrous capsule. The cortex and
medulla have separate embryonic origins and separate
functions and are involved in the synthesis and secretion of
different hormones. The neuroendocrine medullar por-
tion of the gland is not essential for viability. However,
cortical hormones are required for life. Transgenic mouse
models played a major role in establishing the require-
ments for the specific compartments of the adrenal gland.
In rodents, the glucocorticoid (GC) corticosterone is re-
quired for normal lung development of the fetus (1), and
the mineralocorticoid aldosterone regulates sodium reten-
tion and water balance after birth (1–3). Recent research
also highlighted the role for GCs in stress and social adap-
tion during adolescence (4, 5).
The pioneering work by the Parker and Morohashi lab-
oratories (6–8) spearheaded the subsequent research ef-
fort that firmly established the commanding position of
the transcription factor steroidogenic factor 1 (SF1) (SF1/
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in USA
Copyright © 2015 by the Endocrine Society
Received October 6, 2014. Accepted April 20, 2015.
First Published Online May 1, 2015
Abbreviations: BrdU, 5-bromo-2Ј-deoxyuridine; Cyp21a1, Cytochrome P450, family 21,
subfamily a, polypeptide 1; DAPI, 4Ј,6-diamidino-2-phenylindole; DPBS, Dulbecco’s phos-
phate-buffered saline; E, embryonic day; EGFP, enhanced green fluorescent protein;
GATA, GATA binding protein; GC, glucocorticoid; 3␤HSD, 3␤-hydroxysteroid dehydro-
genase/⌬-5-4 isomerase; Ki67, antigen Ki67; IF, immunofluorescence; ISH, in situ hybrid-
ization; Ki67, antigen Ki67; NF, neurofilament; PND, postnatal day; qPCR, quantitative
RT-PCR; SF1, steroidogenic factor 1; TH, tyrosine hydroxylase; TUNEL, Terminal deoxynu-
cleotidyl transferase (TdT)-mediated dUTP nick end labeling; WT1, Wilm’s tumor 1.
O R I G I N A L R E S E A R C H
doi: 10.1210/en.2014-1815 Endocrinology, July 2015, 156(7):2503–2517 press.endocrine.org/journal/endo 2503
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adrenal 4-binding protein/nuclear receptor subfamily 5,
group A, member 1) in adrenal development. SF1 defines
steroidogenic cell identity in the fetal and adult adrenal
cortices, acts to promote steroidogenic cell proliferation,
and activates steroidogenic gene expression. In addition to
SF1, several other transcription factors and paracrine and
morphogenic pathways figure prominently in adrenocor-
tical development (reviewed in Ref. 9). The transcription
factor Wilm’s tumor 1 (WT1) is required for the initial
specification of the adrenocortical cell progenitors but has
to be down-regulated to commence steroidogenic differ-
entiation (10). GATA binding protein (GATA) 4 and
GATA6 transcription factors have long been implicated in
adrenal development (11) and adult adrenal function (12–
14; reviewed in Ref. 15). Recently, a role for GATA4
(along with WT1, zinc finger protein GLI1, and transcrip-
tion factor 21) in the long-living progenitor population
within the adrenal gland has been proposed (10). To de-
termine the function of the GATA proteins in the adrenal
gland, we used a Cre recombination approach to ablate
both Gata4 and Gata6 genes. We determined that adrenal
loss of GATA function is incompatible with adrenocorti-
cal development. Specifically, a combined loss of GATA4
and GATA6 in the precursor cells results in the loss of SF1
expression, decreased adrenocortical proliferation, and
adrenal agenesis in both sexes. The Sf1Cre;Gata4flox/flox
Gata6flox/flox
females (conditional double mutants) har-
boring this double deletion died. However, Sf1Cre;
Gata4flox/flox
Gata6flox/flox
males survived and had normal
lifespans, likely due to the shift of the vital steroidogenic
synthesis to their testes. GATA6 loss alone is compatible
with adrenal development and leads to adrenal hypopla-
sia. In summary, GATA6 serves as a principal driver of
adrenocortical cell maintenance, whereas GATA4 protein
acts in an ancillary role, carrying out basic regulatory
functions to support the requisite number of steroidogenic
cells to assure animal viability in the absence of GATA6.
Materials and Methods
Mouse strains
All animal experiments were approved by the University of
Florida Institutional Animal Care and Use Committee. The
Gata4flox/flox
(16) and Gata6flox/flox
(17) strains were obtained
from The Jackson Laboratory, and the transgenic Sf1Cre
mice were a kind gift from the late Dr Keith Parker (18).
Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
males were backcrossed with
Gata4flox/flox
Gata6flox/flox
(double flox) females to generate
Sf1Cre;Gata4flox/flox
Gata6flox/flox
(conditional double mutant)
animals. ROSAmT/mG
males (19) (also from The Jackson Lab-
oratory) were crossed with double flox females to obtain
ROSAmT/mG
;Gata4 flox/flox
Gata6flox/flox
animals. ROSAmT/mG
;
Gata4flox/flox
Gata6flox/flox
were backcrossed with Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
to generate the Cre-reporter animals
(ROSAmT/mG
;Sf1Cre;Gata4flox/flox
Gata6flox/flox
) with Gata de-
letions. Gata4flox/flox
Gata6flox/flox
(Sf1Cre-negative) animals
were used as experimental the controls. The primers used for
genotyping were obtained from Integrated DNA Technologies
and are shown in Supplemental Table 1.
Immunofluorescence (IF) staining
Torsos and adrenal glands were collected from the controls,
Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
, Sf1Cre;Gata4flox/flox
Gata6flox/flox
,
ROSAmT/mG
, and ROSAmT/mG
;Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
mice at embryonic day (E)13.5, E15.5,
and fixed overnight in 4% (wt/vol) paraformaldehyde. Samples
were dehydrated and then rehydrated in graded methanol series
followed by overnight saturation in 30% (wt/vol) sucrose. Optimal
cutting temperature-embedded sections were processed as previ-
ously described (20, 21). The primary and secondary antibodies
used are listed in the antibody table (Table 1).
RNA extraction and cDNA synthesis
Total RNA was isolated from controls and Sf1Cre;Gata4flox/flox
Gata6flox/flox
testes at E15.5 and 18.5. Similarly, RNA was isolated
from controls, Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
and Sf1Cre;
Gata4flox/flox
Gata6flox/flox
adrenal glands using the TRI Reagent
(Sigma-Aldrich) following the manufacturer’s instructions. The
details of the procedure are described in Supplemental Materials
and Methods.
Quantitative RT-PCR (qPCR)
Power SYBR Green PCR Master Mix (Applied Biosystems)
was used to perform qPCRs in an ABI 7500 system (Applied
Biosystems). The primers (Integrated DNA Technologies) used
for qPCRs are listed in Supplemental Table 2. The details of the
procedure are described in Supplemental Materials and
Methods.
Immunohistochemistry
Ovaries at postnatal day (PND)4 and testes at E18.5 from
controls and Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals were
fixed as described above for the IF experiments. OCT-embedded
samples were sectioned on the cryostat (5–7 ␮m) and mounted
on slides. Sections were blocked with BLOXALL (Vector Lab-
oratories, Inc) for 30 minutes at room temperature followed by
one-hour incubation with a rabbit anti-Cytochrome P450,
family 21, subfamily a, polypeptide 1 (CYP21A2) antibody
(Sigma-Aldrich) in Dako diluting buffer (Dako North Amer-
ica, Inc) at room temperature. CYP21A2 antibody was de-
tected with the immPRESS 3, 3Ј-diaminobenzidine peroxidase
reagent (Vector Laboratories, Inc). Hematoxylin (Fisher Sci-
entific) was used for counterstaining.
Proliferation assays
5-Bromo-2؅-deoxyuridine (BrdU) and antigen Ki67
(Ki67) proliferation assays
Pregnant females were sacrificed after 2 hours of receiving an
ip injection of BrdU (Sigma-Aldrich) at 0.1 mg/g of body weight.
Embryos were harvested and processed as described above for
2504 Tevosian et al GATA Factors in Adrenal Development Endocrinology, July 2015, 156(7):2503–2517

the IF experiments. The staining procedure and statistical anal-
ysis are described in detail in Supplemental Materials and Meth-
ods. For Ki67, IF analysis was performed as described above,
with adrenal sections from the controls and Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
fetuses (n ϭ 3) costained with the antibody against
the proliferation-associated Mki67 protein and the antibodies
against SF1, GATA4, and neurofilament (NF)68 proteins and
counterstained with 4Ј,6-diamidino-2-phenylindole (DAPI).
Terminal deoxynucleotidyl transferase [TdT]-
mediated dUTP nick end labeling (TUNEL) assay
Cryosections from the control, Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
,
and Sf1Cre;Gata4flox/flox
Gata6flox/flox
adrenals at E15.5 were
processed for TUNEL staining using an in situ cell death detec-
tion kit (TUNEL; Roche Diagnostics Corp), as previously de-
scribed (21, 22)
Rescue of Sf1Cre;Gata4flox/flox
Gata6flox/flox
females
by GC supplementation
The controls and Sf1Cre;Gata4flox/flox
Gata6flox/flox
females
were identified by genotyping at PND2 and treated with a rescue
cocktail containing 2 hormone analogs: fludrocortisone acetate
(Sigma-Aldrich) and dexamethasone 21-phosphate (Santa Cruz
Biotechnology, Inc) at 0.025 and 0.02 mg/kg, respectively. The
details of the rescue protocol are provided in Supplemental Ma-
terials and Methods.
Glucose concentration
Glucose concentration was determined from whole blood of
the female animals used in the GC supplementation procedure
and from the control and Sf1Cre;Gata4flox/flox
Gata6flox/flox
males (n ϭ 3 from each genotype) at PND60 using a human
commercial over the counter device (TRUEresult and TRUEtest
system) following the manufacturer’s instructions. The details of
the procedure are described in Supplemental Materials and
Methods.
Plasma and intratesticular corticosterone
concentration
Corticosterone concentration was determined by using the
competitiveCayman’scorticosteronekit(CaymanChemicalCo)
according to the vendor’s specifications. The details of the pro-
cedure and the statistical analysis are described in Supplemental
Materials and Methods.
Table 1. Antibody Table
Peptide/Protein
Target Name of Antibody
Manufacturer, Catalog Number,
and/or Name of Individual
Providing the Antibody
Species Raised in;
Monoclonal or Polyclonal
Dilution
Used
GATA 4 GATA 4 (C-20) Santa Cruz Biotechnology, Inc; SC-
1237
Goat; polyclonal 1:300
GATA 6 GATA 6 Cell Signaling; 5851 Rabbit; monoclonal 1:300
3␤HSD 3␤HSD (P-18) Santa Cruz Biotechnology, Inc; SC-
30820
Goat; polyclonal 1:300
Tyrosine
hydroxylase
Antityrosine
hydroxylase
Millipore; AB152 Rabbit; polyclonal 1:300
Steroidogenic
factor 1 (SF1)
Antimouse
Nr5a1 (Ad4BP/SF-1)
Transgenic, Inc; KO610 Rat; monoclonal 1:300
Neurofilament 68 Anti-68-kDa NF Abcam; ab72997 Chicken; polyclonal 1:300
Bromodeoxyuridine
(BrdU)
Anti-BrdU proliferation
marker
Abcam; ab1893 Sheep; polyclonal 1:300
CYP21A2 Anti-CYP21A2 Sigma-Aldrich; HPA048979 Rabbit; polyclonal 1:300
Ki67 Anti-Ki67 antibody Abcam; 66155 Rabbit; polyclonal 1:300
Goat IgG Alexa Fluor 488 donkey
antigoat IgG
Life Technology; A11055 Donkey; polyclonal 1:500
Goat IgG Alexa Fluor 555 donkey
antigoat IgG
Rabbit IgG Alexa Fluor 488 goat
antirabbit IgG
Life Technology; A11070 Goat; polyclonal 1:500
Rabbit IgG Alexa Fluor 555 goat
antirabbit IgG
Chicken IgG Alexa Fluor 488 goat
antichicken
Chicken IgG Alexa Fluor 555 goat
antichicken
Rat IgG Alexa Fluor 488 donkey
antirat
Life technology; A21208 Donkey; polyclonal 1:500
Rat IgG Alexa Fluor 594 donkey
antirat
Sheep IgG Alexa Fluor 488 donkey
antisheep
doi: 10.1210/en.2014-1815 press.endocrine.org/journal/endo 2505

Results
Mice with SF1Cre-mediated loss of GATA4 and
GATA6 proteins lack adrenal glands.
In the course of experiments aimed at the characteriza-
tion of gonadal development in animals with Sf1Cre-
mediated deletion of Gata4 and Gata6 genes (21, 23),
we crossed Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
males with
Gata4flox/flox
Gata6flox/flox
females. Upon genotyping the
progeny at weaning, we noted that the sex ratio of the
Gata6flox/flox
animals was skewed
from the expected 50:50 Mendelian distribution. Specif-
ically, Sf1Cre;Gata4flox/flox;
Gata6flox/flox
male (XY) ani-
mals were present at the expected ratio (ϳ1/8). However,
Gata6flox/flox
female (XX) animals of
the same genotype were absent. Upon examination of the
genotypes at E15.5, XX animals were also observed at the
expected ratio of approximately 1/8. Because we never
found dead or resorbed fetuses at E19.5 in the females
carrying double mutant litters we concluded that, Sf1Cre;
Gata4flox/flox
Gata6flox/flox
females died between the time
of birth and the weaning (PND21).
The adrenal gland is one of the few organs where the
Sf1Cre transgene is active (18), and both GATA4 and
GATA6 transcription factors are expressed (11). Exami-
nation of animals at PND9 animals revealed that macro-
scopic adrenal glands were absent in both genetic sexes of
the Sf1Cre;Gata4flox/flox
Gata6flox/flox
genotype (Figure 1,
A–D). Therefore, we concluded that adrenal organogen-
esis is incompatible with the loss of both GATA proteins.
Although Sf1Cre;Gata4flox/flox
Gata6flox/flox
females died,
males of the same genotype survived after weaning and
appeared to have a normal lifespan in the absence of the
adrenal glands.
Figure 1. Adrenal gland aplasia in Sf1Cre;Gata4flox/flox
Gata6flox/flox
mice. Representative pictures of torsos from the control (A and B) and Sf1Cre;
Gata4flox/flox
Gata6flox/flox
(C and D) of female (XX; A and C) and male (XY; B and D) mice at PND9. A and B, Adrenal glands are encircled in dashed
orange lines. Note the absence of adrenal glands in Sf1Cre;Gata4flox/flox
Gata6flox/flox
(C and D) animals. RK, right kidney; LK, left kidney; A,
anterior; P, posterior. E–M, Histological appearance of the abdominal area of the control (E, H, and K), Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
(F, I, and L),
Gata6flox/flox
(G, J, and M) fetuses at E15.5 (E–J) and 19.5 (K–M). Sections were stained with hematoxylin and eosin (H&E).
Arrows in panels E–G point to the adrenal gland. Panels H–J are higher magnification of the adrenal gland showed in E–G, respectively. Scale bars,
200 ␮m (E–G), 100 ␮m (K–M), and 50 ␮m (H–J).

Embryonic analysis of adrenal development in
E15.5 Sf1Cre;Gata4flox/flox
Gata6flox/flox
mice
The adrenal development of the control and Sf1Cre;
Gata4flox/flox
Gata6flox/flox
animals was compared at
E15.5.Thecontrolshadclearlyidentifiableadrenalglands
in the characteristic position juxtaposed against the de-
veloping kidneys (Figure 1E). Histological examination of
the adrenals revealed the expected heterogeneous cell
types corresponding to the developing steroidogenic and
adrenergic compartments. The cells with steroidogenic
appearance (large polyhedral eosinophilic cells containing
vacuoles) comprised most the population, with other cell
types also present (Figure 1H). In contrast, Sf1Cre;
Gata4flox/flox
Gata6flox/flox
fetuses had underdeveloped ru-
dimentary tissue located either anterior or medially to the
kidneys at the transverse level where adrenal glands nor-
mally form (Figure 1G). Tight homogenous cell clusters
that appeared nonsteroidogenic occupied the adrenal loca-
tion (Figure 1J). E15.5 fetuses retaining 1 functional allele of
Gata4 in the adrenal (Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
)
were also analyzed (Figure 1, F and I). The adrenal pri-
mordia that developed in these fetuses were greatly dimin-
ishedcomparedwiththecontrolsandwereofasimilarsize
as the adrenal primordia in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
(Figure 1, G and J). However, these adrenals
harbored islands of cells with steroidogenic appearance,
suggesting that, in the absence of Gata6, the remaining
Gata4 allele is sufficient to support some degree of cortical
differentiation (Figure 1I). Furthermore, the examination
of the sections obtained from later stage fetuses (E19.5,
Figure 1, K–M) identified normal organs in the controls
(Figure 1K) and greatly diminished adrenal tissue in the
Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
(Figure 1L); no identifiable
structures were observed in this location in the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
fetuses (Figure 1M). In contrast, an-
imals retaining a single functional Gata6 allele (Sf1Cre;
Gata4flox/flox
Gata6flox/ϩ
) had unremarkable adrenal
development indistinguishable from that in the controls (data
not shown).
Simultaneous deletion of Gata genes leads to a
loss of adrenocortical steroidogenic markers
Expression of Gata genes in the developing adrenal
glands has been previously examined by in situ hybridiza-
tion (ISH) and immunohistochemistry at E17 (11). This
work showed that Gata6 is strongly expressed in the ste-
roidogenic cells in the developing adrenal gland, whereas
Gata4 is mostly restricted to small clusters of subcapsular
steroidogenic cells. As shown here, immunofluorescent
analysis of GATA protein expression in the control E15.5
adrenal was in general agreement with the previously re-
ported data (11) (Figure 2, A–I). In the capsule, most cells
expressed GATA6, with GATA4 present only in a subset
of these cells (Figure 2, A–H). Interestingly, GATA4 ex-
pression appeared to be notably enriched in the anterior
capsular cells compared with the posterior (adjacent to
kidney) layer (Figure 2D). The role of GATA4-positive
cells as a source of the adrenogonadal progenitor-like
population in the adrenal has been recently proposed
(10). Adrenocortical cells in the control gland coexpress
GATA6 with the steroidogenic master regulator SF1
(Figure 2, A–C), and these SF1- and 3␤-hydroxysteroid
dehydrogenase/⌬-5-4 isomerase (3␤HSD)-positive cells
(Figure 2, A–F and I) surround the medulla. 3␤HSD is
a requisite enzyme for corticosteroid synthesis.
The examination of the residual tissue present in the
location corresponding to the adrenal gland in E15.5
Gata6flox/flox
mice showed the pres-
ence of numerous scattered GATA6-positive cells, but the
expression of SF1 and 3␤HSD was not detected (Figure 2,
J, L, M, and O). In the anterior part of the capsule, residual
GATA4-positive cells in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
adrenal were primarily retained (Figure 2, K and N). Ad-
ditionally, adrenocortical protein expression was com-
pared in control and Sf1Cre;Gata4flox/flox
Gata6flox/flox
fetuses at early stages of embryonic development (E13.5)
(Supplemental Figure 1). Similarly, we observed no adre-
nocortical-specific steroidogenic expression in these
Gata6flox/flox
fetuses. We concluded
that Sf1Cre-mediated loss of Gata4 and Gata6 in the ad-
renal cortex leads to an early demise of the steroidogenic
gene expression program.
Medullar cells arrive in the adrenal space in the
Gata6flox/flox
mice
Cells harboring steroidogenic expression were not ob-
served anterior to the kidneys in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals (Figure 2, J–O). However, residual
tissue was present at this location. As previously reported,
the neural crest-derived chromaffin cells migrated into
theirnormalpositionevenintheabsenceofadrenocortical
cells (24, 25), suggesting that the collection of cells in the
GATA4 and GATA6 loss-of-function Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals may represent chromaffin tissue.
The developing neurons express a set of genes that is
similar to the expression observed in adrenal chromaffin
cells. Normally, the intraadrenal location allows one to
easily distinguish between the committed chromaffin cells
and the adjoining neurons. However, in the complete ab-
senceofcorticalmass,thedevelopingmedullarcellswould
no longer be partitioned within the defined space. In a
similar situation, the neuronal cells were previously de-
finitively distinguished from the closely associated sym-
pathetic neurons by tyrosine hydroxylase (TH) immuno-

reactivity in combination with NF mRNA ISH (24). In this
assay, the adrenal chromaffin progenitor cells were TH-
positivebutNF-negative(THϩ/NFϪ).However,thesym-
pathetic neuron progenitors were positive for both mark-
ers (THϩ/NFϩ) as early as E13.5. We conducted a similar
approach to characterize the residual cell clusters in the
Gata6flox/flox
adrenals, except
that the NF68 antibody was used to detect the NF. Unlike
previously described NF RNA hybridization (24), chro-
maffin cell progenitors were also positive for the NF68
pan-neuronal marker, most likely
due to a higher sensitivity of the IF
assay compared with ISH (Figure 3,
A–D). In the control adrenal, the 2
areas were clearly distinguishable as
THhigh
/NFlow
(chromaffin cell loca-
tion)andTHlow
/NFhigh
(sympathetic
neurons). However, in the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
adrenal
location, 2 types of cells could be
identified with the THhigh
group lo-
cated juxtaposed and lateral to the
THlow
cells; both groups equally
stained for NF68 (Figure 3, E–H).
These results are in agreement with
the previous work that established
that the lack of an adrenal cortex
upon Sf1 loss is compatible with the
generation and differentiation of
chromaffin cells (24). In summary,
collective data strongly suggest that
the lateral THhigh
cells are chromaffin
progenitors that, even in the absence of
the adrenocortical cells in the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
mice, arrive
in the correct anatomical location, dif-
ferentiate, and coalesce to form a com-
pactcluster(Figure3,E–H).Noneofthe
NF68-positive cells (whether THlow
or
THhigh
) expressed steroidogenic mark-
ers (Figure 3, I–K).
The loss of Gata6 in
steroidogenic cell progenitors is
compatible with
steroidogenesis
Previous work using Gata4Ϫ/Ϫ
embryonic stem cells in chimera
complementation experiments led to
the conclusion that GATA4 is dis-
pensableforearlyadrenocorticaldif-
ferentiation (11). Our studies have
suggested that Gata4 and Gata6 are
required for the initiation or the early maintenance of ste-
roidogenic fate in the adrenal gland. To better understand
the mechanism underlying GATA protein function, fe-
tuses carrying single deletions of either transcription fac-
tor were examined. The adrenal glands were present in the
fetuses and were indistin-
guishable from the control organs (data not shown). In
contrast, the adrenal glands in the Sf1Cre;Gata6flox/flox
mice were notably smaller compared with the controls
Figure 2. Loss of steroidogenic cells in Sf1Cre;Gata4flox/flox
Gata6flox/flox
adrenals. Representative
sections from controls (A–I) and Sf1Cre;Gata4flox/flox
Gata6flox/flox
(J–O) adrenals at E15.5 were
stained for GATA6 (red) and SF1 (green) (A–C, J, and M); GATA4 (red) and SF1 (green) (D–F, K,
and N); GATA6 (red) and GATA4 (green) (G); and 3␤HSD (red) (I, L, and O) and SF1 (green) (I).
DAPI (blue) was used as nuclear staining. In the control adrenals, most SF1-positive cells express
GATA6 but not GATA4 (compare B and E). Only anterior capsular cells consistently express
GATA4 (arrow in E), whereas most the capsular cells express GATA6 (arrows in B and G). G, A
small subset of subcapsular cells (arrowheads) and rare capsular cells (asterisk) coexpresses both
GATA factors. In J–O, note that steroidogenic (SF1- or 3␤HSD-positive) cells are absent in the
Gata6flox/flox
adrenals. B and C, and E and F, are higher magnifications of A
and D, respectively, and M–O are higher magnifications of J–L, respectively. Scale bars, 100 ␮m
(A and J–L), 50 ␮m (D and M–O), and 20 ␮m (B, C, and E–I).

(Supplemental Figure 2). Similar to the previously re-
ported animals harboring adrenal-specific deletion of
Gata6(12),theSf1Cre;Gata6flox/flox
animalsofbothsexes
were viable and fertile.
To examine the adrenal phenotype in the Sf1Cre;
Gata6flox/flox
animals, the glands in E15.5 ROSAmT/mG
;
fetuses were analyzed. In these ani-
mals, Sf1Cre-mediated recombination of the mT/mG
reporter (19) permanently induced enhanced green fluo-
rescent protein (EGFP) expression. This manipulation en-
abled the tracking of cell fate, in which Cre recombinase
was active (Figure 4, G–L). SF1 staining of the sectioned
adrenal glands from the E15.5 ROSAmT/mG
;Sf1Cre;
Gata6flox/flox
fetuses revealed that steroidogenic (SF1-pos-
itive) adrenocortical cells developed in the absence of
GATA6 (Figure 4, G–L). No ectopic steroidogenic expres-
sion was observed outside of the EGFP-positive cells.
Although numerous GATA6-positive cells were present
in the suprarenal location, the sparse adrenocortical
(EGFP/SF1-positive) cells did not express GATA6
(compare Figure 4, J and K). We concluded that GATA6
expression is required for the development of the adre-
nal cortex and the generation of the full complement of
adrenocortical cells. The presence of SF1-positive;
GATA6-negative cells (Figure 4J, arrows) implies that
GATA6 is not absolutely required for establishing ste-
roidogenic cell fate in the developing adrenal gland (see
also Ref. 12).
Steroidogenic development in animals that retain
a sole functional Gata4 allele
Adrenocortical development in animals with a Gata4
conditional deletion is normal, and in mice with a Gata6
deletion, adrenocortical development is severely impaired
but not completely abrogated. In contrast, the simultane-
ous loss of Gata4 and Gata6 gene expression leads to a
Figure 3. The adrenal area in Sf1Cre;Gata4flox/flox
Gata6flox/flox
fetuses is occupied by medullar cells. Torso sections of control (A–D) and Sf1Cre;
Gata6flox/flox
(E–K) fetuses at E15.5 were stained for TH (green) and NF (NF68) (red) (A, B, E, and F), 3␤HSD (red) and TH (green) (I). In A and E,
adrenals are encircled by a blue dashed line, and B–D and F–H are higher magnifications of A and E, respectively. In the control, the medullar
(THhigh
/NFlow
) and the nervous (THlow
/NFhigh
) zones are separated by the negative adrenocortical mass (A and B). Note that the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
animals lack of adrenocortical cells, these 2 populations are directly juxtaposed (E and F). Also note in I and K the absence
of steroidogenic cells (defined by 3␤HSD staining) in the adrenal glands of Sf1Cre;Gata4flox/flox
Gata6flox/flox
fetuses. cx, cortex; m, medulla; sg,
sympathetic ganglion. Scale bars, 200 ␮m (A and E), 100 ␮m (B–D), and 50 ␮m (F–K).

completedemiseofadrenocorticalcells.Toreconcilethese
2 observations, we hypothesized that in the absence of
GATA6, GATA4 takes over the functions of its primary
adrenal counterpart, GATA6, in organizing cortical
development.
To explore this possibility, animals with a Gata6 dele-
tion that carry only 1 functional allele of Gata4, Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
, were examined. Similar to the
single Gata6 deletion, Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
animals are fertile and were used as breeders to gener-
ate conditional double knockout Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals in this project. At E15.5, Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
fetuses harbored severely under-
developed organs (Figure 1F, arrow) that were similar in
size to residual adrenal tissue in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals (Figure 1G, arrow). However, in
contrast to the Sf1Cre;Gata4flox/flox
Gata6flox/flox
fetuses
(Figure 2, J–O), adrenal glands in animals retaining
GATA4 expression harbored a small number of steroid-
ogenic cells that express SF1 and 3␤HSD (Figure 5, C–J).
Figure 4. Adrenocortical development is severely impaired in the absence of GATA6. A–F, Representative sections from control (A and B) and
ROSAmT/mG
; Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
(C–F) adrenals at E15.5 were stained for SF1 (bright green) (A and B) and GATA4 (red) (A–D and F).
Cells that underwent Sf1Cre-mediated recombination are traced by membrane EGFP (EGFPm
) (dark green). In B note that GATA4 expression is
mostly limited to capsular cells (arrowhead), with rare cortical cells expressing the protein (arrows). In contrast, numerous cortical cells are GATA4-
positive in the ROSAmT/mG
Gata6flox/flox
adrenal (C, D, and F; encircled in C). In C–E only some EGFP-positive cells express GATA4
(see also Supplemental Figure 4). G–L, A limited number of steroidogenic cells is present in the ROSAmT/mG
; Sf1Cre;Gata6flox/flox
adrenals. Adrenal
sections of ROSAmT/mG
; Sf1Cre;Gata6flox/flox
at E15.5 were stained with antibodies against GATA6 (red; G, H, and J–L) and SF1 (bright green) (G, I,
J, and L). EGFPm
expression (dark green) traces SF1Cre-mediated excision (G, I, J, and L). In J, arrows point to SF1-positive nuclei that are strictly
confined to the EGFPm
-positive/GATA6-negative cells. J–L, Higher magnifications of a rectangular area in G. Scale bars, 50 ␮m (A, C, and G–I) and
20 ␮m (B, D–F, and J–L).

These cell clusters developed side-by-side with the
medullar (TH-positive) cells (Figure 5, C–H). Similar to
ROSAmT/mG
adrenals (Figure 4,
G–L), SF1 and 3␤HSD expression in the ROSAmT/mG
;
Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
mutants was confined to
the EGFP-positive cells.
GATA4 expression in the control embryonic adrenal
gland is mostly limited to the SF1-negative capsular cells
(Figure 4, A and B, arrowhead; see also Figure 2, D–H).
Rare doubly GATA4- and SF1-positive cells are clustered
in the subcapsular region of the developing adrenal glands
and likely represent the stem cell/progenitor population
(10, 11, 19) (Figure 4B, arrows). In contrast, in the Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
adrenal, the cells that express
GATA4 protein were more numerous and observed
throughout the adrenal cortex (Figure 4, C–F). GATA4
expression only partially overlaps with the EGFP-positive
staining that marks the cells with steroidogenic fate. In
general, GATA4 presence in the steroidogenic cells is not
required to maintain SF1 expression (Supplemental Figure
3, A–F); in fact, most the residual SF1-positive cells in the
E15.5 Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
adrenals no longer
express GATA4 (Supplemental Figure 3, G–L). The com-
pensatory expression of the single Gata4 allele in Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
adrenals is better revealed at
E13.5 (Supplemental Figure 4). Numerous doubly pos-
itive GATA4; SF1 cells are present in the Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
adrenals (Supplemental Figure
4, E–H) and are not observed in the controls (Supple-
mental Figure 4, A–D). Therefore, GATA6 is likely pro-
viding the necessary GATA function for steroidogenic
differentiation under normal conditions. These findings
further support the hypothesis that GATA-dependent
cell commitment to steroidogenic fate is restricted early
in the adrenal development.
Quantitative analysis of RNA expression upon
Gata genes deletion
To quantify the RNA expression of cortical and med-
ullar enzymes in the adrenal tissue, we performed qPCR.
Thedecreaseintheexpressionofsteroidogenicgenesupon
double deletion was significant (P Ͻ .01 to P Ͻ .001) for
Star, Cyp11a1, Cyp21a1, and Cyp11b1, confirming the
agenesis of the adrenal cortex upon GATA loss (Figure 6).
In animals retaining 1 functional Gata4 allele (Sf1Cre;
Figure 5. The sole functional allele of the Gata4 gene is sufficient to support adrenocortical development and steroidogenesis. Adrenal sections
from control (A and B) and ROSAmT/mG
Gata6flox/flox
(C–J) fetuses at E15.5 were stained for 3␤HSD (red) (A–F) and TH (bright
green) (A–H); SF1 (bright green) (I and J) and 3␤HSD (red) (I and J). In the ROSAmT/mG
Gata6flox/flox
adrenals, EGFP expression
traces SF1Cre-mediated gene excision only by displaying membrane staining (EGFPm
, dark green) (C–J). DAPI was used as nuclear staining (blue).
Scale bars, 200 ␮m (C, E, G, and I), 100 ␮m (A, D, F, and H), 50 ␮m (D, F, and H), and 20 ␮m (B and J).
Figure 6. Gene expression analysis of adrenal glands. RNA expression
analysis via qPCR for the steroidogenic enzymes Star, Cyp11a1,
Cyp11b1, Cyp21a2, and Cyp17a1 and the medullar-specific genes Th
and Nefl. Adrenal glands were obtained from controls (at least n ϭ 5),
Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
(at least n ϭ 4), and Sf1Cre;
Gata4flox/flox
Gata6flox/flox
(n ϭ 3) at E15.5. Bars represent mean Ϯ SEM
of fold change of Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
(black bar) and
Gata6flox/flox
(gray bar) relative to the controls. Data
were analyzed by ANOVA (one-way) followed by Tukey’s multiple
comparisons test, with significance at *, P Ͻ .05; **, P Ͻ .01; and
***, P Ͻ .001. Statistical difference between Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
Gata6flox/flox
is shown by the respective P value.

Gata4flox/ϩ
Gata6flox/flox
) a similar, but less comprehen-
sive, reduction in steroidogenic expression was also ob-
served, corresponding to a drastic decrease in the number
of SF1-positive cells in this genotype (Figure 5). In both
classes of adrenal mutant samples (Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
Gata6flox/flox
), the
expression of medullar Nefl (Nf68) and Th genes was in-
creased (P Ͻ .001 and P Ͻ .05, respectively) correspond-
ing to the greater ratio of medullar cells in these samples
(Figure 3). One gene that was differentially regulated be-
tween the Sf1Cre;Gata4flox/flox
Gata6flox/flox
and Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
was Cyp17a1. The expression of
this gene was decreased in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
but significantly up-regulated in Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
adrenals (P Ͻ .01). This result
agrees with the previous report describing the deletion of
Gata6 in the adult adrenal and likely reflects the acquisi-
tion of a Leydig-like phenotype in these GATA4-positive
cells (12).
Postnatal development of Sf1Cre;Gata4flox/flox
Gata6flox/flox
males is supported by the expansion
of the adrenal-like cell population in the testis
The adrenal gland is absent in Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals regardless of sex (Figure 1, A–D).
However, in contrast to Sf1Cre;Gata4flox/flox
Gata6flox/flox
females, male animals of the same genotype (Sf1Cre;
Gata4flox/flox
Gata6flox/flox
) have a normal lifespan. The
fetal rodent testis has been recently shown to harbor a
small number of adrenal-like cells (26–28). We hypothe-
sized that in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
male
mice, this population is activated, thus supporting animal
viability. Therefore, we examined the testes of the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
males during embryonic devel-
opment for the presence of adrenal-like cells. We observed
a remarkable expansion of the cell population positive for
the adrenal specific marker, a key adrenocortical enzyme
CYP21A2 (Figure 7). At E18.5, the number of CYP21A2-
positive cells in the control testis is quite limited, with rare
isolated CYP21A2-positive cells residing in the interstitial
Figure 7. Presence of adrenal-like cells in the embryonic Sf1Cre;Gata4flox/flox
Gata6flox/flox
testes. Representative testicular sections from controls (A
and B) and Sf1Cre;Gata4flox/flox
Gata6flox/flox
(C and D) males at E18.5 were stained for the adrenal enzyme CYP21A2. B and D, Higher
magnification of A and C, respectively. In B and D, arrowheads point to CYP21A2-positive cells. Scale bars, 100 ␮m (A and C) and 50 ␮m (B and
D). E and F, Quantitative changes in the expression of Insl3, Star, Cyp11a1, Hsd3b6, Mc2r, Cyp21a1, Cyp11b1, Cyp11b2, Gata4, and Gata6 in
Gata6flox/flox
testes at E15.5 (E) and E18.5 (F). The results are plotted as the mean Ϯ SEM of the fold change relative to the
controls from n ϭ 3 biological replicates of each genotype. G, Glucose concentration (mg/dL) from whole blood of the controls (n ϭ 3; black bar)
Gata6flox/flox
(n ϭ 3; gray bar) males at PND60. H and I, Corticosterone concentration in plasma (ng/mL) from the controls
(black bar) and Sf1Cre;Gata4flox/flox
Gata6flox/flox
(gray bar) male mice at PND4–PND10 (n ϭ 4) (L) and 60 (n ϭ 3) (M). J, Intratesticular
corticosterone concentration (ng/mL) of the controls (n ϭ 3; black bar) and Sf1Cre;Gata4flox/flox
Gata6flox/flox
(n ϭ 3; gray bar) mice at PND45.
Results are shown as the mean Ϯ SEM, and datasets were analyzed by Student’s t test, with significance considered at *, P Ͻ .05; **, P Ͻ .01;
and ***, P Ͻ .001.

space (Figure 7, A and B). In contrast, in the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
testis, the number of CYP21A2
cells is greatly increased, and clusters of positive cells are
widely distributed throughout the entire organ (Figure 7,
CandD).Geneexpressionanalysisoftheadrenalsteroidogenic
pathwayinembryonicSf1Cre;Gata4flox/flox
Gata6flox/flox
testes
revealed a significant up-regulation of Cyp21a1 (P Ͻ .05) and
Hsd3b6 (P Ͻ .001) at E15.5 (Figure 7E) and Mc2r (P Ͻ .05),
Cyp21a1 (P Ͻ .01), and Hsd3b6 (P Ͻ .001) at E18.5 (Figure
7F).
In addition, we determined the intratesticular
corticosterone concentrations in Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals using ELISA (Figure 7J). In
Gata6flox/flox
testes, it was signifi-
cantly elevated (ϳ20 ng/mL; P Ͻ .001) when compared
with the control group (ϳ2 ng/mL). Additionally,
plasma corticosterone was measured at PND4–PND10
and PND60 (Figure 7, H and I, respectively). The
plasma corticosterone level in Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals was still significantly decreased
compared with the controls. However, the circulating
level of corticosterone corresponded with the level syn-
thesized by the testis (Figure 7J). In the absence of a
functional adrenal gland, the regulation of glucose me-
tabolism by the adrenal hormones is compromised (re-
viewed in Ref. 9). We determined glucose concentration
in whole blood from the controls and Sf1Cre;Gata4flox/flox
Gata6flox/flox
males at PND90, with no difference be-
tween the 2 groups (Figure 7G). We concluded that the
amount of corticosterone synthesized by the testis is
likely sufficient to sustain the life of the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
males.
GC supplementation rescues
Gata6flox/flox
females
Analysis of adrenal development and steroidogenic
gene expression in fetuses harboring the Sf1Cre-mediated
double deletion of both Gata genes strongly suggests that
adrenocortical function in these animals is completely im-
paired. Absence of the adrenal gland at the time of birth is
currently recognized to be lethal in rodents (7), and this
premise is true for Sf1Cre;Gata4flox/flox
Gata6flox/flox
fe-
males in which the deletion of both Gata4 and Gata6 leads
to death before weaning. We determined corticosterone
concentration in plasma from controls and Sf1Cre;
Gata4flox/flox
Gata6flox/flox
females between PND4 and
PND9 by ELISA (Figure 8A). The mean concentration of
corticosterone in Sf1Cre;Gata4flox/flox
Gata6flox/flox
was
significantly lower (ϳ3.4 ng/mL; P Ͻ .05) when compared
with the control group (ϳ52 ng/mL). To examine whether
Figure 8. Rescue of Sf1Cre;Gata4flox/flox
Gata6flox/flox
female mice with GC treatment. A, Plasma corticosterone concentration (ng/mL) of the
controls (n ϭ 3; black bar) and Sf1Cre;Gata4flox/flox
Gata6flox/flox
(n ϭ 3; gray bar) females between PND4 and PND9. Results are shown as the
mean Ϯ SEM, and the data were analyzed by Student’s t test, with significance considered at *, P Ͻ .05. B, Percent survival of the controls treated
with DPBS vehicle (n ϭ 7; dashed line), Sf1Cre;Gata4flox/flox
Gata6flox/flox
females that were injected daily with a cocktail of GCs (fludrocortisone
acetate ϩ dexamethasone 21-phosphate) (n ϭ 7; dotted line), and nontreated Sf1Cre;Gata4flox/flox
Gata6flox/flox
females (n ϭ 6; solid line). C,
Glucose concentration (mg/dL) in whole blood from the controls treated with DPBS vehicle (black bar), treated Sf1Cre;Gata4flox/flox
Gata6flox/flox
females (light gray bar), and noninjected Sf1Cre;Gata4flox/flox
Gata6flox/flox
females (dark gray bar). The data are presented as the mean Ϯ SEM, and
datasets were analyzed with ANOVA (one-way) followed by Tukey’s test for multiple comparisons. Bars with different superscripts differ
significantly (***, P Ͻ .001). D–G, Representative ovarian sections from the controls (D and E) and Sf1Cre;Gata4flox/flox
Gata6flox/flox
(F and G) at
PND4. Sections were stained for the adrenal enzyme CYP21A2. E and G, Higher magnification of D and F, respectively. Note the lack of CYP21A2-
positive cells in the ovaries of both genotypes. Pr, primordial; P, primary; PA, preantral. Scale bars, 50 ␮m (D and F) and 20 ␮m (E and G).

the lethality of the Sf1Cre;Gata4flox/flox
Gata6flox/flox
fe-
males is due to a lack of hormonal synthesis, we performed
a rescue experiment with a steroid supplementation reg-
imen initiated shortly after birth (based on Ref. 3). All of
the untreated Sf1Cre;Gata4flox/flox
Gata6flox/flox
females
died between PND11 and PND15. However, all of the
treated Sf1Cre;Gata4flox/flox
Gata6flox/flox
females were
alive at weaning and then required no further supple-
mentation (Figure 8B). Four of these females survived
the 3- to 4-month mark, and 2 remained alive at
PND150. This result confirms that the lethality of the
Gata6flox/flox
females is due to ad-
renal hormone insufficiency.
The glucose concentration was determined in whole blood
from the Dulbecco’s phosphate-buffered saline (DPBS)-vehicle
control, rescued Sf1Cre;Gata4flox/flox
Gata6flox/flox
and nonin-
jected Sf1Cre;Gata4flox/flox
Gata6flox/flox
females 2 weeks after
birth.Thecontrolfemaleshadanaverageglucoseconcentration
of 93 mg/dL, which is within the normal range. In contrast,
the glucose concentration in the nontreated Sf1Cre;
Gata4flox/flox
Gata6flox/flox
females was significantly reduced to
29 mg/dL (P Ͻ .001), which was barely within the limit of
detection. Similarly, glucose levels were measured in the hor-
mone-injectedfemales(Figure8C).Themeanconcentrationof
glucose in the treated group between PND30 and PND90 (82
mg/dL) was improved compared with the nontreated Sf1Cre;
Gata4flox/flox
Gata6flox/flox
females (29 mg/dL; P Ͼ .001).
Thus, glucose level maintenance is compromised in Sf1Cre;
Gata4flox/flox
Gata6flox/flox
females in the absence of hormonal
supplementation. We also performed IHC experiments to de-
terminethepresenceofCYP21A2-positivecellsintheovariesof
the controls and Sf1Cre;Gata4flox/flox
Gata6flox/flox
females at
PND4 and detected no CYP21A2-positive cells in either group
(Figure 8, D–G).
In summary, the rescue experiments performed in
Gata6flox/flox
females along with the
analysis of CYP21A2 expression in Sf1Cre;Gata4flox/flox
Gata6flox/flox
animals strongly suggest that the demise of
the adrenal cortex in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
males is adequately compensated by the expansion of the
adrenal-like cell population in their testis. These testicular
cells are capable of corticosterone synthesis and could be
sufficient to sustain life in the absence of the functional
adrenal cortex.
Proliferation and apoptosis of the adrenal cells
upon GATA protein loss
A dramatic decrease in adrenal size reflected either de-
creased proliferation or increased cell death upon Gata
gene deletion. Adrenocortical cell proliferation in Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
fetuses was analyzed using the
BrdU cell proliferation assay (Supplemental Figure 5). Sta-
tistical analysis showed that the ratio between total and
proliferating cells was similar in the control and Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
mice adrenals (Supplemental
Figure 5, A and B). However, the ratio of steroidogenic
cells among the proliferating cells was significantly de-
creased (P Ͻ .001) (Supplemental Figure 5, C and D).
Additionally, we observed a higher number of TUNEL-
positive cells in the adrenal location in the Sf1Cre;
Gata4flox/flox
Gata6flox/flox
adrenal (Supplemental Figure
5, H–J). At E15.5, most capsular/subcapsular adrenal cells
remained highly proliferative in the control and mutant
adrenals and many of these were GATA4(ϩ) (Supplemen-
tal Figure 6). However, examination of proliferation in
Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
adrenals in cells located
outside of the subcapsular area showed no correlation
between the cell proliferation status (Ki67-positive) and
GATA4 expression (Supplemental Figure 6). The bulk of
highly proliferative cells in the E15.5 control and Sf1Cre;
Gata4flox/ϩ
Gata6flox/flox
adrenal core consists of medullar
NF68(ϩ)/TH(ϩ) cell (Supplemental Figure 7). We con-
cluded that, upon GATA6 loss, the sole functional
GATA4 allele is insufficient to support the normal prolif-
eration of SF1-positive adrenocortical cells. In the absence
of the steroidogenic cells, cell death is likely a contributing
factor in the ultimate adrenal demise during embryogen-
esis (eg, Ref. 25) and likely accounts for the absence
of medullar components in the Sf1Cre;Gata4flox/flox
Gata6flox/flox
adrenals at E19.5 (Figure 1M).
Discussion
GATA transcription factors (GATA1–GATA6) execute
transcriptional control of critical developmental decisions
in multiple tissues in vertebrates where they often conduct
functions that partially overlap but are not completely
redundant (29–32; reviewed in Refs. 15, 33, 34; see also
Refs. 35, 36). Although GATA proteins are broadly ex-
pressed, SF1 is a master regulator of steroidogenic expres-
sion in the adrenal cortex and is required for normal
adrenocortical differentiation (7). During adrenal devel-
opment, the transcription factor WT1 likely serves to ini-
tially up-regulate Sf1 expression, and the ability of WT1 to
directly activate Sf1 transcription in the testis was de-
scribed in the past (37).
Hormonal synthesis by the adrenal cortex is required for
animal viability, and mice with dysfunctional adrenal glands
normally do not survive after 2 weeks after birth without
hormonal supplementation (3, 25, 38–40). Unlike their fe-
male littermates that die of hormonal insufficiency and sub-
sequent hypoglycemia, Sf1Cre;Gata4flox/flox
Gata6flox/flox
males appear to have a normal lifespan. Both the embryonic

and postnatal (23) Sf1Cre;Gata4flox/flox
Gata6flox/flox
testes
harbor an expanded adrenal-like cell population that ex-
presses adrenocortical enzymes and is capable of synthesiz-
ing GCs. Cells with adrenal-like properties are normally
found in the fetal testis. However, their number is very lim-
ited (26–28). Furthermore, it has been long suggested (41)
that these cells result from the incomplete partition of the
adrenal and gonadal progenitor populations during adreno-
gonadal progenitor separation. However, this hypothesis
has not been experimentally proven. The population of cells
presentintheSf1Cre;Gata4flox/flox
Gata6flox/flox
testesisrem-
iniscent of a testicular tumor caused by adrenogenital syn-
drome or testicular adrenal rest tumor cells in human con-
genital adrenal hyperplasia patients and is the primary
reason for their infertility (42). Steroidogenic cells invading
Wnt4 mutant ovaries have adrenal-like properties (43),
which led to the hypothesis that the WNT pathway is ac-
countable for repelling these cells from entering the ovary.
This may explain the absence of adrenal-like cells in the fe-
male organ. In contrast, the fate mapping of adrenal cells
indicates that the same pathway is unlikely to function in the
testis (44).
Previous research has convincingly demonstrated that
Nr5a1/Sf1 expression in steroidogenic organs is guided by
at least 3 conserved regulatory elements. The proximal Sf1
promoter located within the first 674 bp upstream of the
Sf1 transcription site drives Sf1 expression in indifferent
(undifferentiated) gonad. The regulatory role of this ele-
ment appears to be limited to Sertoli and granulosa cell
expression (37). Pioneering studies in the Morohashi lab-
oratory identified a fetal adrenal enhancer in the fourth
intronoftheSf1gene.Thisshort(ϳ650bp)fragmentfrom
the forth intron is sufficient to drive cortico-specific ex-
pression of the LacZ reporter in the fetal adrenal gland
(45). Lastly, the third element active in fetal Leydig cells
has also been described (46).
Our inspection identified a highly conserved GATA site
in all of the 3 regulatory elements guiding Sf1 gene ex-
pression. The functional significance of GATA binding
sites in Sf1 regulatory elements has not been determined
yet. The ability of GATA proteins to activate Sf1 expres-
sion through different enhancer elements at different de-
velopmental times is likely accountable for the survival of
adrenal-like population in the testis, but not in the adrenal
itself. In this respect, although SF1-positive cells are no
longer present in the Sf1Cre; Gata4flox/flox
; Gata6flox/flox
adrenals (Figure 2, J–O), testicular cells in these animals
retain SF1 expression (23).
The most parsimonious scenario for the emergence of
the adrenal-like cells in the testis is similar to the pathway
presumed to be active in congenital adrenal hyperplasia
patients. Namely, the demise of adrenal cortex and GC
synthesisleadstoalossofGC-inducedfeedbackinhibition
of ACTH production. High ACTH concentration in the
fetus stimulates the expansion of the ACTH-responsive
progenitor cell population in the testis. At present, the
mechanism underlying the execution of this transition is
not well understood. Similarly, the relationship between
these cells and the fetal or adult Leydig cell progenitors
remains to be established. The misallocated adrenal pro-
genitors residing in the testis may be destined to expand
into adrenocortical-like cells under certain conditions. Al-
ternatively, the adrenal-like and Leydig cells may share a
common progenitor that is directed into an adrenal fate
under high ACTH conditions. In addition, whether the
loss of GATA factors in the testis is a contributing factor
in actively promoting this transition remains unknown.
Recapitulating chronic ACTH exposure is technically
challenging in neonatal mice, and poor hormonal control
is likely to be another factor that provides favorable con-
ditions for the expansion of the adrenal-like testis popu-
lation.Inotherwords,theanimalswithfunctionaladrenal
glands will respond with increased GC synthesis in re-
sponse to high ACTH and prevent the expansion of the
adrenal tissue in the testis. An increase in the expression of
an adrenal marker was observed in the testis of Cyp11a1
null mice (40). However, these animals (being deficient in
a key steroidogenic enzyme) did not survive and did not
synthesize active hormones.
We believe that Sf1Cre;Gata4flox/flox
Gata6flox/flox
pres-
ents a unique experimental model and the opportunity to
produce testicular adrenal rest tumor-like tissue in rodents
and to understand its origin. This work provides conclu-
sive evidence that the formation of the functional adrenal
gland in mice is incompatible with the simultaneous de-
letion of GATA4 and GATA6 factors in adrenocortical
cells. The functions of the 2 proteins in the adrenal do not
completely overlap. Loss of GATA6 alone impairs but
does not completely preclude adrenocortical formation
and steroidogenesis. Hypoplastic adrenal glands in the
mice contain a diminished number of
active steroidogenic cells that express the master regulator
SF1 and sustain viability. In the Sf1Cre; Gata6flox/flox
ad-
renal, steroidogenic cells that are GATA6-negative are in-
termixedwithnumerousGATA6-positivecellsthatdonot
express the steroidogenic markers SF1 or 3␤HSD. In con-
trast, Gata4 deletion is dispensable for adrenal organo-
genesis. In summary, GATA6 protein is a principal driver
of adrenocortical cell maintenance and fully compensates
for the absence of GATA4 protein. GATA4 protein is not
required for adrenocortical steroidogenic differentiation
and hormone synthesis. However, this protein performs
essential regulatory functions in the absence of GATA6
and supports the requisite number of adrenal steroido-

genic cells to assure viability of Sf1Cre;Gata4flox/ϩ
Gata6flox/flox
females. The conditional double mutant
Gata6flox/flox
males appear to live
normal lifespans even in the absence of both GATA pro-
teins as vital steroidogenic synthesis shifts to their testes.
In addition to the adrenal phenotype described in this
work, Sf1Cre;Gata4flox/flox
Gata6flox/flox
males exhibit a
lack of testis functionality, with a loss of normal steroid-
ogenic testis function (23).
Acknowledgments
Address all correspondence and requests for reprints to: Dr
Sergei G. Tevosian, Department of Physiological Sciences, Col-
lege of Veterinary Medicine, University of Florida, Gainesville,
FL 32610. E-mail: stevosian@ufl.edu; or Dr Maria B. Padua,
Department of Physiological Sciences, College of Veterinary
Medicine, University of Florida, Gainesville, FL 32610. E-mail:
mpadua@ufl.edu.
Present address for M.B.P.: Department of Surgery, School of
Medicine, Indiana University, Indianapolis, IN 46202.
This work was supported by University of Florida and by
National Institutes of Health Grant R01HD042751.
Disclosure Summary: The authors have nothing to disclose.
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