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Am J Physiol Gastrointest Liver Physiol 306: G474–G490, 2014.
First published January 16, 2014; doi:10.1152/ajpgi.00119.2013.
Review
Role of GATA factors in development, differentiation, and homeostasis
of the small intestinal epithelium
Boaz E. Aronson,1,2 Kelly A. Stapleton,1 and Stephen D. Krasinski1,3
1
Division of Gastroenterology and Nutrition, Department of Medicine, Children’s Hospital Boston, and Harvard Medical
School, Boston, Massachusetts; 2Department of Pediatrics, Emma Children’s Hospital, Academic Medical Center, University
of Amsterdam, Amsterdam, The Netherlands; and 3Gerald J. and Dorothy R. Friedman School of Nutrition Science
and Policy, Tufts University, Boston, Massachusetts
Submitted 11 April 2013; accepted in final form 7 January 2014
endoderm; GATA; intestinal development; intestinal differentiation; intestinal
epithelium
GATA FACTORS ARE AN ANCIENT family of transcription factors
that mediate processes of development, differentiation, and
gene expression in multiple tissues and cell types. Six GATA
factors are conserved in all vertebrates. On the basis of mutational analyses of GATA1 (70) and GATA4 (84), vertebrate
GATA factors contain transcriptional activation domains localized to the NH2-terminal region, two highly conserved zinc
fingers and adjacent basic regions, a nuclear localization signal
sequence, and a COOH-terminal domain of unknown function
(Fig. 1A). GATA zinc fingers comprise four cysteine residues
(type IV), Cys-X2-Cys-X17-Cys-X2-Cys, that coordinate a divalent zinc ion (Fig. 1B) and, together with the adjacent basic
regions, direct binding to the nucleotide sequence element
WGATAR (W ⫽ A or T, R ⫽ A or G) and also interact with
other regulatory proteins to modulate GATA transcriptional
control of target genes.
Vertebrate GATA factors are separated into two subfamilies:
1) GATA1, GATA2, and GATA3 and 2) GATA4, GATA5,
and GATA6. Members of the GATA1/2/3 subfamily are expressed in developing blood cells, where they regulate differentiation-specific gene expression in hematopoiesis (reviewed
in Ref. 93). GATA2 also functions in urogenital development,
pituitary cell fate specification, and generation of V2 interneuronal cells in the spinal cord (reviewed in Ref. 21). GATA3 is
Address for reprint requests and other correspondence: S. D. Krasinski,
GI/Cell Biology, EN651, Children’s Hospital Boston, 300 Longwood Ave.,
Boston, MA 02115 (e-mail: [email protected]).
G474
also expressed in the adrenal glands, kidneys, central and
peripheral nervous systems, inner ear, hair follicles, skin, and
breast tissue (reviewed in Ref. 51). Members of the GATA4/
5/6 subfamily are expressed in various mesoderm- and endoderm-derived tissues, such as heart, liver, pancreas, lung,
gonad, and gut, where they play critical roles in regulating
tissue-specific gene expression (reviewed in Ref. 78). Here we
review the role of GATA factors in the evolution, development, and function of the small intestinal epithelium.
GATA Factors Are Expressed in Endoderm of Ancient
Metazoa
Gut formation is one of the earliest outcomes of multicellularity (reviewed in Refs. 20 and 107). It occurs by gastrulation,
a process in which invagination of the blastocyst lining at the
blastopore leads to the formation of a gastrovascular cavity,
called the archenteron, that is lined by two epithelial sheets, the
outer ectoderm and the inner endoderm (reviewed in Refs. 72
and 110). In the transition from protist to metazoan, thought to
occur during the Cambrian explosion 542 million years ago,
selective pressures forced the division of labor among cells: the
ectoderm became specialized for protection, locomotion, and
sensing, and the endoderm became specialized for digestion
and absorption of nutrients. Diploblastic animals possessing
two germ layers, ectoderm and endoderm, exhibit radial (rather
than bilateral) symmetry and have a single opening that serves
as both mouth and anus. They are found exclusively in the
phylum Cnidaria, which includes corals, sea anemones, jelly-
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Aronson BE, Stapleton KA, Krasinski SD. Role of GATA factors in development, differentiation, and homeostasis of the small intestinal epithelium. Am J
Physiol Gastrointest Liver Physiol 306: G474 –G490, 2014. First published January
16, 2014; doi:10.1152/ajpgi.00119.2013.—The small intestinal epithelium develops from embryonic endoderm into a highly specialized layer of cells perfectly
suited for the digestion and absorption of nutrients. The development, differentiation, and regeneration of the small intestinal epithelium require complex gene
regulatory networks involving multiple context-specific transcription factors. The
evolutionarily conserved GATA family of transcription factors, well known for its
role in hematopoiesis, is essential for the development of endoderm during
embryogenesis and the renewal of the differentiated epithelium in the mature gut.
We review the role of GATA factors in the evolution and development of endoderm
and summarize our current understanding of the function of GATA factors in the
mature small intestine. We offer perspective on the application of epigenetics
approaches to define the mechanisms underlying context-specific GATA gene
regulation during intestinal development.
Review
G475
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
A
B
Transcriptional
activation
AD
DNA binding and
protein-protein
interactions
Zn BR Zn BR
C
CTD
C
NLS
C
Zn2+
C
Fig. 1. Vertebrate GATA factors contain highly conserved zinc fingers. A: general structure of vertebrate GATA factors. Vertebrate GATA factors contain
activation domains (AD) at the NH2 terminus (yellow), 2 highly conserved zinc fingers (pink) with adjacent basic regions (BR, light blue), a nuclear localization
signal (NLS), and a COOH-terminal domain (CTD). B: general structure of GATA zinc fingers. GATA zinc fingers consist of 4 cysteine residues (type IV),
Cys-X2-Cys-X17-Cys-X2-Cys, that coordinate a divalent zinc ion.
of which express at least two GATA factors, generally, a
single, conserved GATA1/2/3 ortholog that is expressed in
ectoderm and at least one, but usually multiple, GATA4/5/6
orthologs that are expressed in mesoderm and endoderm derivatives. These findings suggest that the two vertebrate GATA
factor classes originated from distinct ancestral orthologs and
that the GATA gene duplication and functional divergence that
led to these two ancestral GATA factors occurred after the split
from diploblasts (Fig. 2). These studies also aligned the vertebrate GATA4/5/6 subfamily with endodermal differentiation.
Members of the phylum Echinodermata, the second-largest
grouping of deuterostomes, after chordates, express two Gata
Single cell
(choanoflagellates)
?
Multicellular
(poriferae)
?
Diploblasts
(cnidarians)
Endoderm
specification
Triploblastic protostomes
(arthropods, nematodes,
annelids)
Mesoderm
specification
Ancient Gata
ortholog
Gata1/2/3
Triploblastic deuterostomes
(echinoderms, chordates)
(vertebrates)
Gata4/5/6 ortholog
Gata4/5/6
ortholog
Gata4/5/6 ortholog
ortholog
Gatac
Gatae
Gata1/2/3
Gata4/5/6
Fig. 2. Evolution of GATA factors in Metazoa coincides with evolution of
endoderm. Although GATA factors have not been identified in choanoflagellates or members of the phylum Poriferae, a single, ancient GATA factor was
characterized in cnidarian diploblasts (orange). Evolution of GATA1/2/3 (red)
and GATA4/5/6 (yellow) orthologs coincided with evolution of mesoderm and
bilateral symmetry in triploblastic protostomes. Noteworthy was a biased
expansion of GATA4/5/6 orthologs. A single member of each GATA subfamily is present in early triploblastic deuterostomes, which eventually expanded
to 3 members in each subfamily in vertebrates.
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fish, and hydra. Mesoderm, or the middle layer, thought to be
a derivative of endoderm, arose ⬃40 million years after the
emergence of ectoderm and endoderm. Triploblastic animals
possessing three germ layers are linked to the evolution of axis
formation and bilateral symmetry. In contrast to diploblastic
animals, triploblastic bilaterians (with the exception of a few
highly reduced forms) have complete digestive tracts with
separate mouth and anus. The through-gut system enabled the
directional movement of nutrients, facilitating the diversification of cells along the digestive tract to control the processing
of food at different stages of digestion. This, in turn, allowed
for the utilization of a wider assortment of food types, enabling
an expanded range of mobility. Thus the through-gut digestive
system was one of the earliest and key innovations in the
evolution of metazoans.
Triploblastic bilaterians are further subdivided into protostomes and deuterostomes on the basis of how mouth and anus
develop. During gastrulation, invagination occurs at the blastopore, and the two opposing poles of the blastopore margin
close, forming a slitlike structure that defines the ventral
midline. In protostomes (including arthropods, nematodes, and
annelids), the openings at each end become mouth or anus,
whereas in deuterostomes (including echinoderms and chordates), the formation of the mouth occurs by a secondary
breakthrough of the archenteron.
Although multiple GATA factors have been identified in
plants, fungi, and molds (66), no GATA factors have been
reported for members of the phylum Porifera (sponges), the
earliest members of the animal kingdom, or their hypothesized
single-cell evolutionary predecessors the choanoflagellates.
However, Martindale et al. (73) cloned and characterized the
developmental expression of the only GATA factor in the
starlet sea anemone Nematostella vectensis, the first report of a
GATA factor in a cnidarian diploblast. NvGata, with homology
to the vertebrate GATA1/2/3 and GATA4/5/6 subfamilies (47),
is first detected in individual cells throughout the blastula,
which coalesce at the blastopore and ingress into the blastocoel
(cavity in blastocyst). By the late gastrula stage, all the gastrodermal cells lining the gastrovascular cavity (called the
coelenteron in diploblasts) express NvGata, and expression
continues into the polyp stage. This report demonstrates a
pattern of expression of an ancient GATA factor that intersects
with the evolutionary emergence of gastrulation and endoderm
specification (Fig. 2).
Expression of GATA factors has also been reported for
developing bilaterian protostome flatworms (17, 18, 46, 71), all
Review
G476
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
Divergent GATA Factors Act Sequentially in Endoderm
Formation and Gut Development in Invertebrates
The function of GATA factors and their placement in the
regulatory cascade of endoderm formation and gut development in the invertebrate protostomes Caenorhabditis elegans
and Drosophila melanogaster (Drosophila) have been well
reviewed (reviewed in Refs. 55, 87) and are updated here.
Seven GATA orthologs specify endoderm fate and terminal
differentiation of the C. elegans intestine. C. elegans expresses
no less than 11 orthologous GATA factors, of which 7 have
been implicated in endoderm and/or gut formation (reviewed in
Ref. 55). The sequences of these GATA factors display significant divergence, with only the elt-1/Gata1/2/3 ortholog
containing complete dual zinc finger domains. The remaining C. elegans GATA factors, all lacking the first zinc
finger, group with the GATA4/5/6 subgroup and, like lower
Metazoa, display a biased expansion of this subgroup of
GATA factors (47).
Embryogenesis in C. elegans (reviewed in Ref. 99) begins
when the asymmetric first cleavage produces a large anterior
daughter called AB and a smaller posterior daughter called P1.
P1 then divides to produce the anterior germline and mesectodermal precursor cell P2 and the posterior mesendodermal
precursor cell EMS (Fig. 3A). The anterior daughter of EMS,
called MS, gives rise to many mesodermal cell types, while the
posterior daughter of EMS, the E blastomere, is the progenitor
of the sole endoderm-derived organ, the intestine. Early embryonic patterning events are essentially completed by the
28-cell stage, when gastrulation begins with the ingression of
the intestinal precursor cells. The mature intestine comprises
the midgut of the C. elegans alimentary tract, connecting to the
pharynx (or foregut, derived partly from MS) and rectum (or
hindgut, derived from P2).
Tissue specification of the EMS blastomere is controlled by
the maternal bzip homeodomain transcription factor SKN-1,
the translation of which is confined to EMS by maternally
provided mRNA only to the posterior embryo. SKN-1 directly
activates transcription of the genes encoding MED-1 and
MED-2, two nearly identical and functionally redundant
GATA-like transcription factors (Fig. 3A). The med-1 and
med-2 genes are unique in that they lack introns and express
GATA-like factors that contain a single type IV zinc finger that
differs from classical GATA zinc fingers by a single amino
acid. This alteration results in recognition of the noncanonical
DNA sequence RAGTATAC, suggesting that med-1 and
med-2 have diverged in function and may be a nematode
invention (67). MED-1 and MED-2 play essential roles in
mesoderm specification by activating the T-box gene tbx-35 in
the MS blastomere (19) but play only a minor role in endoderm
specification and gut development (25, 68).
Specification of the E blastomere fate is determined by
expression of the genes encoding the redundant pair of GATA
factors, END-1 and END-3, which are activated by SKN-1
and, to a lesser extent, MED-1/MED-2 (Fig. 3A). While individual end-1 or end-3 knockouts develop normally, double
knockouts of end-1 and end-3 do not form an intestine (94).
Ectopic expression of either factor converts other embryonic
cells to an endodermal fate (69, 126). End-1 and end-3 appear
to be the result of a recent duplication, with each diverging to
activate two distinct, but overlapping, E lineage regulatory
pathways, perhaps through differences in their DNA-binding
domain specificities (11). End-1 and end-3 are also expressed
differently; end-3 is expressed prior to that of end-1 (and may
activate end-1 gene expression), and single end-3 knockouts
reveal a delay in E lineage activation that is not apparent in
single end-1 knockouts. Expression of both end-1 and end-3 is
extinguished prior to terminal gut differentiation.
The major target of END-1/END-3 in the early intestine (2E)
is the gene encoding yet another GATA factor, ELT-2 (43)
(Fig. 3A). Deletion of elt-2 results in a lethal arrest at birth; the
newly hatched larvae have a malformed, but clearly specified,
intestine (43, 74, 75). Promoters of genes that are exclusive to,
or highly enriched in, the intestine contain GATA binding sites
(compared with ⬍5% for control promoters), and most of these
genes are downregulated in elt-2 null worms. ELT-2 may
regulate these target genes in cooperation with Notch signaling
by physically interacting with the Notch-dependent effector
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genes (50, 63), Gatac and Gatae, which are orthologous to
vertebrate Gata1/2/3 and Gata4/5/6, respectively. The Gatae
mRNA is first detected in a ring around the vegetal pole of the
blastula. During gastrulation, transcripts are detected surrounding the blastopore, in the posterior archenteron, and in the
anterior mesoderm of the archenteron. In the late gastrula and
early larval stages, expression is localized to the midgut and
hindgut and to the developing coelomic pouches.
Using a combination of large-scale perturbation analyses,
computational methodologies, genomic data, cis-regulatory
analyses, and molecular embryology, Davidson et al. (30)
derived a mesoderm/endoderm gene regulatory network
(GRN) for the sea urchin embryo. In the 24-h blastula, all the
genes known to execute mesoderm and endoderm fate are
active. Early inputs are provided by the B lymphocyte-induced
maturation protein-1 (blimp1, formerly called krox1) and orthodenticle homolog-1 (otx1) genes, which regulate virtually
all the genes that specify mesendoderm, a transient precursor
cell population that subsequently differentiates into mesoderm
and endoderm. The principal target is gatae, which contributes
to activation of six other transcriptional regulatory genes of the
mesendoderm and also feeds back to the otx1 cis-regulatory
system, permanently locking down the mesendodermal specification state.
In summary, expression of GATA factors in metazoans can
be traced back to the evolution of gastrulation and the development of endoderm (Fig. 2). In a diploblast, a single GATA
factor that may be ancestral to the vertebrate GATA1/2/3 and
GATA4/5/6 subfamilies is expressed at the blastopore prior to
gastrulation and then in all endodermal cells after gastrulation.
In triploblasts, at least two GATA orthologs are expressed,
generally corresponding to the vertebrate GATA1/2/3 and
GATA4/5/6 subfamilies. Triploblastic protostomes generally
have a single GATA1/2/3 ortholog, but multiple GATA4/5/6
orthologs, whereas early triploblastic deuterostomes have a
single member of each subfamily that eventually expanded into
three members of each GATA subfamily in vertebrates. In all
cases, the GATA4/5/6 ortholog is expressed at the blastopore
prior to gastrulation, in ingressing cells during gastrulation, in
all endodermal cells after gastrulation, and in cells along the
digestive tract at maturity. GRN analysis in the sea urchin
indicates a pivotal role for GATA4/5/6 orthologs in the development of endoderm.
Review
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
A
B
C. elegans
G477
Drosophila
EMS
Tll
SKN-1
Hkb
Tll
Tor
MED-1/2
Hkb
Stage 5
Tor
E
MS
END-3
Endoderm
specification
TBX-35
END-1
Srp
Srp
Byn
2E
Mesoderm
specification
Byn
ELT-7
ELT-4?
dGATAc ? dGATAe
ELT-2
Terminal
differentiation
of the intestine
Stage 13
Terminal
differentiation
genes
Terminal Morphology
differentiation
genes
Intestine
Stage 16
LAG-1/CSL and, together, selecting target genes for endodermal expression (89). Thus ELT-2 plays a central role in the
establishment and maintenance of most aspects of terminal C.
elegans intestinal physiology.
Although the GATA orthologs ELT-4 and ELT-7 are also
expressed in the mature C. elegans intestine, deletion of either
elt-4 or elt-7 alone results in essentially wild-type intestine (5,
42, 104). ELT-4 is a very small GATA factor, barely the size
of a single zinc finger, and has no discernible function as
determined by deletion and overexpression experiments (42).
Deletion of elt-4 in the context of other elt deletions, however,
has not been reported; thus redundant functions with ELT-2
and/or ELT-7 remain a possibility. Simultaneous deletion of
elt-2 and elt-7, but not of either alone, eliminates all morphological features of differentiation in patches of the gut, suggesting cross-regulatory functions between ELT-2 and ELT-7
in gut development (104). Elt-7, like elt-2, is activated by
END-1/END-3, is expressed before elt-2, and activates its own
expression and expression of elt-2, constituting an apparent
positive-feedback system that locks down gut differentiation.
In summary, up to seven GATA4/5/6 orthologs play cooperative and sequential roles in guiding cell fate specification,
endoderm commitment, and terminal differentiation of the
nematode intestine (Fig. 3A).
Three GATA orthologs are necessary for endoderm development in Drosophila melanogaster. Five GATA genes are
found in the Drosophila genome, only one of which (dGATAc,
grain) is orthologous to the GATA1/2/3 subgroup (47). Three
GATA orthologs, serpent (srp, dGATAb), dGATAc (grain), and
dGATAe, are related to endoderm development (reviewed in
Ref. 87). With the exception of srp, which lacks an NH2terminal zinc finger due to a splicing isoform, all the Drosophila GATA factors have two zinc fingers associated with basic
domains.
In Drosophila, the digestive tract develops by invaginations
of prospective endoderm at the anterior and posterior regions
of the early blastoderm (stage 7– 8) that migrate bilaterally
along the yolk toward the trunk, encounter each other in the
middle of the trunk (stage 9 –10), and spread over the yolk to
form a continuous epithelial tube that encloses the yolk (stage
12–13), eventually giving rise to the epithelial lining of the
midgut (stage 15–16) (reviewed in Ref. 87). The prospective
endodermal regions of the early blastoderm (stage 5–6) are specified
by graded, maternally derived signaling from the transmembrane receptor tyrosine kinase, Torso (Tor) (Fig. 3B). Tor
signaling activates the two earliest zygotic gap genes tailless
(tll) and huckebein (hkb), which are expressed in a nested
pattern, with the Hkb regions included in Tll-positive domains.
While the Tll-positive, Hkb-negative domains specify ectodermal foregut and hindgut, Hkb-positive, Tll-positive regions
specify endoderm, although Tll itself is not necessary for this
process.
In anterior and posterior terminal regions, Hkb activates srp,
the earliest GATA gene expressed in Drosophila endoderm (95,
98) (Fig. 3B). Expression of srp begins at an early blastoderm
stage in the prospective endodermal regions but disappears in
the endoderm around stage 10 –11, long before terminal differentiation of the gut occurs. Loss of Srp activity results in a
transformation of prospective endoderm into ectodermal foregut and hindgut (98), while overexpression of srp ectopically
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Stage 7
Fig. 3. Multiple GATA factors act sequentially in endoderm and gut development in
protostome invertebrates. A and B: GATA
regulatory pathways in gut development for
Caenorhabditis elegans and Drosophila melanogaster. For consistency, A and B, were generated from the foundation established in
Kormish et al. (reviewed in Ref. 55) and
Murakami et al. (reviewed in Ref. 87), respectively. Endoderm and its progenitors and
terminal gut derivatives are shown in pale
yellow. GATA1/2/3 orthologs are indicated
in red, GATA4/5/6 orthologs in yellow, and
non-GATA factors in blue. Interactions are
described in detail in the text.
Review
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GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
Conserved GATA4/5/6 Orthologs Are Necessary for
Endoderm Development in Vertebrates
Deuterostome vertebrates possess a remarkably conserved
molecular program in which the Nodal signaling pathway is
necessary and sufficient to initiate mesendoderm development
(reviewed in Refs. 102, 106, 127, and 128). In general, high
levels of Nodal signaling promote endoderm development,
whereas lower levels specify mesoderm development. Nodal
signaling stimulates the downstream expression of a conserved
group of transcription factors, including Mix-like homeodomain proteins, Sry-related high-mobility group box (SOX)
factors, forkhead box (FOX) factors, and GATA factors. Generally, these factors together function to segregate endoderm
from mesoderm and to lock down endodermal fate. In this
section, we update the specific functions of GATA factors in
endoderm specification and intestinal development in Xenopus
laevis (Xenopus) and Danio rerio (zebrafish).
Gata4/5/6 regulate endodermal gene expression in X. laevis.
The early Xenopus embryo forms two distinct halves, the
pigmented animal pole and the yolky vegetal pole. Fertilization
occurs at the animal pole and triggers a displacement of egg
cytoplasm, establishing an asymmetric distribution of morphogenetic factors. In the early blastula, the yolky vegetal cells
along the blastocoel floor are fated to become endoderm. In the
midblastula stage (⬃4,000 cells), the so-called midblastula
transition, in which the cell cycle lengthens and zygotic transcription begins, occurs. A blastopore forms by invagination of
local endodermal cells on the anterior-dorsal side of the embryo, marking the beginning of gastrulation. As gastrulation
proceeds, the endoderm and mesoderm become internalized,
and the leading edge of the dorsal-anterior endoderm migrates
to the position of the ventral foregut. In the postgastrula
Xenopus embryo, the majority of the endoderm mass is located
ventrally, and the presumptive organ domains are arranged
along the anterior-posterior axis, consistent with their final
position in the gut. The mass of endodermal tissue elongates
and eventually cavitates to form a primitive gut tube. The
digestive tract then undergoes extensive remodeling from larva
to adult to adapt from an aquatic herbivorous to a terrestrial
carnivorous life.
Nodal signaling in Xenopus is induced in the late blastula
stage by the maternally inherited T-box transcription factor
VegT, which is localized to the vegetal region, where it directly
activates the transcription of Nodal-related genes (Fig. 4A). At
this stage, gata4, gata5, and gata6 are induced in the yolky
vegetal cells of the prospective endoderm (1, 29, 118). Induction of gata gene expression is abrogated by VegT loss of
function (120), indicating that gata expression is dependent on
Nodal signaling. Knockdown of Gata6 activity using morpholinos leads to a decrease in endodermal gene expression and
defects in gut morphology, while forced ectopic expression of
gata4, gata5, or gata6 in ectoderm induces endodermal markers such as sox17, hepatocyte nuclear factor-1␤ (hnf1␤), and
foxa2 (1, 118). These data suggest that Nodal signaling acts
through gata4/5/6 to establish and maintain endodermal gene
expression.
Active migration of the leading edge of mesendoderm across
the blastocoel roof of the Xenopus embryo is necessary for the
development of tissues such as head mesoderm, heart, blood,
and liver. Gata4 and gata6 are expressed in this migratory
tissue during gastrulation. Gain-of-function experiments with
these Gata factors reveal an induction of cell spreading and
migration in nonmotile cells of presumptive ectoderm, while
expression of a dominant-negative form of Gata6 severely
impairs the ability of the dorsal leading edge of the mesendoderm to spread (40). These studies implicate a critical role for
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induces endodermal genes (87). Thus srp, like end-1/end-3 in
C. elegans, is necessary for endoderm specification but is not
required later to maintain terminal gut differentiation.
Recently, it was shown that Srp induces an endodermal
epithelial-mesenchymal transition (EMT) (22). During formation of the Drosophila endoderm, epithelial cells adopt a
mesenchymal state and migrate through the embryo and later
reepithelialize to give rise to a large portion of the intestinal
tract. In srp mutants, endodermal cells retain epithelial shape
and polarity and do not migrate, and dE-cadherin, a junctional
protein that mobilizes in EMT, remains tightly localized to the
apical domain of cells. Thus Srp confers critical migratory
capabilities on epithelial cells during intestinal development.
dGATAc is expressed in the head, posterior spiracles, and
central nervous system, as well as in developing endoderm
beginning at stage 12, and continues to be expressed in anterior
regions of midgut at least to stage 15 (reviewed in Ref. 87). In
nongut tissue, dGATAc affects organ shape by locally controlling cell rearrangement and morphogenetic movement, but
neither deficiency nor overexpression of dGATAc affects
midgut morphology or endodermal gene expression. Thus the
function of dGATAc in endoderm development remains unknown.
dGATAe is expressed specifically in the anterior and posterior endoderms at stage 8, inversely correlating with weakening srp expression, and continues to be expressed in the larval
and adult midgut (91) (Fig. 3B). dGATAe is not expressed in
srp mutants and is induced when srp is overexpressed, indicating that srp is required for dGATAe expression. Loss of
dGATAe causes a general downregulation of genes associated
with terminal differentiation of the midgut, while dGATAe
overexpression induces ectopic expression of these genes (91).
However, a subset of terminal differentiation genes activated
by Srp, but not dGATAe, suggests that dGATAe-independent
pathways that are downstream of Srp regulate some intestinal
genes (92). In embryos deficient in srp or dGATAe, a master
gene of ectodermal hindgut, brachyenteron (byn), is ectopically expressed in innate endodermal regions. Taken together,
these data indicate that Srp specifies endodermal fate and,
together with its target dGATAe, activates the terminal gut
differentiation program while suppressing an ectodermal fate.
In summary, sequential expression of discrete GATA4/5/6
orthologs mediates endoderm specification and intestinal development in C. elegans and Drosophila. In both cases, a
maternally derived process asymmetrically activates the early
and transient expression of a GATA factor (end-1/end-3 in C.
elegans and srp in Drosophila) in prospective endoderm that
specifies endodermal fate. This GATA factor, in turn, activates
a later GATA factor (elt-2/elt-7 in C. elegans and dGATAe in
Drosophila) that triggers a terminal gut differentiation program. Thus the sequential functions of distinct GATA factors
in the gene regulatory pathways of endoderm development are
well conserved in the protosome invertebrates C. elegans and
Drosophila.
Review
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
B
VegT
Zygotic Nodals
Otx2?
Mesendoderm
Xenopus
Gata4
Mix-like
transcription
factors
Zebrafish
YSL
?
Zygotic Nodals
Otx2
Gata5
Bon
Gata5
Gata6
Gata6
Hnf1β
Casanova
Sox17
FoxA2
Fig. 4. Gata4/5/6 regulate endoderm development in Xenopus and zebrafish. A
and B: placement of Gata factors in the progressive commitment of mesendoderm to endoderm for Xenopus laevis and Danio rerio (zebrafish). For
consistency, A and B were generated from the basis established by Shivdasani
(reviewed in Ref. 102). Gata4/5/6 orthologs are depicted in yellow and
non-Gata factors in blue. Interactions are described in detail in the text.
Gata factors in cell migration similar to that of SRP in EMT
during Drosophila development.
GRN analysis indicates that GataE in sea urchin and Gata
factors in Xenopus share common downstream targets, including other gata factors and hnf1␤ (65). Although blimp and otx
homologs have been identified in Xenopus, they have not been
indicated as inducers of Xenopus endoderm by GRN analysis.
However, injection of mouse otx2 mRNA into Xenopus embryos induces expression of the endodermal marker endodermin (27), suggesting that Otx function in endoderm development may indeed be conserved.
Gata factors regulate endoderm formation in zebrafish. In
zebrafish embryos, the epiblast represents the cells that lie on
top of a large yolk cell prior to gastrulation. Ectoderm is
derived from the animal portion of the epiblast, whereas
endoderm emerges from the four rows of cells closest to the
yolk; mesoderm precursors are intermingled with endoderm
progenitors but extend up to eight cells from the yolk margin.
During gastrulation, prospective endodermal cells involute
first, migrate anteriorly under the epiblast, and eventually form
a monolayer of cells dispersed with mesoderm precursors.
During early somite stages of development, the endodermal
sheet converges on the dorsal midline to form a rod of cells
from which organ buds eventually emerge and which later
cavitates to form a gut tube.
While maternally inherited VegT directly stimulates transcription of Nodal genes in Xenopus, an as-yet-unidentified
maternal signal, likely originating from the maternal extraembryonic yolk syncytial layer, is thought to activate Nodal
signaling in zebrafish (reviewed in Refs. 102, 106, 127, and
128) (Fig. 4B). In endodermal progenitors during late blastula
stages, Nodal activates zebrafish faust (fau), which encodes
Gata5 (96, 111). Both loss- and gain-of-function experiments
reveal that Gata5 regulates the amount of endoderm formed
and promotes the expression of sox17 and foxa2, markers of
specified endoderm (96, 97). Combinatorial mutant analysis
demonstrates that Gata5 cooperates with Bon, a Mix-like
homeobox transcription factor, downstream of Nodal signaling
and upstream of casanova (a sox17-related gene) to regulate
endoderm formation (97). Gata5 and Bon may synergize with
eomesodermin, a localized maternal determinant required for
endoderm induction, to induce cas, possibly by facilitating the
assembly of a transcriptional activation complex on the cas
promoter (10).
The lateral-to-medial migration of endoderm during gastrulation, thought to be analogous to the movement of mammalian
rostral endoderm as it folds to form the foregut (119), is
impaired by selective inhibition of gata5 translation in the yolk
syncytial layer, the tissue ortholog of the mammalian extraembryonic visceral endoderm (115). Since extraembryonic GATA4,
and not GATA5, is required for ventral folding morphogenesis
in mice (59, 79), zebrafish Fau/Gata5 has been suggested to be
a functional ortholog of mammalian GATA4 (115). In contrast
to fau/gata5 mutants in which endoderm specification is impaired, zebrafish gata4 mutants show normal endoderm specification, differentiation, and morphogenetic movement, but
subsequent organogenesis fails, in that the intestine lacks
normal epithelial folds, and liver and exocrine pancreatic
tissues fail to form (52).
Zebrafish gata6 morphants express early endoderm markers,
indicating that the endoderm is specified, but die prior to gut
development. Titration of the gata6 morpholino to allow lowlevel expression of Gata6 and embryonic survival reveals a
specific defect in liver development (52), similar to that in mice
(125). Interestingly, loss of microRNA-145, which targets
gata6 in zebrafish gut smooth muscle, results in defects in
smooth muscle function as well as gut maturation (124),
implicating a role of mature smooth muscle in promoting
epithelial differentiation, and an importance for nonepithelial
Gata factors in gut homeostasis, an area that has not been well
studied.
Morpholino knockdown, chromatin immunoprecipitation,
and GRN analyses revealed that Otx2 is necessary for endoderm specification in zebrafish and likely directly activates
gata5 and gata6 (111). Gata5 and gata6 then activate each
other, forming an autoregulatory positive-feedback system that
locks down endoderm specification. Thus the Otx-Gata regulatory loop found in sea urchin (30) is conserved in fish and
possibly frogs.
GATA4 and GATA6 Are Required for Extraembryonic
Endoderm Development in Mice
Amniotes, including reptiles, birds, and mammals, are vertebrates whose embryos are totally enclosed in a fluid-filled
sac, an evolutionary advantage that enabled breeding away
from water. During embryogenesis, amniotes develop extraembryonic structures, called extraembryonic endoderm, that
protect and nourish the developing embryo. Extraembryonic
endoderm is distinguished from definitive endoderm, the germ
layer precursor of the gut and its accessory organs, although
both share many developmental features and molecular markers. Since most of the GATA work has been conducted on
mice, this section focuses on the role of GATA factors in
murine endoderm development.
Although Nodal signaling is essential for specification of the
definitive endoderm in the mouse embryo (101) and GATA
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Sox17
Endoderm
A
G479
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GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
tions to a columnar epithelium, cytodifferentiation occurs, and
villi form. Crypts develop during the first 2 wk after birth, in
contrast to crypt development in humans, which occurs before
birth (reviewed in Ref. 81). The small intestine undergoes
“re”-differentiation during the weaning transition, when expression of enzymes and transporters shifts to accommodate
the change from a milk-based to a solid-food diet.
In contrast to Xenopus and zebrafish, in which GATA5 is
essential for endoderm and gut development (96, 97, 118),
GATA5 is not expressed in mouse embryonic stem (ES) cells
(24) or primitive or definitive endoderm (83). Gata5 null mice
are sterile and show abnormalities in the female urogenital
systems (80), but defects in endoderm or gut development have
not been reported. However, Gata5 is upregulated in Gata4
null (Gata4⫺/⫺) or Gata6 null (Gata6⫺/⫺) ES cells (24),
suggesting that GATA5 is redundant with GATA4 and
GATA6 during early development. Resolution of the function
of GATA5 in endoderm development must await combination
knockout studies.
Gata6⫺/⫺ mice die at E5.5–E6.5 due to defects in PrE
formation and subsequent extraembryonic endoderm development (56, 86). Gata6⫺/⫺ ES cells fail to develop extraembryonic endoderm in vivo and in vitro, and the expression of early
and late markers of VE is significantly downregulated or
abolished (56, 86), while forced overexpression of Gata6 in ES
cells promotes differentiation toward extraembryonic endoderm (41). These data demonstrate that GATA6 plays a critical
role in differentiation of the PrE and survival of the embryo
past the early primitive streak stage.
A model is emerging for the role of GATA6 in PrE specification (reviewed in Refs. 100 and 109) (Fig. 5). At the
eight-cell stage (E2.0), all blastomeres retain the potential to
form all cell lineages, but following compaction, segregated
expression of Pou domain, class 5, transcription factor 1
(Pou5f1, formerly known as Oct4) and caudal type homeobox
2 (Cdx2) accompanies specification of the ICM and TE, respectively. At E3.5, the cells of the ICM in the preimplantation
blastocyst, although morphologically indistinct, are already
heterogeneous. At this stage, Nanog, which encodes a homeodomain protein necessary to maintain pluripotency in ES cells
in vitro (77), and Gata6 are expressed in the ICM in a random,
mutually exclusive “salt-and-pepper” pattern (26) (Fig. 5A).
Lineage-tracing experiments showed that Nanog-positive cells
specify Epi, while Gata6-positive cells become PrE. By E4.5,
overt segregation of PrE and Epi has occurred in the ICM, in
which Gata6-positive PrE cells localize to the surface of the
ICM facing the blastocoel cavity, while the Nanog-positive
cells of the Epi are confined inside the ICM (Fig. 5A).
The segregation of Epi and PrE lineages in the ICM is
regulated by FGF signaling (61). FGF4, FGF receptor (FGFR)
2, and SH2/SH3 adaptor (GRB2), which together activate the
MAPK signaling pathway, are necessary for PrE formation
(Fig. 5B). In embryos lacking Grb2, Gata6 expression is lost,
all ICM cells become Nanog-positive, and no PrE is formed
(26), indicating that all ICM cells have adopted the Epi fate at
the expense of PrE. ES cells overexpressing a dominantnegative FGFR cannot differentiate into PrE, but this phenotype can be rescued by overexpression of GATA factors (64),
suggesting that activation of Gata factor expression is the key
PrE-promoting event downstream of FGF signaling. The precise mechanism that underlies Gata6/Nanog segregation is
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transcription factors are necessary downstream Nodal effectors
of endoderm development in Xenopus and zebrafish, it remains
uncertain whether GATA factors have an analogous role in the
development of the definitive endoderm in mice. However, it is
clear that GATA factors are critical for extraembryonic endoderm specification and mouse embryo viability.
After fertilization and zygotic cleavage of the early mouse
embryo, a growing mass of undifferentiated cells, the morula,
is formed by the second embryonic (E) day (reviewed in Refs.
100 and 109). Each of the eight cells of the morula, called a
blastomere, increases its surface contact with its neighbor
through a process called compaction, resulting in a polarization
of cells. By E3.5, a blastocyst of ⬃32 cells is established in
which ⬃12 internal cells comprise the new inner cell mass
(ICM) and the remaining 20 cells comprise the surrounding
trophoectoderm (TE), the first cell lineage to be specified in the
developing mouse embryo. After implantation at E4, the blastocyst elongates, and the placenta develops around the egg
cylinder and begins to provide maternal blood to the embryo.
At this stage, the primitive endoderm (PrE) lining the blastocoel cavity is specified from the ICM. The TE and PrE
contribute mainly to extraembryonic tissues that form the
placenta and yolk sac, respectively, whereas the pluripotent
cells of the ICM, called the epiblast (Epi), give rise to all the
cell lineages of the future embryo, including the definitive
endoderm.
Prior to gastrulation, PrE differentiates into visceral endoderm (VE), which forms from cells that remain in contact with
the Epi, and parietal endoderm (PE), which forms from cells
that migrate over the inner surface of the TE. VE cells have
microvilli and synthesize proteins involved in nutrient transport and also function in patterning the early embryo by
participating in primitive streak formation and embryonic mesoderm induction by antagonizing Nodal signaling. Distinct
from the VE, the PE cells produce a large amount of extracellular matrix that is deposited along the inner surface of the TE
to form Reichert’s membrane, a thick basement membrane-like
structure that separates the yolk cavity from the maternal
tissues. Extraembryonic endoderm thus provides critical
patterning cues and essential maternal-fetal connections required to sustain and protect the embryo after implantation.
Disruption of extraembryonic endoderm development results
in arrest at gastrulation (34), demonstrating the importance of
extraembryonic endoderm for fetal development.
The definitive endoderm, from which the gut and its accessory organs are formed, arises from the Epi at gastrulation
(beginning at E6.5). Cells in the Epi undergo EMT, migrate
through the primitive streak, and differentiate into mesoderm
and endoderm. The presumptive definitive endoderm cells
invade and displace cells in the VE, although lineage-tracing
experiments suggest that the definitive endoderm at least partly
comprises VE cells (60). Morphogenesis begins at E7.5, when
the epithelial sheet folds over at the anterior and posterior ends,
forming foregut and hindgut pockets, termed the anterior
intestinal portal and caudal intestinal portal. The folding continues caudally and rostrally until the folds meet, completing
tubulogenesis. During somite stages, the mass of endodermal
tissue elongates and cavitates to form a primitive gut tube
(E8.5). At E9.5, endodermal organ buds form, and from E9.5
to E13.5, the gut tube lengthens and the circumference increases. From E14.5 to E17.5, the stratified epithelium transi-
Review
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
G481
A
Early blastocyst
E3.5 (32-64 cells)
Late blastocyst
E4.5 (~120 cells)
TE
Epi
CDX2-positive
Sorting
Nanog-positive
GATA6-positive
Blastocoel
ICM
PrE
FGF4
FGFR2
GRB2
MAPK
FGF4
NANOG >>> GATA6
GATA6 >>> NANOG
Fgf4
Epiblast
Fgfr2
Primitive endoderm
unknown, although an inverse correlation in expression of Fgf4
and Fgfr2 in the early ICM (48) suggests a mechanism in
which Fgf4 expression from presumably Epi-fated cells generates a lateral inhibitory signal to Fgfr2-expressing, PrE-fated
cells (Fig. 5B), resulting in mutually exclusive specification of
Epi and PrE.
The sorting mechanism responsible for the relocation of
randomly positioned PrE progenitors to the surface of the ICM
facing the blastocoel remains unknown. While overexpression
of Gata6 in deeper ICM cells did not significantly change their
position, a combination of Gata6 and Wnt9A, which encodes a
Wnt ligand known to be expressed in the surface ICM, facilitates repositioning of Gata6-expressing cells (76). One of the
GATA6-induced genes is disabled homolog-2 (Dab2), which
encodes a phosphoprotein involved in endocytosis. Dab2 is
detectable in PrE of the implanting blastocyst at E4.5 and is
downregulated in Gata6 mutants (85). Homozygous Dab2deficient mutants are embryonic lethal by E6.5 due to defective
relocation of the PrE progenitors (121). Thus DAB2 is an
essential downstream effector of GATA6 that may function in
PrE progenitor positioning in the blastocyst. The relationship
of DAB2 to WNT9A-faciliated sorting remains to be determined.
Although targeted deletion of Gata4 is embryonic lethal at
E9.5 (see below), long after the PrE has been specified,
GATA4 may still function in PrE development. Cultured
Gata4⫺/⫺ mouse ES cells show a defective formation of PrE
(88, 105), while forced overexpression of Gata4 in ES cells is
sufficient to induce the proper differentiation program toward
extraembryonic endoderm (41). Gata6⫺/⫺ ES cells reveal a
decrease in Gata4 expression (56, 86), while Gata4⫺/⫺ em-
bryos show an increase in Gata6 expression (59, 79). These
data suggest a linear pathway in which GATA4 is downstream
of GATA6 in PrE differentiation.
Although GATA6 and, possibly, GATA4 function in PrE
specification and, thus, VE and PE formation, little is known
about the specific roles of GATA4 or GATA6 in the further
differentiation or maintenance of VE or PE. Using embryoid
bodies derived from mouse ES cells in which both copies of the
Gata4 gene were disrupted, Soudais et al. (105) found that, in
contrast to Gata4-sufficient embryoid bodies, which were covered by a layer of VE, Gata4⫺/⫺ embryoid bodies formed no
VE. Gata4⫺/⫺ ES cells also showed defects in VE development but can be induced to undergo epithelial differentiation
by retinoic acid (23). Since Gata4⫺/⫺ ES cells have the
capacity to differentiate along other lineages, it was concluded
that Gata4 was specifically required for VE formation (105).
PE produces large amounts of basement membrane components, such as laminin-1 and collagen IV. Mouse F9 embryonal
carcinoma cells can be induced to differentiate into PE-like
cells, including expression of basement membrane components, by treatment with retinoic acid and dibutyryl cAMP.
Silencing of Sox7 or combined silencing of Gata4 and Gata6
results in suppression of PE differentiation. Although overexpression of Sox7 alone was insufficient to induce PE differentiation, overexpression of Gata4 or Gata6 in Sox7-silenced F9
cells restored the differentiation into PE (44). These data
suggest that Sox7 is required for the induction of Gata4 and
Gata6, and the interplay among these transcription factors
plays a crucial role in PE differentiation.
Gata4⫺/⫺ mice die between E7.5 and E9.5, with a migration
and folding defect in ventral morphogenesis that results in a
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B
Fig. 5. GATA6 specifies primitive endoderm in the early
mouse blastocyst. A: distribution of primitive endoderm
(PrE) and epiblast (Epi) progenitors in early and late
mouse blastocyst. PrE (yellow) and Epi (light blue) progenitors are randomly distributed in the inner cell mass
(ICM) at embryonic day 3.5 (E3.5). Trophoectoderm (TE,
pink) cells make up the peripheral cell layer. By E4.5, PrE
cells are localized to the surface of the blastocoel cavity;
Epi cells are restricted to the inner region of the ICM.
B: model for FGF signaling in PrE formation. Fgf4 expression is upregulated in Epi progenitor cells; FGF receptor 2 (Fgfr2) expression is increased in PrE progenitors. FGF4 secreted by Epi progenitors is thought to
interact with FGFR2, which, in turn, activates GRB2 and
MAPK in PrE progenitors, resulting in induction of Gata6
expression and simultaneous repression of Nanog expression, promoting PrE fate while inhibiting Epi fate. For
consistency, A and B were generated from the foundation
published by Takaoka and Hamada (reviewed in Ref.
109).
Review
G482
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
functions in PrE development prior to gastrulation, perhaps
downstream of GATA6 (56, 59, 79, 86, 100, 109), but its
expression in extraembryonic endoderm is essential for ventral
folding morphogenesis and primitive heart and gut tube formation after gastrulation (59, 79, 88). The precise role of
GATA factors in definitive endoderm remains to be determined. Further investigation using combination Gata deletion
and/or alternative Cre drivers is necessary to define the specific
and overlapping functions of GATA factors in early intestinal
development and their function in definitive endoderm.
GATA4 and GATA6 Show Specific and Redundant Functions
in the Mature Mouse Small Intestine
The mature mammalian small intestine is lined by a highly
specialized epithelium that exhibits a wide-ranging, yet tightly
regulated, functional diversity along its cephalocaudal axis
(Fig. 6A), resulting in an exquisite efficiency in the absorption
of dietary nutrients. The functional diversity is linked to a
continuous renewal process in which stem cells located at or
near the base of the crypts of Lieberkühn produce proliferating
transit-amplifying progenitor cells that ultimately differentiate
into five principal postmitotic cell types comprising one type of
absorptive cell (absorptive enterocytes) and four types of
secretory cells (enteroendocrine, goblet, Paneth, and tuft cells)
(reviewed in Ref. 90) (Fig. 6B). Absorptive enterocytes, goblet
cells, enteroendocrine cells, and tuft cells migrate up the crypt
to populate the villi, fingerlike structures that protrude into the
lumen to increase absorptive surface area, whereas Paneth cells
migrate to the base of crypts. The differentiated cells are
eventually removed by apoptosis and/or extrusion. Cells of the
villus epithelium turn over in 3– 4 days, whereas Paneth cells at
the base of crypts turn over in 3– 6 wk.
Current models of intestinal epithelial differentiation support the existence of two populations of stem cells, rapidly
dividing crypt base columnar cells marked by leucine-rich
repeat-containing G-protein coupled receptor 5 (LGR5), and
slow-cycling, quiescent cells that reside at the so-called ⫹4
position (reviewed in Refs. 90 and 122). The stem cells
produce proliferating transit-amplifying cells that undergo a
series of transitions, ultimately giving rise to the differentiated
cell lineages of the small intestinal epithelium (Fig. 6C). The
Wnt and bone morphogenetic protein (BMP) signaling pathways are implicated in crypt morphogenesis and proliferation,
whereas the Notch signaling pathway plays a critical role in
determining epithelial cell fate by regulating the balance of
absorptive vs. secretory cells (reviewed in Refs. 90 and 122).
Notch signaling activates the expression of hairy and enhancer
of split 1 (Hes1), which encodes a transcription factor that
specifies the absorptive enterocyte lineage. Progenitor cells
that elude Notch signaling express atonal homolog 1 (Atoh1,
formerly called Math1), which encodes a transcription factor
that designates the secretory lineages. Within secretory progenitors, growth factor-independent 1 (Gfi1) distinguishes enteroendocrine from goblet/Paneth progenitors; neurogenin 3 (Neurog3) specifies the enteroendocrine lineage; sam pointed domain-containing Ets transcription factor (Spdef), a GFI1 target,
promotes goblet differentiation; and the Wnt targets SRY
box-containing gene 9 (Sox9) and ephrin type B receptor 3
(Ephb3) are necessary for the differentiation of Paneth cells
and their localization to the crypt base, respectively. The role
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failure to form a primitive heart tube and foregut (59, 79). At
E8.0, Gata4 is expressed in cardiogenic splanchnic mesoderm
and in definitive and extraembryonic endoderm. To define the
cause of the Gata4⫺/⫺ defect, chimeric mice were produced in
which Gata4⫹/⫹ cells were restricted to extraembryonic endoderm and small portions of foregut and hindgut definitive
endoderm (88) but deleted in all other cells of the developing
embryo. Heart and foregut developed normally, indicating that
expression of GATA4 in extraembryonic or definitive endoderm, rather than mesoderm, is required for ventral morphogenesis. Tetraploid embryo complementation, in which tetraploid embryos contribute cells to the extraembryonic endoderm,
while the fetus, including definitive endoderm, is derived
solely from ES cells, was used to produce Gata4⫺/⫺ embryos
with wild-type extraembryonic endoderm (116). Despite an
absence of GATA4 in all other cells of the developing embryo
including definitive endoderm, ventral folding morphogenesis
and heart tube formation occurred normally, indicating that the
GATA4 in extraembryonic endoderm, rather than mesoderm or
definitive endoderm, is required for ventral morphogenesis and
heart development. Thus, although GATA4 may have overlapping functions with GATA6 in PrE formation at ⬃E6, it is
specifically required later in extraembryonic endoderm for
definitive endoderm migration and ventral folding morphogenesis at around E8.
Tetraploid embryo complementation was also used to determine the importance of GATA factors in extraembryonic
endoderm for later endoderm organ development. Gata4⫺/⫺
embryos with wild-type extraembryonic endoderm form the
dorsal pancreas normally but show defects in ventral pancreatic
development (117). Gata6⫺/⫺ embryos with wild-type extraembryonic endoderm specify hepatic cells normally, but the
specified cells fail to differentiate, and the liver bud does not
expand (125). In vivo footprinting showed that a DNA-binding
site for GATA factors is occupied on a liver-specific, transcriptional enhancer of the serum albumin gene long before albumin
or other liver-specific genes are expressed, suggesting that
GATA factors at target sites in chromatin may potentiate gene
expression and tissue specification in endoderm development
(12). GATA4 was one of a select few factors capable of
binding to sites in compacted chromatin (28), suggesting that
GATA factors may act as pioneering factors in liver development.
Conditional deletion of Gata factors in the intestinal epithelium has been established using villin-Cre transgenes in which
Cre is expressed in the epithelial cells of the small and large
intestine, including stem cells (37). Using a tamoxifen-inducible model in which Gata4 was deleted prior to cytodifferentiation and villus formation and during the suckling period,
only modest alterations in gene expression, but no overt
changes in intestinal development, were noted (13).
In summary, Nodal signaling is essential for endoderm and
gut development in vertebrates. In Xenopus and zebrafish,
Nodal signaling stimulates the downstream expression of
members of the Gata4/5/6 subfamily (96, 111, 120), which, in
turn, contribute to endoderm development (1, 96, 97, 111,
118). In mice, GATA5 is not necessary for mouse endoderm
development (80), while GATA6 is essential for PrE development in the blastocyst (56, 86), possibly for mediating migration of committed PrE cells from the ICM to the blastocoel
surface (reviewed in Refs. 100 and 109). GATA4 likely also
Review
pylorus
ligament
of Treitz
A
ileocecal
valve
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
stomach
colon
duodenum
jejunum
Iron
absorption
ileum
Fat, protein,
carbohydrate
absorption
Bile acid,
vitamin B12
absorption
B
C
migration
Villus
Absorptive enterocyte
Enteroendocrine cell
SOX9, EPHB3
Goblet cell
Paneth cell
Tuft cell
NEUROG3
Apoptotic cell
GFI1, SPDEF
?
‘+4’ (quiescent) stem cell
CBC (cycling) stem cell
TA/progenitorcell
ATOH1, DLL1
(Secretory
progenitor)
HES1
(Absorptive
progenitor)
Notch
Crypt
Wnt
BMP
of GATA factors in the topographic patterning and regulation
of intestinal proliferation, lineage commitment, differentiation,
and gene expression in the mature mouse small intestine is the
focus of this section.
GATA4/5/6 are expressed in distinct and overlapping patterns in the mature mouse small intestine. Gata4, Gata5, and
Gata6 mRNAs are expressed in mature mouse small intestine
(2, 62, 82, 83). Along the proximal-distal gradient, Gata4
mRNA is generally constant but declines sharply in the distal
ileum, while Gata5 mRNA shows a generally increasing proximal-distal pattern and Gata6 mRNA is generally constant
throughout the small intestine (7, 14, 35, 112). The distal
decline in Gata4 expression was confirmed for GATA4 protein
in mice (14, 112) and humans (14, 49). A tracing approach, in
which Cre excision resulted in expression of alkaline phosphatase under the control of the endogenous Gata4 promoter, was
used to determine that Gata4 is expressed in the proximal 85%
of small intestine and is sharply downregulated in the distal
15% (9), coincident with the distal ileum.
Immunohistochemical analysis of GATA protein expression
in crypt and villus compartments, and in specific lineages, is
highly variable, probably owing to differences in the specific
antibodies used. GATA4 is expressed in the nuclei of epithelial
cells in crypts and on villi (14, 16, 32, 49, 112), and costaining
with proliferation or lineage markers indicates that GATA4 is
expressed in proliferating cells in crypts and in absorptive
enterocytes on villi, but not in goblet or enteroendocrine cells;
it remains unclear whether GATA4 is expressed in differentiated Paneth cells (14, 36, 49); expression of GATA4 in
Fig. 6. Regional function and lineage differentiation of mouse small intestinal epithelium
are tightly controlled by multiple regulatory
proteins. A: specific functions of the small
intestine are regionally segregated. The main
function of the jejunum (blue) is general absorption of fat, protein, and carbohydrate.
Although these functions are conducted
throughout the small intestine, the duodenum
(green) is specifically tasked with iron absorption, while the ileum (purple) is responsible
for bile acid and vitamin B12 absorption.
B: crypt-villus structure enables the small
intestinal epithelium to regenerate itself with
a precise distribution of highly diverse cell
lineages. Stem cells in crypts, including rapidly cycling crypt base columnar cell (CBC)
and quiescent cells located at the ⫹4 position,
produce proliferating transit-amplifying (TA)
cells that differentiate as they migrate out of
the crypt and onto villi or, in the case of
Paneth cells, as they migrate to the base
of crypts. C: stem/progenitor cells in crypts
undergo a series of decisions as they terminally differentiate. Regulatory proteins are
shown next to cells in which they are expressed and/or have a role in the differentiation process. HES1, hairy and enhancer of
split 1; ATOH1, atonal homolog 1; DLL1,
delta-like 1; GF1, growth factor-independent
1; SPDEF, sam pointed domain-containing
Ets transcription factor; SOX9, SRY boxcontaining gene 9; EPHB3, ephrin type B
receptor 3; NEUROG3, neurogenin 3; BMP,
bone morphogenetic protein. The function of
these regulatory proteins is described in detail
in the text.
intestinal stem cells or tuft cells has not been reported. GATA5
has been suggested to be specific for secretory cells (36) and
has also been reported to be transiently expressed in the nuclei
of absorptive enterocytes before weaning in mice (⬃2 wk of
age) (33), but further work is necessary to confirm these
patterns of expression. GATA6 is expressed in the nuclei of
cells in crypt and villus epithelium in mice (7, 14, 32, 103) and
humans (49). GATA6 is expressed in proliferating crypt cells
and in all differentiated lineages tested in mice (7). GATA6 is
expressed in all but enteroendocrine cells in humans (49),
suggesting a possible species difference in intestinal GATA6
expression. It should be noted that the field is hampered by
antibodies for GATA5 and GATA6 that cross-react with other
proteins and that, unlike GATA4, immunostaining for GATA5
or GATA6 has not been verified using conditional knockout
mice as controls.
GATA factors bind and activate intestinal genes. The realization that GATA factors were expressed in the mature small
intestine led to a search for putative GATA target genes. Using
EMSAs and transient cotransfection assays in cultured cell
lines, multiple groups showed that GATA4, GATA5, and/or
GATA6 bind and activate the promoters of multiple intestinespecific genes (16, 31, 32, 38, 39, 45, 54, 58). Mutations in
these GATA motifs generally abrogated binding and transactivation. Dusing et al. (35) showed that a transgene constructed
from the intestine-specific enhancer in the adenosine deaminase (Ada) gene that contains consensus GATA binding sites
had high expression levels in murine duodenum, but a homologous transgene with selective mutations in all three enhancer
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Secretory
Absorptive
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GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
GATA binding sites had markedly reduced duodenal expression and alterations in the proximal-distal and cell-specific
expression patterns, demonstrating the importance of GATA
motifs for transcriptional activation in vivo.
GATA4 mediates regional identities in absorptive enterocyte
gene expression and function. GATA4 ACTIVATES A SUBSET OF
ABSORPTIVE ENTEROCYTE GENES IN THE PROXIMAL SMALL
INTESTINE. Gata4 is expressed in the proximal 85% of small
Proximal
Distal
Slc10a2, Fabp6
Si, Fabp2
Lct, Fabp1
Gata4 expression
Gata6 expression
Fig. 7. Gata4 expression coincides with transitions in absorptive enterocyte
gene expression in the distal ileum. Proximal-distal expression patterns of 3
distinct gene sets are shown. “Intestinal” gene set, expressed throughout the
small intestine (black), is exemplified by sucrase isomaltase (Si) and fatty
acid-binding protein 2 (Fabp2); “jejunal” gene set (blue) is exemplified by
lactase (Lct) and fatty acid-binding protein 1 (Fabp1); “ileal” gene set (purple)
is exemplified by solute carrier family 10, member 2 (Slc10a2) and fatty
acid-binding protein 6 (Fabp6).
GATA4 REPRESSES THE EXPRESSION OF A SUBSET OF ABSORPTIVE
ENTEROCYTE GENES, LIMITING THEIR EXPRESSION TO THE DISTAL
ILEUM. Conditional deletion of Gata4 also revealed an upregu-
lation in jejunum of absorptive enterocyte genes, the expression of which is normally restricted to the distal ileum (4, 9,
14), including solute carrier family 10 member A2 [Slc10a2,
which encodes the apical sodium-dependent bile acid transporter (Asbt)] and fatty acid-binding protein 6 [Fabp6, which
encodes the ileal lipid-binding protein (Ilbp)]. ASBT was
induced on villi within 24 h of induction of Gata4 deletion,
indicating that GATA4 represses Slc10a2 gene expression
within differentiated absorptive enterocytes, rather than by a
process that originates in progenitor cells in crypts (8). Zinc
finger protein, multitype 1 [Zfpm1, also known as friend of
GATA type 1 (Fog1)] is coexpressed with Gata4 in absorptive
enterocytes and, like Gata4, shows a declining proximal-distal
pattern of expression (8). Conditional expression of a knockin
Gata4 mutant that is unable to bind FOG cofactors, but
otherwise functions normally, leads to a significant upregulation of Slc10a2 expression (8), suggesting that FOG1 may
participate in the repressive effect of GATA4 on certain target
genes. Comparison of the global gene expression profiles of
control jejunum, control ileum, and Gata4⫺/⫺ jejunum revealed that Gata4⫺/⫺ jejunum gained expression of 47% of the
ileal-specific genes (genes normally expressed in distal ileum,
but not jejunum) (4).
Together, these findings indicate that GATA4 has a specific
function, distinct from other intestinal GATA factors (GATA4specific pathway), in which it both activates and represses
subsets of absorptive enterocyte genes in the proximal intestine
(Fig. 8). GATA4 activates a discrete subset of genes that is
normally not expressed in the distal ileum (“jejunal” gene set),
likely in cooperation with HNF1␣, and represses a specific
subset of genes in the proximal intestine that are normally
expressed in the distal ileum (“ileal” gene set), possibly in
cooperation with FOG1. Although it remains to be determined
how GATA4 regulates its target genes, whether by directly
binding their promoters and/or enhancers or by indirect mechanisms, GATA4 nonetheless is responsible for maintaining the
regional identity in absorptive enterocyte gene expression between the proximal intestine and the distal ileum.
GATA4 MAINTAINS REGIONAL FUNCTION IN THE MATURE SMALL
INTESTINE. Conditional Gata4 deletion also results in alterations
in intestinal physiology. Using a conditional, noninducible
knockout approach in which Gata4 is inactivated early in
intestinal development (⬃E12.5), Battle et al. (4) showed that
the intestinal deletion of Gata4 resulted in smaller mice that
weighed consistently less than their gender-matched littermate
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intestine but is not expressed in the distal ileum (9), coinciding
with transitions in the expression of subsets of absorptive
enterocyte genes (Fig. 7) and suggesting a role in defining the
topographic patterning of gene expression in the small intestine. GATA4 was the preferred GATA factor in nuclear extracts from murine jejunum that bound the GATA motifs in the
regulatory regions of sucrase-isomaltase (Si) (16) and lactase
(Lct) (112), two absorptive enterocyte genes. In isolated
Gata4⫺/⫺ cells in the small intestines of chimeric mice, fatty
acid-binding protein 1 (Fabp1) was downregulated (31). Conditional inactivation (14) or deletion (4) of Gata4 using villinCre transgenes did not reveal major changes in structure,
proliferation, or lineage specification in the small intestine but
showed a general downregulation in jejunum of a specific
subset of absorptive enterocyte genes, including Lct and
Fabp1. Interestingly, Si and fatty acid-binding protein 2
(Fabp2) gene expression was unaffected by conditional Gata4
deletion. Closer scrutiny revealed that while Lct and Fabp1 are
not expressed in distal ileum, Si and Fabp2 are expressed in
this region of small intestine (57, 108) and, thus, are discordant
with Gata4 expression (Fig. 7), suggesting that Si and Fabp2
are not GATA4 targets. Comparison of the global gene expression profiles of control jejunum, control ileum, and Gata4⫺/⫺
jejunum revealed that Gata4⫺/⫺ jejunum lost expression of
53% of the “jejunal-specific” genes (i.e., genes normally expressed in jejunum, but not distal ileum) (4). Gata5 and Gata6
were expressed normally when Gata4 was deleted (4, 14, 32),
indicating that neither GATA5 nor GATA6 is able to compensate for GATA4 loss.
Multiple studies support a mechanism in which GATA4
cooperates with HNF1␣ to activate specific absorptive enterocyte genes. In transient cotransfection experiments, GATA4
acts synergistically with HNF1␣ to transactivate the Lct and
Fabp1 intestinal gene promoters (31, 32, 58, 112, 113).
GATA4 physically associates with HNF1␣, and this associa-
tion is necessary for synergistic Lct or Fabp1 transactivation.
Physical association was mapped to the COOH-terminal zinc
finger in GATA4 and the homeodomain in HNF1␣ (112, 113),
indicating that the protein-protein interaction domains are
colocalized with domains responsible for site-specific DNA
binding. Parallel mechanisms in other tissues as well as in
Drosophila suggest that zinc finger/homeodomain interactions
are an efficient mechanism for cooperative activation of gene
transcription that has been conserved throughout evolution
(113). Lct and Fabp1, but not Si or Fabp2, were downregulated
in Hnf1␣⫺/⫺ mice (15), indicating that GATA4 and HNF1␣
have common activation targets in the small intestine, further
supporting a mechanism of cooperativity.
Review
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
GATA4
FOG1
ಫIlealಬ gene set
ಫJejunal ಬ gene set
HNF1α
GATA4specific
pathway
GATA6
GATA4/GATA6redundant
pathway
Absorptive enterocyte
gene expression
Absorptive enterocyte
gene expression
Enteroendocrine
cell commitment
ಫGobletಬ program
Crypt cell
proliferation
ಫPaneth ಬ program
NEUROG3
Paneth cell
differentiation
Fig. 8. GATA4 and GATA6 regulate multiple processes in the mature mouse
intestine. Two pathways have been identified: one is mediated by GATA4
(GATA4-specific pathway) and the other by GATA4 or GATA6 (GATA4/
GATA6-redundant pathway). In the GATA4-specific pathway, GATA4 activates the jejunal gene set, likely in cooperation with hepatocyte nuclear
factor-1␣ (HNF1␣), while repressing the ileal gene set, possibly in cooperation
with friend of GATA type 1 (FOG1). In the GATA4/GATA6-redundant
pathway, GATA4 and GATA6 are redundant for 4 main small intestinal
functions: 1) absorptive enterocyte gene expression by activating lipid-binding
proteins and apolipoproteins (“lipid gene set”) and repressing specific colonic
genes (“colonic gene set”), 2) crypt cell proliferation, likely in cooperation
with CDX2, 3) enteroendocrine cell commitment by promoting Neurog3
expression, and 4) Paneth cell differentiation by promoting a terminal Paneth
program while inhibiting a goblet program in committed (SOX9-, EPHB3positive) Paneth progenitors. GATA factors are indicated in yellow and
non-GATA factors in blue.
controls. Lipid metabolism was identified as the molecular/
cellular function most affected by loss of GATA4 in the
intestinal epithelium. Plasma levels of glucose were not different, but the levels of cholesterol and phospholipids were
significantly reduced in the mice lacking GATA4 in their
intestine. These mice also showed a 21% reduction of fat
absorption and a 93% reduction of cholesterol absorption.
Global profiling analysis of jejunum showed a downregulation
of genes associated with lipid transport and an upregulation of
genes critical for bile acid uptake, including Fgf15, a key bile
acid signaling molecule. Hepatic cholesterol 7 alpha-hydroxylase (Cyp7a1) expression was downregulated in liver, supporting changes in bile acid uptake. Thus decreases in lipid and
cholesterol absorption could be due to a loss of jejunal-specific
proteins necessary for absorption and/or an ectopic absorption
of bile acids, making them unavailable for efficient solublization of luminal lipid components.
Using a conditional, inducible model, we showed that proximal bile acid absorption was indeed induced by intestinal
Gata4 deletion (9). Although total bile acid excretion remained
unchanged, taurocholate transport from the mucosal to the
serosal fluid in everted gut sacs was increased in proximal
segments and bile acid concentration in luminal contents was
depleted in distal segments when intestinal Gata4 was deleted,
confirming a proximal induction of bile acid absorption. Bile
acid pool size was not different, but, owing to a relative
increase in absorption of specific bile acids along the length of
the small intestine, the makeup of the pool was shifted to a
more hydrophobic composition. These data were the first to
show that bile acid absorption can be induced in absorptive
enterocytes that normally do not exhibit this characteristic and
that the restriction of Slc10a2 expression and active bile acid
absorption to the distal small intestine by GATA4 plays a key
role in determining the composition, but not the size, of the bile
acid pool.
We also tested the hypothesis that an induction of Slc10a2
gene expression and bile acid absorption in proximal small
intestine by conditional mutation or deletion of Gata4 is
sufficient to correct bile acid malabsorption resulting from
ileocecal resection (ICR) (9). We utilized two Gata4 mutant
models, each producing a moderate (22% of ileal levels) or
high (69% of ileal levels) proximal induction of Slc10a2
mRNA expression. ICR in wild-type mice resulted in an
anticipated increase in bile acid excretion, reduction of the bile
acid pool size, and compensatory increase in transcription of
hepatic bile acid biosynthetic enzymes compared with shamoperated controls, reflecting bile acid malabsorption. ICR in
the Gata4 mutant mice showed an induction of bile acid
absorption in the proximal small intestine that was sufficient to
reduce bile acid excretion, maintain the bile acid pool size, and
reduce the compensatory upregulation of hepatic bile acid
biosynthetic enzymes. Lipid absorption was not examined in
this study. This is the first example of a nontransplant intervention capable of restoring intestinal bile acid absorptive
function following ileectomy and establishes the concept that
reducing GATA4 activity may be clinically useful for restoring
lost ileal function due to disease or resection.
GATA4 and GATA6 modulate crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene
expression. To define the function of GATA6 in the small
intestine, we compared the intestinal phenotype produced
among single and double Gata4/Gata6 conditional knockout
mice using the villin-CreERT2 driver (7). In the distal ileum,
where Gata6 is expressed but Gata4 is not (Fig. 7), conditional
deletion of Gata6 resulted in a decrease in crypt cell proliferation, decreases in differentiated enteroendocrine and Paneth
cells, and alterations in absorptive enterocyte gene expression.
This altered phenotype was not present in the proximal intestine, where Gata4 is expressed. Instead, Paneth cells were
increased, perhaps as a compensatory response to the loss of
Paneth cells in distal ileum. When Gata4 and Gata6 (herein,
Gata4/Gata6) were conditionally deleted, the proximal intestine showed all the changes in proliferation, differentiation, and
gene expression found in the distal ileum of single Gata6
conditional knockout mice. These data indicate that, in addition
to the GATA4-specific functions in maintaining jejunoileal
distinctions in absorptive enterocyte gene expression, GATA4
and GATA6 share common functions (GATA4/GATA6-redundant pathway) in regulating proliferation, differentiation,
and gene expression in the mature small intestine (Fig. 8).
Cellular proliferation in intestinal crypts is necessary for the
continuous renewal of the intestinal epithelium. Gata4/Gata6
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ಫLipidಬ gene set
ಫColonic ಬ gene
set
CDX2
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GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
by mechanisms that involve differential recruitment of specific GATA factors.
GATA6 Regulates Colonic Epithelial Homeostasis
Although the focus of this review is on the role of GATA
factors in small intestinal homeostasis, we recently reported
that GATA6 is necessary for normal colonic epithelial differentiation (6). The colonic epithelium, also a derivative of
endoderm, has crypts and a surface epithelium, but no villi. It
is maintained through a process of continuous cellular renewal
in which crypt stem cells give rise to three differentiated cell
lineages, colonocytes, goblet cells, and enteroendocrine cells.
Although neither GATA4 nor GATA5 is expressed in colon,
GATA6 is expressed in colonic epithelium of humans (49, 53)
and mice (6). Using improved GATA6 antibodies, validated
using conditional Gata6 knockout mice, we showed that
GATA6 is expressed in all proliferating and differentiated cells
in the mature mouse colon (6). Conditional, inducible deletion
of Gata6 in the small intestinal and colonic epithelium using
the villin-CreERT2 driver resulted in an altered crypt structure,
a decrease in crypt proliferation, and a delayed crypt-to-surface
epithelium migration rate (6). Specific colonocyte genes were
significantly downregulated, goblet cells were immature, and
the mRNAs for specific hormones produced by endocrine cells
were altered. Gata6 knockdown in human LS174T cells, a
colonic adenocarcinoma cell line, resulted in the downregulation of a similar subset of colonocyte genes. GATA6 occupied
active loci in enhancers and promoters of some of these genes,
suggesting that they are direct targets of GATA6. These data
demonstrate that GATA6 is necessary for proliferation, migration, lineage maturation, and gene expression in the mature
mouse colonic epithelium. A role in supporting colonic epithelial proliferation is consistent with the suggestion that GATA6
may promote colorectal cancer (reviewed in Ref. 3).
Summary and Perspectives
In this review, we show that GATA factors are essential for
the earliest designation of presumptive endoderm and the
establishment and maintenance of homeostasis in the mature
small intestine. Invertebrates demonstrate sequential expression of divergent GATA factors in which early orthologs
essential for endoderm development are extinguished, giving
way to other orthologs critical for later intestinal development.
Vertebrates, on the other hand, show a continuum of expression of the same members of the Gata4/5/6 subfamily throughout development, suggesting an evolutionary advance in the
ability of the same GATA factor to regulate different functions
dependent on the developmental time frame. GATA6 is essential for the earliest designation of PrE, possibly by regulating
the migration of PrE-committed cells to the blastocoel surface
in the early blastocyst. GATA4 may also participate in PrE
development, along with GATA6, but is essential for ventral
folding morphogenesis after gastrulation. In vertebrates and
invertebrates, cell migration of presumptive or established
endoderm is emerging as a key function of GATA factors in
embryogenesis. The role of GATA factors in the differentiation
of PrE into VE and PE (23, 44, 105) and in the regulation of
cytodifferentiation and villus formation (13) has received some
attention, but additional work using combination knockouts
with different Cre drivers is necessary to establish the individ-
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deletion results in a reduction of the number of Ki67- and
bromodeoxyuridine-positive cells in crypts, as well as a decrease in villus height and epithelial cell number, demonstrating a requirement for GATA factors in supporting intestinal
renewal and the maintenance of the absorptive surface area (7).
In Caco-2 cells, an intestinal cell line that proliferates rapidly
until reaching confluence and then undergoes differentiation,
CDX2, a key transcriptional regulator of intestinal genes,
occupies a specific set of gene loci in proliferating cells but a
different set in differentiated cells (114). GATA motifs were
the most highly represented motif (other than CDX motifs) in
the CDX2 occupancy sites in proliferating cells, and GATA6
was found to preferentially co-occupy these sites, suggesting
that CDX2 co-regulates intestinal proliferation with GATA4/
GATA6 (Fig. 8).
Conditional deletion of Gata4/Gata6 also showed an altered
secretory cell phenotype. Enteroendocrine cell number and
Neurog3 mRNA abundance were decreased, suggesting a
GATA requirement for enteroendocrine cell specification in
ATOH1-secretory progenitors. The number and morphology of
goblet cells on villi were normal, but Paneth cells were replaced by a goblet-like cell type that expresses abundant mucin
2 (Muc2), but no defensins. Paneth cell loss and goblet-like cell
gain at the base of crypts did not occur until ⱖ2 wk after the
induction of Gata6 deletion, consistent with the slower turnover rate of Paneth cells. The goblet-like cells also express
genes that promote Paneth cell differentiation and their crypt
base localization, including SOX9 and EPHB3, respectively,
which are not normally expressed in mature goblet cells. Thus
the goblet-like cells that accumulate in the crypts are likely
committed Paneth cells that, in the absence of GATA4/
GATA6, default to a goblet-like cell type. These data suggest
that GATA4/GATA6 act within secretory progenitors to promote enteroendocrine cell commitment and the terminal differentiation of Paneth cells (Fig. 8).
GATA4/GATA6 also activate and repress specific absorptive enterocyte genes (Fig. 8). Many of the genes downregulated by Gata4/Gata6 deletion encode lipid transporters and
apolipoproteins (“lipid” gene set) but are distinct from the
GATA4-specific lipid transporters and carriers discussed
above. Many of the genes upregulated by conditional Gata4/
Gata6 deletion are normally more highly expressed in colon
than small intestine (“colonic” gene set). Thus the GATA4specific and GATA4/GATA6-redundant pathways both function in the proximal-distal patterning of absorptive enterocyte
gene expression.
In summary, these studies outline two fundamental pathways of GATA regulation in the mature mouse small intestine, a GATA4-specific pathway and a GATA4/GATA6redundant pathway (Fig. 8). In the GATA4-specific pathway, GATA4 regulates the proximal-distal transcriptome in
the gastrointestinal tract by distinguishing jejunal vs. ileal
gene expression and function (4, 9, 14). GATA6 is unable to
compensate for this pathway, supporting a mechanism in
which GATA4 is specifically selected by its target genes for
regulation. In the GATA4/GATA6-redundant pathway,
GATA4 or GATA6 is capable of regulating multiple processes, including proliferation, lineage specification, and
terminal differentiation (7). These findings indicate that
GATA target genes in the intestinal epithelium are regulated
Review
GATA FACTORS AND INTESTINAL EPITHELIAL HOMEOSTASIS
ACKNOWLEDGMENTS
We thank Drs. E. Beuling, T. Bosse, M. P. Verzi, and R. J. Grand for
reading the manuscript and providing helpful suggestions.
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant RO1-DK-061382 to S. D. Krasinski and grants
from the Nutricia Research Foundation, KWF Kankerbestrijding, Prins Bernhard Cultuurfonds (The Netherlands), and the European Society for Pediatric
Research (Switzerland) to B. E. Aronson.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
B.E.A. and S.D.K. drafted the manuscript; K.A.S. and S.D.K. prepared the
figures; B.E.A., K.A.S., and S.D.K. edited and revised the manuscript and
approved the final version of the manuscript.
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ual and overlapping functions at these stages of development.
Conditional knockout approaches in adult mice have revealed
two fundamental pathways of GATA regulation: 1) a GATA4specific pathway that functions to demarcate the differences in
absorptive enterocyte gene expression between proximal intestine and distal ileum (4, 9, 14) and 2) a GATA4/GATA6redundant pathway that regulates crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene
expression (7).
While the general function of GATA factors at various
stages of development can be gleaned from the downstream
effect of Gata deletion, we do not yet know the first-line direct
targets of individual GATA factors that put these downstream
outcomes in play or how GATA factors endow embryonic
germ layers to gain competence to differentiate into distinct
cell types or how they modulate chromatin in vivo to control
transcriptional activation or repression. Almost 15 years ago,
Zaret utilized in vivo footprinting to show that GATA factors
occupy GATA motifs in chromatin in genes silent in endoderm, providing these genes with the potential to be activated
later in development, a phenomenon called genetic potentiation
(reviewed in Ref. 123). Using chromatin pull-down assays and
global epigenetics approaches in vivo, we now have the ability
to determine the occupancy sites of GATA factors within
chromatin in vivo and map these sites to genes that change in
gene knockout studies, providing a reasonable account of
first-line direct targets of specific GATA factors at various
stages of development. We can also characterize histone modifications at GATA occupancy sites and the effect of GATA
deletion on these modifications, establishing a fundamental
basis of how GATA factors modulate chromatin to effect
transcriptional potentiation, activation, or repression. Finally,
characterization of DNA motifs and occupancy of other transcriptional regulators at GATA-occupancy sites, as well as
identification of partner proteins that physically interact with
GATA factors using immunoprecipitation/proteomics techniques, will allow us to define distinct pathways of GATA
regulation. Application of these techniques at different developmental time frames will establish the fundamental underpinnings of GATA regulation throughout the continuum of intestinal development.
G487
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