The Fox Genes in the Liver: From Organogenesis
to Functional Integration
JOHN LE LAY AND KLAUS H. KAESTNER
Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania
II. Organogenesis of the Liver
A. The Foxa family in early embryogenesis
B. Liver organogenesis and developmental competence
C. Foxa1/a2 deficiency and the liver-less mouse
III. Foxa and Foxo: Integrating Transcription and Metabolism in the Mature Hepatocyte
A. Fox genes and glucose homeostasis
B. Foxa factors control bile acid metabolism and biliary development
IV. Foxl1: A Marker of Adult Hepatic Stem Cells?
V. Foxm1b and Liver Cancer
Le Lay J, Kaestner KH. The Fox Genes in the Liver: From Organogenesis to Functional Integration. Physiol Rev
90: 1–22, 2010; doi:10.1152/physrev.00018.2009.—Formation and function of the liver are highly controlled, essential
processes. Multiple signaling pathways and transcriptional regulatory networks cooperate in this complex system.
The evolutionarily conserved FOX, for Forkhead bOX, class of transcriptional regulators is critical to many aspects
of liver development and function. The FOX proteins are small, mostly monomeric DNA binding factors containing
the so-called winged helix DNA binding motif that distinguishes them from other classes of transcription factors. We
discuss the biochemical and genetic roles of Foxa, Foxl1, Foxm1, and Foxo, as these have been shown to regulate
many processes throughout the life of the organ, controlling both formation and function of the liver.
The liver is the body’s largest internal organ, com-
prising ?5% of body mass in mammals. As examples of its
tremendous functional diversity, the liver secretes essen-
tial serum components and clotting factors; regulates glu-
cose, protein, and lipid metabolism; and detoxifies xeno-
biotics, drugs, and other chemicals. Impaired liver func-
tion is associated with multiple disease states. In addition,
the development of the vertebrate liver has served as a
paradigm for understanding fundamental mechanisms of
organogenesis. Initial analyses of liver-specific gene ex-
pression in fetal and adult liver and hepatoma cell lines
led to the identification of a number of liver-enriched
transcription factors containing various structural motifs.
Among these are the divergent homeodomain proteins
HNF1? and HNF1?; the winged helix proteins Foxa1,
Foxa2, and Foxa3 (previously termed HNF-3?, -?, and -?);
the leucine zipper proteins C/EBP? and -?; the orphan
nuclear receptor HNF4?; and the PAR protein DBP (re-
viewed in Refs. 32, 44, 207). While none of these factors is
exclusively expressed in the liver, the combinatorial ac-
tions of tissue-specific and hormone-dependent transcrip-
tion factors collaborate to achieve the stringency and
dynamic regulation of gene expression required for the
proper development and function of the organ.
Twenty years ago, the biochemical characteriza-
tion of proteins that bind to the promoters of genes
expressed in a liver-specific, or at least liver-enriched,
fashion led to the discovery of the Foxa or hepatic
nuclear factor 3 (HNF-3) transcription factors (42, 95–
97). Shortly thereafter, Weigel and Ja ¨ckle and co-
workers (194, 195) discovered that the central domain
of the nuclear protein encoded by the Drosophila mela-
nogaster gene fork head, which is essential for the
proper formation of the foregut and hindgut in the fly,
is closely related to the Foxa/HNF-3 proteins. In fact,
this ?100-amino acid motif, termed Forkhead or
winged helix domain, is conserved among all Fox genes
and forms the basis of their classification (Fig. 1). The
Physiol Rev 90: 1–22, 2010;
www.prv.org1 0031-9333/10 $18.00 Copyright © 2010 the American Physiological Society
mutant phenotype of the fork head fly, together with the
observation that the Foxa genes are expressed very
early during the formation of definite endoderm in the
mouse, led to the hypothesis that the Foxa proteins
function in mammalian liver development (5, 122, 151,
154), which will be discussed in detail in section II.
The metabolic role of the Fox proteins is the subject
of section III. Both Foxa and Foxo proteins have been
shown by biochemical and genetic means to play a major
role in orchestrating the metabolic functions of the liver.
Especially instructive here is the story leading to the
discovery that Foxo proteins are major transcriptional
mediators of insulin signaling. As it turns out, this discov-
ery might not have been possible without the seminal
work in genetic model systems, specifically the nematode
Caenorhabditis elegans and the fruit fly Drosophila mela-
nogaster. This section also highlights the remarkable evo-
lutionary conservation of these signaling systems, despite
the fact that the final outcome of pathway activation
differs between worm, fly, and human.
In section IV, the recent discovery of an important
function of another member of the Fox gene family,
termed Foxl1, in the liver will be discussed. From genetic
lineage tracing experiments, it appears that the Foxl1
gene is expressed specifically in facultative progenitor
cells of the liver, the so-called “oval cells.” These cells
have been difficult to identify in the normal, quiescent
liver but are abundant after certain types of liver injury.
Foxl1 appears to be a marker of at least a subset of these
bipotential progenitor cells, as cells expressing Foxl1-Cre
track to both hepatocytes and cholangiocytes following
bile duct ligation.
Finally, section V details the role of Foxm1b in
hepatic carcinogenesis. This fascinating protein is ex-
pressed in many proliferating cell types but is extin-
guished upon terminal differentiation. In the liver,
Foxm1b is highly induced after partial hepatectomy,
when hepatocytes in the remaining liver lobes reenter
the cell cycle to restore liver mass. Foxm1b is required
for hepatocyte proliferation in these conditions as
FIG. 1. Amino acid sequence alignment of the DNA-binding domains of selected Fox proteins relevant to this review. A: schematic representation of
the domain structure of selected murine Fox proteins. FKH, winged helix or Forkhead domain; TA, transactivation domain. For FOXL1, no biochemical
studies have identified transactivation or transrepression domains thus far. For FOXO1, important phosphoacceptor sites are indicated. B: the forkhead
domain is the only consistently conserved portion of the protein across all members of the family, whereas there are limited similarities in other regions
among Fox subfamilies. The color coding of the amino acids is based on the physicochemical properties provided by the alignment editor Jalview. CE,
Caenorhabditis elegans; DM, Drosophila melanogaster; HS, human. We thank Dr. Sridhar Hannenhalli for contributing this figure.
LE LAY AND KAESTNER
Physiol Rev • VOL 90 • JANUARY 2010 • www.prv.org
shown by loss of function, while gain of function for
Foxm1b can restore the regenerative capacity of the
aging liver. But, most excitingly, Foxm1b-deficient
hepatocytes are resistant to hepatic carcinogenesis,
suggesting inhibition of Foxm1b as a potential thera-
peutic approach for the treatment of liver cancer.
Multiple excellent reviews on the Forkhead Box
gene family have appeared over the past decade, and
the reader is referred to these for further detailed
information on the role of Fox genes in cancer, pan-
creas development, or in evolution, for instance (1, 7, 8,
57, 81, 82, 91, 98, 134, 141, 189, 197). In addition, two
“snapshots” have appeared that contain salient infor-
mation on all mammalian Fox genes in tabular format
(183). Members of this diverse gene family, with 42
members in mammals, have been shown to be ex-
pressed in all cell types and organ systems. Fox genes
function in speech acquisition in humans and vocal
learning in song birds; they control developmental pro-
cesses from pharynx development in the worm to chon-
drogenesis in zebrafish to iris development in humans,
to just name a few examples. While this review is
focused on the function of Fox genes in the liver, we
would like to point out that this covers only a small part
of the impressive range of biology that is influenced by
members of this gene family.
II. ORGANOGENESIS OF THE LIVER
A. The Foxa Family in Early Embryogenesis
During mouse development, the first Foxa gene to
be activated is Foxa2, whose mRNA and protein gene
expression are first detectable at embryonic day 6.5
(E6.5) in the node and the anterior primitive streak (5,
122, 151, 154). The node of the mammalian embryo is
critical to gastrulation, the process that first establishes
each of the three fundamental germ layers (ectoderm,
mesoderm, and endoderm) and is equivalent to the
dorsal blastopore lip in Xenopus and Hensen’s node in
chicken. By E7.5, Foxa2 expression is found through-
out the definitive endoderm and persists into adulthood
in endodermal derivatives such as the liver, pancreas,
lung, thyroid, and prostate (41, 87, 97, 120, 122, 154,
155, 203). The importance of Foxa2 to early embryo-
genesis has been demonstrated by targeted gene abla-
tion. Mouse embryos homozygous for a null mutation in
Foxa2 die by E11 due to severe defects in structures
related to all three germ layers, abnormalities of the
neural tube and somites, absence of the notochord, and
failure to form a gut tube (4, 196). A summary of this
and several other Fox family mutant phenotypes rele-
vant to this review is described in Table 1.
Foxa1 is also broadly expressed in the early em-
bryo, in a very similar pattern to Foxa2, although Foxa1
mRNA is not detectable until E7.0 in the late primitive
streak and is not found in the node (5, 122). With some
notable exceptions, the expression domains of Foxa1
and Foxa2 largely overlap in adulthood, suggesting
likely functional redundancy for these two factors (15,
137). Foxa3 expression does not initiate in the
endoderm until E8.5 and is not present in the primitive
streak or axial mesoderm (122). However, by E10.5,
Foxa3 expression is found in the liver primordium and
persists throughout development into the adult liver
where it is the most highly expressed member of the
Foxa family, at least at the mRNA level (84, 122). In
contrast to Foxa2, neither Foxa1 nor Foxa3 is required
during early mouse embryogenesis as both Foxa1?/?
and Foxa3?/?embryos appear normal and develop to
birth (12, 13, 85, 86, 166), likely owing to differences in
their spatial and temporal expression domains as well
as functional compensation by the remaining Foxa fac-
B. Liver Organogenesis and
The developing endoderm initially consists of an ep-
ithelial sheet that lines the ventral surface of the embryo.
Shortly after specification, the endodermal epithelium in-
vaginates anteriorly to form the ventral foregut, which is
the region that gives rise to the liver, lung, thyroid, and
ventral pancreas (Fig. 2) (206, 208, 213). Posteriorly, a
more dorsal domain of the definitive endoderm exists that
develops into the intestines and the dorsal bud of the
pancreas. The liver primordium is first delineated at ap-
proximately E8.5 with the expression of hepatocyte-spe-
cific genes in cells termed hepatoblasts (65, 80, 210).
These hepatoblasts remain multipotent, as they will fur-
ther differentiate into the hepatocytes and cholangiocytes
(the epithelial cells of the bile duct) that make up the bulk
of the mature liver (206). Fate mapping studies have
revealed that the liver bud is derived from laterally
positioned precursor populations within the foregut
endoderm, which migrate toward the ventral midline to
meet a spatially distinct domain also harboring hepatic
The specification and development of these domains
are controlled by an array of inductive and inhibitory
signals originating from the adjacent mesoderm (100–
103). Studies performed nearly 30 years ago showed that
cardiac mesoderm from several species maintained the
capacity to induce hepatic epithelium when transplanted
into the chick endoderm (58). It has since been confirmed,
in multiple systems, that fibroblast growth factor (FGF)
signaling from the cardiac mesoderm is necessary for
both the induction of hepatic cell fate as well as the
expansion of these cells once specified and that the mech-
FOX GENES IN THE LIVER
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anism involves activation of the mitogen-activated protein
kinase (MAPK) pathway (29, 37, 80, 162, 167, 212). FGF
signaling simultaneously suppresses the ventral pancre-
atic program, thus directing the early patterning of the
foregut endoderm (51). Bone morphogenetic proteins
(BMPs), secreted from the nearby septum transversum
mesenchyme, have also been shown to act coordinately
as inductive signals mediating both the hepatic and ven-
tral pancreatic fates (149, 167, 212). Interestingly, sup-
pression of other signaling events is also required for the
early stages of endoderm specification. Wnt signaling, for
instance, must initially be inhibited to allow proper he-
patic specification, although its reactivation is required at
later stages to support expansion of the liver bud and liver
organogenesis (6, 115, 127, 130, 177).
It is now believed that these signals influence cell-
autonomous factors such as transcriptional regulators to
initiate highly specific gene expression programs. How-
ever, it has also been suggested that these inductive sig-
nals alone are not sufficient to elicit the desired impact on
their target cells in the developing endoderm. Rather,
specific molecular events within the receiving cells must
first occur before the tissue can become competent to
respond to the instruction of the signal (Fig. 3) (205). This
model is based on the observation that dorsal endoderm,
which does not normally differentiate into liver cells, can
be induced to express the liver marker albumin if dis-
sected between E8.5 and E11.5 and cultured in the pres-
ence of FGF (23). This competence is lost, however, if the
dorsal endoderm is isolated at E13.5 or later, suggesting
that factors required for competence are no longer
present at this stage or have ceased to disseminate the
directive of the signal. Interestingly, there is a direct
correlation between the ability of FGF to induce hepatic
gene expression in the dorsal endoderm and the binding
activity of Foxa and GATA proteins to an albumin gene
enhancer region. Thus the loss of competence was ac-
companied by the loss of Foxa and GATA binding in the
more mature dorsal endoderm (23). In support of this
model, earlier studies from the Zaret lab had demon-
strated selective binding of Foxa and GATA transcription
factors to this enhancer in the endoderm, even prior to
activation of the albumin gene, further implicating Foxa
and GATA proteins as important factors involved in the
establishment of developmental competence (22, 65).
Traditionally, transcription factors are thought to
promote gene activation via intrinsic transactivation do-
mains or through the recruitment of cofactors that en-
hance their activation potential. For example, this has
been shown for GATA1 and cAMP response element bind-
ing protein (CREB) binding protein (CBP), a cofactor
with histone acetyltransferase properties that augments
Targeted deletions of Foxa and Foxo family members in mice
Foxa GenotypeCre Transgene PhenotypeReference Nos.
Death at P2–P12
Abnormal prostate morphology
Delayed respiratory cell maturation and alveolar morphogenesis
Death at E10–E11
Severe defects in node, notochord, neural tube, and gut tube
Normal morphology and life span
Hypoglycemia after prolonged fast
Rescue of diabetic phenotype
Restored insulin sensitivity
Rescue of diabetic phenotype
Partial restoration of B cell mass
Death at E10.5
Defects in embryonic and yolk sac vasculature
Abnormal ovarian follicular development
No noticeable defects
12, 15, 86, 120, 166
Conditional null alleles and compound mutants
Diminished induction of gluconeogenic enzymes during fasting
Death at P0–P5
Death at E9.5–E10.5
Loss of liver specification
Perinatal and fasting hypoglycemia
Foxa3-Cre (endoderm) 104
Diminished hepatic glucose production
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