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| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 21 December 2006
doi: 10.1242/dev.02770
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Review |
University of Utah, Department of Human Genetics, 15 N. 2030 E. Room 2100, Salt Lake City, UT 84112, USA.
e-mail: murtaugh{at}genetics.utah.edu
SUMMARY
The development of insulin-producing pancreatic beta (ß)-cells represents the culmination of a complex developmental program. Cells of the posterior foregut assume a pancreatic identity, cells within the expanding pancreatic primordia adopt an endocrine fate, and a subset of these precursors becomes competent to generate ß-cells. Postnatally, ß-cells are primarily maintained by self-duplication rather than new differentiation. Although major gaps in our knowledge still persist, experiments across several organisms have shed increasing light on the steps of ß-cell specification and differentiation. Increasing our understanding of the extrinsic, as well as intrinsic, mechanisms that control these processes should facilitate efforts to regenerate this important cell type in humans.
Introduction
The pancreas serves two major functions: (i) the production of digestive
enzymes, which are secreted by exocrine acinar cells and routed to the
intestine by a branched ductal network; and (ii) the regulation of blood
sugar, which is achieved by endocrine cells of the islets of Langerhans. Type
1 diabetes results from the autoimmune destruction of one islet cell type -
the insulin-producing ß-cells. Although insulin treatment has saved
countless diabetics from early death, it represents an ameliorative treatment
rather than a cure. A true cure for diabetes - and a triumph for the concept
of `regenerative medicine' - might be achieved by replacing lost ß-cells.
This was demonstrated in an animal model several decades ago: rats rendered
diabetic by the ß-cell toxin streptozotocin could be cured by injection
of isogeneic islets (Ballinger and Lacy,
1972
).
Clinical diabetes researchers have since made considerable progress in
translating this approach to human patients, although numerous hurdles remain
(Naftanel and Harlan, 2004).
Not least of these is the scarcity of transplantable islets, which explains
the interest in generating ß-cells artificially and the corollary
interest in understanding how ß-cells normally develop. Here, I review
pancreas development from the perspective of the ß-cell; several
additional reviews should be consulted for a more comprehensive view
(Edlund, 2002;
Jensen, 2004;
Slack, 1995). In addition, as
the mouse provides the pre-eminent model for pancreas development, I will
focus on this species, although not exclusively. In utero development of the
human pancreas has obviously received less attention, but studies suggest that
it resembles that of the mouse (Piper et
al., 2004).
Because of its obvious medical importance, the pancreas has been subject to
decades of close study. The differentiation of exocrine, and to a lesser
extent endocrine, cells can be observed in simple histological sections, which
allowed these processes to be studied and manipulated before the availability
of immunohistochemical methods. As discussed below, the pancreas was among the
first organs in which the importance of epithelial-mesenchymal interactions
was recognized (Golosow and Grobstein,
1962), and the development of enzymatic and radio-immunoassays for
pancreatic gene products conferred remarkable quantitativeness to studies of
the differentiation in this organ (Pictet
and Rutter, 1972). The pancreas is also among the relatively few
organ systems in which systematic promoter-mapping has informed embryology:
two of the transcription factors most crucial for pancreas development,
Pdx1 (pancreatic and duodenal homeobox 1; also known as
Ipf1) and Ptf1a (pancreas specific transcription factor 1a;
also known as PTF1-p48), were identified as DNA-binding proteins on
pancreatic promoters (Krapp et al.,
1996; Ohlsson et al.,
1993). As we shall see, few of the unsolved problems in pancreas
and ß-cell development are conceptually unique, and further work on this
fascinating (and frustrating) organ should shed a more general light on the
processes of specification, differentiation and regeneration.
The anatomy of pancreas development
The pancreas is often described as two organs in one, due to the distinct
function and organization of its endocrine and exocrine components. In higher
vertebrates, it might more properly be thought of as four organs, as it
comprises anatomically distinct dorsal and ventral lobes. Referred to in
humans as the tail and head, respectively, these two pancreatic lobes arise as
thickenings along the dorsal and ventral surfaces of the posterior foregut,
near the prospective hepatic endoderm (Fig.
1A). These thickenings are histologically recognizable by
approximately 9 days of development (E9.0-E9.5) in the mouse
(Wessells and Cohen, 1967).
Retaining luminal continuity with the gut tube, these structures evaginate
into the surrounding mesenchyme as dense epithelial buds, which subsequently
expand, branch and differentiate to yield a fully functional organ system
prior to birth (Fig. 1A,
Fig. 2). Gut rotation brings
the two lobes into close apposition; in humans, their ductal systems undergo
partial fusion, although this process is less obvious in rodents.
The bulk of the mature pancreas is comprised of acinar cells, connected to
the intestine via a highly branched ductal tree, while islets are primarily
scattered through the central regions of the organ. Several separate endocrine
cell types comprise the islet: ß-cells are the most prominent (50-80% of
the total, depending on species) (Brissova
et al., 2005; Cabrera et al.,
2006), and tend to segregate to the islet core, with other cell
types arranged closer to the mantle (see
Fig. 3R). Glucagon-producing
-cells are the next most-common cell type; the remaining islet cells,
each comprising a small minority of the total, include 
-cells, which
produce somatostatin, PP cells, which produce pancreatic polypeptide, and the
recently-described 
-cells, which produce ghrelin
(Prado et al., 2004;
Wierup et al., 2002).
The visible anatomy of the developing pancreas is prefigured by the
molecular anatomy of differential gene expression, and few genes are better
studied in this regard than the homeodomain transcription factor
Pdx1. The initial expression of Pdx1 (E8.5-E9.0) marks the
pre-pancreatic endoderm before it has visibly thickened
(Ahlgren et al., 1996;
Guz et al., 1995;
Offield et al., 1996)
(Fig. 2), and corresponds to
the classically defined period of pancreatic specification
(Wessells and Cohen, 1967).
Early Pdx1 expression is therefore a useful marker of pancreatic
identity, although it expands over the next several days of development to
encompass the posterior stomach, duodenum and bile duct. Another transcription
factor, Ptf1a, is also expressed in the early pancreas, and its
endodermal expression remains pancreas-specific throughout development
(Kawaguchi et al., 2002).
|
)
(Fig. 1B). Pdx1 is
expressed broadly in the pancreas during the first several days of pancreas
development, as the organ grows and branches (Figs
2,
3). Ptf1a expression
is similarly broad at the early stages, and Kawaguchi et al.
(Kawaguchi et al., 2002) have
found, through lineage tracing with a Ptf1aCre knock-in
allele, that Ptf1a+ cells contribute to all three mature
lineages. These authors also observed that a minor population of duct and
islet cells was not labeled by Ptf1aCre, possibly due to
the fact that Ptf1a expression becomes restricted to acinar precursor
cells by approximately E13.5 (Fig.
2). | Box 1. Lineage tracing and pancreas development.
Genetic-lineage-tracing techniques have produced considerable insight into the
development of the pancreas and other tissues. The most common lineage-tracing
approach uses Cre recombinase, which can delete DNA segments that are flanked
by loxP (so-called floxed) sites
(Branda and Dymecki, 2004).
Mouse strains have been developed in which the ubiquitous expression of a
reporter gene, such as LacZ, is prevented by a floxed sequence being
placed between the promoter and the reporter
(Soriano, 1999); Cre-mediated
deletion of this sequence results in heritable marking of the Cre-expressing
cell. When Cre expression is driven by a promoter that is active in progenitor
cells, the descent of those cells in various classes of differentiated
offspring can be traced (Gu et al.,
2002; Kawaguchi et al.,
2002) (see Fig. 1).
Conversely, if Cre is co-expressed with a differentiation marker, such as
insulin, one can determine whether differentiated cells change their phenotype
in the course of embryogenesis or adulthood. Such an approach was used to
refute the hypothesis that mature - and ß-cells are derived from
progenitors that co-express glucagon and insulin
(Herrera, 2000). Lineage
tracing can therefore offer valuable insights that are not obvious from
histological studies. A major limitation to the technique, however, is its
relatively low resolution: if a particular Cre driver labels multiple
differentiated cell types, it is impossible to distinguish whether that driver
was active in multipotent progenitors or in separate classes of
tissue-restricted precursor cells. A further limitation is that recombination
converts an analog input (i.e. the level of Cre expression per cell), into a
digital output (either the reporter gene is activated or not). A given Cre
transgene can therefore fail to label a tissue of interest either because its
promoter is completely silent in the progenitors of that tissue, or because
the level of its expression falls below a crucial threshhold for
recombination. Cre transgenes often give incomplete labeling of specific
tissues or cell types, possibly due to this sensitivity threshhold.
|
Ptf1a and Pdx1 each play crucial roles in pancreas
specification, as discussed below, yet were identified for their roles in
adult cell type-specific gene expression. Consistent with its later phase of
expression, Ptf1a was identified as an acinar gene activator
(Krapp et al., 1996), and
Ptf1a-deficient pancreata entirely lack acinar cells
(Krapp et al., 1998).
Pdx1, meanwhile, was identified as a regulator of the rat insulin 1
(Ins1) gene (Ohlsson et al.,
1993), and from E15.5 onwards its expression becomes mainly
restricted to ß-cells. (Note that rodents have two insulin genes, whereas
primates have only one; as the regulation and pancreatic expression of these
genes are nearly identical, I refer to them collectively as insulin.)
The transitions of Pdx1 and Ptf1a expression coincide with
the overall conversion of progenitors to mature endocrine and exocrine cells
(Figs 2,
3). This conversion is also
reflected in the dynamic expression of the bHLH transcription factor
neurogenin 3 (Neurog3, also known as Ngn3), which
specifically marks precursors of islet cells
(Gu et al., 2002)
(Fig. 1).
Ngn3+ cells appear in small numbers in the early organ,
dramatically increase during mid-embryogenesis, and finally decline towards
birth (Figs 2,
3)
(Gradwohl et al., 2000;
Schwitzgebel et al., 2000).
Similar to neurons, therefore, islet cells are ordinarily generated during a
restricted developmental window; this process is termed neogenesis, in order
to distinguish it from the proliferation of pre-existing islet cells.
|
|
; Schwitzgebel et al.,
2000). Importantly, the spectrum of endocrine differentiation
changes through embryogenesis, with -cells being born quite early (see
Box 2) and other cell types,
including ß-cells, not being generated in significant numbers until E13.5
or later (Herrera et al.,
1991; Pictet and Rutter,
1972) (Figs 2,
3). Interestingly, ß-cell
differentiation occurs simultaneously with that of acinar cells, albeit in a
different region of the organ (Fig.
2), during a period termed the `secondary transition'
(Pictet and Rutter, 1972).
Newly differentiated islet cells are non-dividing, although they resume low
levels of proliferation towards birth
(Sander et al., 2000). The observation that islets arise from duct-like progenitors has led to the longstanding hope that the duct of the mature pancreas could be coaxed into renewed ß-cell neogenesis. As we shall see, it remains unclear whether this occurs in the normal pancreas, or whether it can be induced in vitro. The same uncertainty applies to essentially all potential sources of transplantable ß-cells. The only tissue that unambiguously exhibits ß-cell neogenesis is the embryonic pancreas, and reproducing this feat elsewhere will probably require that we understand how it happens in situ.
Specification of the early pancreas
Local versus global signals
As early Pdx1 expression marks the newly-specified pancreas, one
approach to the problem of pancreas specification is to ask what lies upstream
of Pdx1. Promoter-mapping has identified putative activators of
Pdx1, including the winged-helix transcription factor forkhead box A2
(Foxa2) (Wu et al., 1997) and
the one cut domain, family member 1 (Onecut1, also referred to as
Hnf6) (Jacquemin et al.,
2003). Deletion of Foxa2 throughout the early mouse
endoderm does not impair Pdx1 activation, however, although
subsequent -cell differentiation is severely impaired
(Lee et al., 2005), whereas
Hnf6-knockout mice still activate Pdx1 expression, albeit
with a slight delay (Jacquemin et al.,
2003). Mouse embryos lacking the transcription factors SRY-box
containing gene 17 (Sox17)
(Kanai-Azuma et al., 2002) or
the homeodomain factor Tcf2 (transcription factor 2, also known as
Hnf1ß) (Haumaitre et al.,
2005; Sun and Hopkins,
2001) also exhibit absent or severely reduced Pdx1
expression, respectively, although it is unknown whether either factor
directly regulates Pdx1. As none of these transcription factors are
expressed in a domain as restricted as that of Pdx1, it is most
likely that they act cooperatively, potentially in concert with additional
intrinsic and extrinsic regulators that are, as yet, undiscovered.
The first extrinsic signals to be implicated in pancreas specification were
transforming growth factor-ß (TGFß) proteins of the activin or nodal
families, and retinoic acid (RA). Experiments in the frog Xenopus
laevis (Gamer and Wright,
1995; Henry et al.,
1996), later confirmed in the mouse
(Tremblay et al., 2000), show
that TGFß signaling is required for endoderm formation. Experiments in
frog embryos have also shown that transient exposure to activin and RA can
induce pancreas development from isolated animal cap ectoderm
(Moriya et al., 2000). Recent
studies suggest that TGFß signaling induces definitive endoderm in mouse
and human embryonic stem (ES) cells
(D'Amour et al., 2005;
Kubo et al., 2004;
Yasunaga et al., 2005), and
that RA treatment promotes Pdx1 expression and pancreas specification
in ES cell-derived endoderm (D'Amour et
al., 2006; Micallef et al.,
2005). These results strongly support the idea that studying early
pancreas development can inform efforts to generate new ß-cells.
| Box 2. Distinct regulation of early and late islet cell development.
Glucagon+ cells appear as early as E9.5 in the developing mouse
pancreas, and a subset of these cells appears to co-express insulin
(Teitelman et al., 1993). As
noted in Box 1, lineage tracing
indicates that these double-positive cells are not precursors of mature
- and ß-cells (Herrera,
2000), and their functional significance remains unclear. These
early islet cells do not express the complement of markers typical of their
mature counterparts (Pang et al.,
1994; Wilson et al.,
2002), and they still form in several knockout mice in which later
islet development is impaired (Ahlgren et
al., 1996; Collombat et al.,
2003; Offield et al.,
1996; Sander et al.,
2000). A distinct population of early islet cells, expressing
insulin exclusively, also forms in zebrafish and Xenopus
(Afelik et al., 2006;
Field et al., 2003), whereas
early islet cells are not found in the human embryonic pancreas
(Piper et al., 2004).
Together, these observations suggest that the secondary transition represents
the major phase of islet development, and that regulatory programs governing
early and late islet populations are only partially overlapping.
|
RA is widely considered to be a mediator of global anteroposterior (AP)
patterning, and RA-deficient zebrafish exhibit endoderm anteriorization, which
eliminates the liver as well as the pancreas
(Stafford and Prince, 2002).
In Xenopus and mouse, however, RA is required only for dorsal
pancreas development, and is dispensable for the development of both the
ventral pancreas and liver (Chen et al.,
2004; Martin et al.,
2005; Molotkov et al.,
2005). Although embryologists like to think globally, the
best-characterized interactions in pancreas specification seem to act locally.
A possible exception, recently described, is fibroblast growth factor 4
(Fgf4), which is expressed in the posterior mesoderm, following gastrulation,
and promotes posteriorization of the endoderm
(Wells and Melton, 2000). The
pancreas appears to arise from cells that receive intermediate levels of Fgf4,
and manipulations of Fgf4 levels in the early embryo can expand or contract
the pre-pancreatic domain (Dessimoz et
al., 2006).
Distinctive dorsal and ventral specification programs?
Both lobes of the pancreas are sensitive to local signals, positive and
negative. RA, synthesized by the product of the aldehyde dehydrogenase family
1, subfamily A2 (Aldh1a2, also referred to as Raldh2) gene,
represents one of these positive signals, although its target may actually be
outside the endoderm. Tissue-recombination studies have shown that expression
of the homeodomain factor ISL1 transcription factor, LIM/homeodomain
(Isl1) in the peripancreatic mesenchyme is essential for dorsal
pancreas development (Ahlgren et al.,
1997). This mesenchymal expression is lost in Raldh2
mutants, possibly accounting for their lack of dorsal pancreas
(Martin et al., 2005). The
precise spatial relationships between endoderm and mesoderm are also crucial,
as revealed by studies of mice lacking the homeodomain transcription factors
bagpipe homeobox 1 (Bapx1)
(Asayesh et al., 2006) and
hematopoietically-expressed homeobox (Hhex)
(Bort et al., 2004). These
genes act outside the pancreas, but mediate morphogenetic movements that
separate the dorsal (Bapx1) and ventral (Hhex) pancreata
from inhibitory cues. Other genes that are differentially required for dorsal
bud and ventral bud development, including N-cadherin and the receptor
tyrosine kinase Kdr (previously known as Flk1), also act
outside the endoderm (Esni et al.,
2001; Yoshitomi and Zaret,
2004). Hlxb9 (homeobox gene B9)-knockout mice lack a
dorsal pancreas, and, because this gene is co-expressed with Pdx1 in
the early pancreas (albeit ventrally as well as dorsally), its knockout is
often cited as evidence for intrinsic differences in the specification of the
dorsal and ventral lobes (Harrison et al.,
1999; Li et al.,
1999). Hlxb9 is also expressed in the notochord, however,
which itself is required for dorsal pancreas development
(Kim et al., 1997), and the
knockout phenotype may actually be due to defects in this tissue, rather than
in the pancreas itself.
The idea of distinctive dorsal and ventral programs is also supported by
Pdx1 and Tcf2 knockouts - in which the dorsal bud forms and
expands somewhat into the mesenchyme, whereas the ventral bud is undetectable
(Haumaitre et al., 2005;
Offield et al., 1996) - as
well as by Ptf1a knockouts, in which at least a fragment of the
dorsal bud forms and begins to expand while the prospective ventral bud ends
up contributing to intestine (Kawaguchi et
al., 2002). It should be emphasized, however, that much of the
Ptf1a-/- dorsal bud assumes an intestinal fate as well,
suggesting that the difference between the buds is of degree rather than kind.
Indeed, the dorsal pancreatic bud starts out physically larger than the
ventral, and may therefore have a `head start' in the tissue movements that
separate prospective pancreatic tissue from nearby inhibitory cues. In
Pdx1, Tcf2 and Ptf1a mutants, this slight edge may suffice
for some of the dorsal pancreas to expand into the permissive mesenchyme,
while the ventral pancreas cannot escape signals that divert it towards an
intestinal or biliary fate. Future work will probably identify additional
signals that act directly on the endoderm, and these findings might be used to
achieve the directed differentiation of pancreas and ß-cells.
Master regulators of pancreas specification
Perhaps the need for signals that promote pancreas and ß-cell
development could be bypassed altogether, if we could transduce cells from
another organ with a `master regulator' of pancreas development. A number of
investigators have taken this approach with the adult liver, using
Pdx1 as a candidate master regulator. Initial work indicated that
adenoviral misexpression of Pdx1 can induce a small number of
insulin+ cells in the liver
(Ferber et al., 2000);
subsequent studies have extended these findings, in some cases demonstrating
limited activation of exocrine gene expression as well
(Ber et al., 2003;
Kojima et al., 2003;
Miyatsuka et al., 2003;
Sapir et al., 2005). The Pdx1
protein directly binds and activates the insulin promoter
(Ohlsson et al., 1993), as
well as that of at least one acinar enzyme gene
(Swift et al., 1998), and it
remains unclear whether the observed induction of pancreatic genes by
Pdx1 represents true transdifferentiation or an artificial `forcing'
of pancreas-specific gene expression.
From a therapeutic standpoint, this may be irrelevant, and reprogramming of
liver cells by the expression of Pdx1 and/or other ß-cell
regulatory factors (described below) remains an attractive possibility. From
our perspective as developmental biologists, however, these studies cannot
settle the question of whether Pdx1 acts as a true specification
factor for the pancreas. Indeed, efforts to induce ectopic pancreas via
Pdx1 misexpression in the embryo have proven unsuccessful
(Afelik et al., 2006;
Grapin-Botton et al., 2001;
Heller et al., 1998;
Horb et al., 2003). By
contrast, expression of the hybrid protein Pdx1-VP16, in which Pdx1 is fused
to the strong transcriptional activator VP16, can fully convert embryonic
Xenopus liver to pancreas, including both exocrine and endocrine
components (Horb et al.,
2003). Similar results were obtained upon transducing human
hepatoma cells with Pdx1-VP16, and this fusion construct is now being
successfully applied by liver-to-ß-cell researchers
(Imai et al., 2005;
Kaneto et al., 2005;
Li et al., 2005).
Endogenously, of course, Pdx1 does not have the benefit of a VP16
activation domain, but it may exert similar effects via collaboration with
Ptf1a. This is suggested by recent studies of mice that lack hairy and
enhancer of split 1 (Hes1), a transcriptional repressor that mediates
Notch signaling in the pancreas and elsewhere
(Fukuda et al., 2006;
Sumazaki et al., 2004). In
addition to their intra-pancreatic phenotype, as discussed below,
Hes1 mutants develop patches of ectopic pancreas tissue in posterior
stomach, duodenum and bile duct. These tissues normally express Pdx1,
and, in the absence of Hes1, they also express Ptf1a,
precisely where pancreatic tissue later forms. Moreover, Ptf1a is
required for ectopic pancreas development in Hes1 mutants, suggesting
that its co-expression with Pdx1 may be necessary and sufficient for
pancreatic specification; this hypothesis is supported by a recent
misexpression study in Xenopus
(Afelik et al., 2006). The fact
that Hes1 normally represses Ptf1a suggests that manipulation of the
Notch pathway could provide a tool for the directed development of pancreas in
vitro, although it remains to be shown that the requirement for Hes1
in the early gut reflects the action of Notch signaling per se.
Establishing endocrine and exocrine compartments: everything in its right place
Are cells of the early pancreas multipotent, capable of contributing to
both the endocrine and exocrine compartments, or do they arise already
committed to one or the other lineage? The only direct evidence bearing on
this question is a single report, based on retroviral `tagging' in vitro,
which showed that single cells in the E11.5 dorsal bud can give rise to both
acinar and islet descendents (Fishman and
Melton, 2002). This conclusion is supported by indirect evidence
from embryological and genetic manipulations that alter the balance of
endocrine and exocrine development. These include studies of
epithelial-mesenchymal interactions, which for decades have been known to
promote growth and acinar differentiation of the bud-stage pancreas
(Golosow and Grobstein, 1962).
Cultured without mesenchyme, pancreatic epithelium shows little proliferation
and fails to produce acinar cells (Horb
and Slack, 2000); more-recent studies have shown that endocrine
differentiation is actually enhanced in the absence of mesenchyme, as though
multipotent progenitors choose islet fates by default
(Duvillie et al., 2006;
Gittes et al., 1996;
Miralles et al., 1998).
Alternatively, the removal of mesenchyme may enhance the proliferation of an
endocrine-restricted progenitor population; improved lineage-tracing
techniques, permitting robust labeling of single cells and their progeny, will
be required to distinguish between these possibilities.
A similar enhancement of endocrine development is observed in pancreata
lacking one of several Notch signaling components [see Lai
(Lai, 2004), for a recent
review of the Notch pathway]. The overall role of Notch signaling in the
pancreas appears to be to delay the differentiation of progenitor cells until
the secondary transition, when an unknown mechanism renders them competent to
assume ß-cell, acinar and other later-born fates. Knockouts of the
Deltafamily ligand delta-like-1 (Dll1), the Notch DNA-binding partner
recombining binding protein suppressor of hairless (Rbpsuh, also
referred to as RBPJ
and CBF1) or the target gene
Hes1 all exhibit pancreatic hypoplasia due to premature
differentiation of progenitor cells into endocrine -cells
(Apelqvist et al., 1999;
Jensen et al., 2000).
Sustained Notch signaling, by contrast, inhibits acinar, as well as islet,
differentiation (Esni et al.,
2004; Hald et al.,
2003; Murtaugh et al.,
2003). The mesenchyme appears to enhance Notch activity within the
epithelium through the secretion of growth factors such as Fgf10
(Bhushan et al., 2001;
Duvillie et al., 2006;
Hart et al., 2003;
Miralles et al., 2006;
Norgaard et al., 2003). By
extending the temporal window of epithelial Notch signaling, the mesenchyme
thus indirectly promotes acinar and ß-cell development, although it may
also produce additional signals that more directly influence the
exocrine-endocrine decision (Li et al.,
2004).
Whereas Notch signals negatively regulate endocrine development, certain
members of the TGFß superfamily appear to promote endocrine
specification, possibly at the expense of exocrine. Thus, early pancreatic-bud
explants treated with the ligand TGFß1 in vitro develop increased numbers
of islet cells, and fewer acini (Sanvito
et al., 1994). TGFß1 activates the MAD homolog 2 (Smad2) and
Smad3 signal transduction pathway (reviewed by
Massague et al., 2005), which
is also activated by activin family ligands, and treatment of early buds with
the activin antagonist follistatin enhances acinar development, while
inhibiting that of islets (Miralles et
al., 1998). This result suggests that endogenous activin-family
ligands normally promote endocrine development; consistent with this
hypothesis, the developing pancreata of mice that lack the type IIB activin
receptor have severely reduced islet mass, but apparently normal acinar tissue
(Kim et al., 2000).
Paradoxically, however, mouse embryos heterozygous for the Smad2
gene, which should be impaired for activin and TGFß signaling, exhibit
increased numbers of Ngn3+ islet precursor cells
(Harmon et al., 2004).
Precisely how and when activin/TGFß-family members act in the developing
pancreas therefore remain important open questions.
|
;
Gradwohl et al., 2000;
Grapin-Botton et al., 2001;
Schwitzgebel et al., 2000)
(although see Box 3). The
scattered distribution of Ngn3+ cells within the
epithelium (see Fig. 3G-L)
reflects, in part, repression by Notch activity, because
Ngn3+ cell numbers increase in the absence of
Dll1 or Rbpsuh, prefiguring precocious endocrine
differentiation (Apelqvist et al.,
1999; Fujikura et al.,
2006). Furthermore, Ngn3 is a target for direct
repression by Hes1 (Lee et al.,
2001). In addition to Dll1, the developing pancreatic
epithelium expresses Notch ligands of the Serrate/Jagged family, and two
recent studies confirm that this ligand family is involved in endocrine
development. First, morpholino knockdown studies in zebrafish embryos show
that loss of specific Jagged-family ligands causes ectopic islet cell
differentiation (Zecchin et al.,
2006). Notch activation by Serrate/Jagged ligands is inhibited by
glycosyltransferases of the Fringe family
(Haltiwanger and Stanley,
2002), and a second recent study demonstrates that the expression
of one family member, manic fringe homolog (Mfng), partially overlaps
with that of Ngn3 in the developing pancreas
(Xu et al., 2006). Moreover,
misexpression of Mfng was found to induce endocrine cells (primarily
-cells), apparently via the inhibition of Notch and the upregulation of
Ngn3. Together, these studies emphasize the crucial role of the
Notch-Ngn3 regulatory axis in islet development, and highlight the need to
better understand Notch ligand distribution and function in the developing
pancreas. | Box 3. Islet development without Ngn3? A recent human
genetics study raises the startling possibility that human ß-cells can
develop without Ngn3 function
(Wang et al., 2006). Three
unrelated children were identified with congenital malabsorptive diarrhea, and
biopsies revealed an almost complete absence of endocrine cells in their
intestines, a phenotype similar to that of Ngn3-null mice
(Jenny et al., 2002).
Sequencing revealed that these patients carry homozygous mis-sense mutations
in the bHLH region of the human NGN3 gene, which render NGN3
completely non-functional in overexpression experiments. Importantly, the
affected children were not hyperglycemic at birth, although two of them later
(aged 8 years) developed type 1 diabetes of unknown etiology. (The third child
developed severe liver disease and died at age 3.) The pancreata of these
children were not examined in infancy to confirm the presence of ß-cells,
and genetic experiments (e.g. targeted replacement of wild-type mouse
Ngn3 with one of these NGN3 mutants) will be required to
confirm that these mutations represent nulls. Nonetheless, the most
straightforward interpretation is that another pathway can compensate for the
lack of NGN3 in human pancreas development
(Wang et al., 2006).
Ngn3-null mice are completely devoid of islet cells at birth, and die
shortly thereafter (Gradwohl et al.,
2000). If this early death, possibly caused by malabsorptive
diarrhea, as in humans, could be prevented, it is formally possible that a
novel pathway of Ngn3-independent islet development could be
uncovered postnatally (Wang et al.,
2006). Such experiments would be especially important if the
NGN3 mutants identified in this study prove to be true nulls.
|
Transcription of Ngn3 presumably reflects the input of positive,
as well as negative, regulators, the best-characterized of which is Hnf6
(Jacquemin et al., 2000). Hnf6
directly binds and activates the Ngn3 promoter in vitro and is
genetically required for its expression. Hnf6 is expressed throughout
the developing pancreas, however, as well as in domains of the foregut that
lack Ngn3 expression (Landry et
al., 1997; Rausa et al.,
1997), and Hnf6-/- mutants exhibit numerous
additional defects in pancreatic organogenesis, to which their loss of
Ngn3 may be secondary (Jacquemin
et al., 2003; Pierreux et
al., 2006).
Neither Notch activity nor Hnf6 expression can obviously account for another aspect of Ngn3 expression, rarely discussed but easy to see: its segregation into a central, `pro-endocrine' domain, and exclusion from a peripheral `pro-exocrine' region (Fig. 3). Although there are many candidates, no signals have yet been identified that can account for such a restricted expression pattern, or indeed for the overall morphological organization of the developing pancreas.
Ngn3+ cells rapidly activate a battery of transcription factors
that constitute a `core program' of endocrine development, in that they appear
to be expressed in all endocrine precursors, and are required, quantitatively
or qualitatively, for the development of many or all islet cells
(Fig. 4A,
Table 1). These include
Isl1, neurogenic differentiation 1 (Neurod1) and
insulinoma-associated 1 (Insm1/IA1)
(Ahlgren et al., 1997;
Gierl et al., 2006;
Naya et al., 1997).
Ngn3 is required for the expression of all known islet-specific
transcription factors (Collombat et al.,
2003; Gradwohl et al.,
2000; Mellitzer et al.,
2006), and presumably their collective absence accounts for the
lack of islet cells in Ngn3-/- pancreata. Precisely how
these factors individually promote endocrine development is not well
understood. For instance, Neurod1 is thought to activate the insulin
promoter, yet Neurod1-/- pancreata still generate
insulin-expressing ß-cells, which are later eliminated by apoptosis
(Naya et al., 1997;
Naya et al., 1995).
|
ß-cell precursors: what do they know and when?
Table 1 lists genes that
exhibit expression patterns and knockout phenotypes consistent with their
having a role in islet subtype specification. These are distinguished from the
core program genes discussed above, perhaps arbitrarily, in that their
deletion causes a very discrete loss of cell types [e.g. NK6 related, locus 1
(Nkx6.1, also known as Nkx6-1)], or results in reciprocal
increases in one or another cell type, at the expense of others [e.g. paired
box gene 6 (Pax6)]. As the roles of these genes in endocrine
development have recently been reviewed
(Collombat et al., 2006;
Servitja and Ferrer, 2004), I
will focus on only one aspect of endocrine subtype specification: when does it
happen?
Some insight is offered by the expression, regulation and function of the
transcription factor paired box gene 4 (Pax4) in ß-cell
development. First, Pax4 is expressed exclusively during
embryogenesis, and tracing the initiation and perdurance of its expression (by
staining for ß-galactosidase protein in Pax4LacZ/+
mice) indicates that it is expressed strongly in ß-cell precursors but
only transiently, if at all, in the -cell lineage
(Wang et al., 2004). Second,
Pax4 is not expressed in the absence of Ngn3
(Gradwohl et al., 2000), and
Ngn3 appears to be a direct upstream activator of the Pax4 promoter
(Smith et al., 2003).
Immediately upon activation, Pax4 appears to be co-expressed with
another transcription factor, aristaless related homeobox (Arx), also
an Ngn3 target gene, but thereafter Pax4 and Arx each begin to repress the
expression of the other. Precursor cells subsequently partition into separate
populations that express either Pax4 or Arx, and these
populations develop into ß- or -cells or -cells,
respectively (Collombat et al.,
2003). Finally, Pax4 mutants develop an excess of
-cells at the expense of ß- and -cells
(Sosa-Pineda et al., 1997).
Together, these results support the conventional model of ß-cell
specification, depicted in Fig.
4, in which Ngn3+ cells adopt a ß-cell
fate, as opposed to an -cell fate, partly due to maintained
Pax4 expression [the role of Pax4 in the somatostatin lineage is
apparently more complex; Collombat et al.
(Collombat et al., 2005)].
The major unknown in this scheme is how the antagonism between
Pax4 and Arx is resolved in such a way as to generate a
reproducible balance between - and ß-cell numbers. One possibility
is that, once Ngn3 activates expression of Pax4 and
Arx, extrinsic signals regulate the repressive activity of these
transcription factors, tipping the balance of their mutual repression and
favoring one lineage over another. Identifying such signals would certainly
enhance the prospects for controlled ß-cell development in vitro. Another
possibility is that the expression levels of these two factors are subtly
biased from the outset, such that ß-cells develop from precursors that
initiate higher levels of Pax4, and vice-versa for -cells and
Arx expression. According to this hypothesis,
Pax4-Arx cross-repression would essentially refine a
pre-existing pattern established before Ngn3 expression. There are no
strong candidate molecules that would support either hypothesis (e.g.
transcription factors that would cooperate with Ngn3 in activating higher
levels of Pax4 in ß-cell precursors), but further investigation
of Pax4 and Arx should continue to illuminate islet subtype
specification.
The question of when ß-cell specification begins is relevant to other
factors involved in the process. Several transcription factors thought to act
relatively late in ß-cell specification, including Nkx2.2,
Nkx6.1 and Hlxb9, are expressed in not only ß-cell
precursors but also widely throughout the early progenitor population
(Harrison et al., 1999;
Li et al., 1999;
Sander et al., 2000;
Sussel et al., 1998). This
early expression may be an epiphenomenon, or it may represent the first stage
of a progressive process by which Ngn3+ cells acquire
ß-cell differentiation competence. In liver development, by analogy,
transcriptional activators of hepatocyte genes actually begin to modify the
chromatin of their targets within multipotent progenitor cells, well before
those cells have committed to a hepatocyte fate
(Zaret, 2002). In the case of
Nkx2.2, separate regulatory elements appear to drive its expression
in early progenitor cells, in Ngn3+ precursors and in
differentiated ß-cells (Watada et
al., 2003). If Nkx2.2 is required prior to Ngn3
expression, then deleting the progenitor-specific element - which has not yet
been precisely mapped - should reproduce the ß-cell deficiency observed
in Nkx2.2-/- mice. If similar elements can be defined for
Nkx6.1 and Hlxb9, the same prediction holds for these
factors as well.
The pancreas is not unique in generating different cell types over time
from a common progenitor population: a similar phenomenon occurs in retinal
development, where an intrinsic `clock' appears to control the changing
developmental competence of progenitor cells
(Livesey and Cepko, 2001).
Studies of retinal development have benefited from the use of very fine-scale
lineage-tracing techniques (Turner and
Cepko, 1987), and analogous approaches might be used to discern
whether a similar timing mechanism exists in the pancreas, as well as to
illuminate other mysteries of endocrine specification and differentiation.
Adult ß-cells: keep on keeping on
A flurry of papers in the past several years has shown that the maintenance
of ß-cell function and numbers in postnatal life relies on mechanisms
that appear to be distinct from those used to generate these cells in utero.
With respect to function, ß-cell differentiation is accompanied by
activation of a novel transcriptional network that is involved in the
maintenance of ß-cell-specific gene expression
(Boj et al., 2001).
Haplo-insufficient mutations in components of this network cause the human
syndrome maturity-onset diabetes of the young (MODY), which is characterized
by the progressive impairment of insulin secretion (reviewed by
Servitja and Ferrer, 2004).
Among the most crucial members of the MODY gene network is Pdx1
(Stoffers et al., 1997),
which appears to activate a number of ß-cell-specific genes, in addition
to insulin (Carty et al.,
1997; Chakrabarti et al.,
2002; Waeber et al.,
1996; Watada et al.,
1996). Consistent with these observations, ß-cell function is
also rapidly lost upon ß-cell-specific inactivation of Pdx1 in
mice (Ahlgren et al., 1998;
Holland et al., 2005),
highlighting the crucial role that this developmental factor plays in
adulthood. (Indeed, while the targets of Pdx1 in mature ß-cells are
relatively well-characterized, those mediating its functions in the developing
organ are unknown.)
Additional regulators of ß-cell function include Neurod1, of
which heterozygous mutations also produce MODY in humans
(Malecki et al., 1999), as
well as the basic leucine-zipper (bZIP) transcription factor Mafa
(v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A)
(Zhang et al., 2005). It
should be emphasized that, although both of these genes were originally
implicated in pancreas function through their binding to essential regulatory
elements of the insulin promoter
(Kataoka et al., 2002;
Matsuoka et al., 2003;
Naya et al., 1995;
Olbrot et al., 2002), neither
gene is absolutely required for insulin expression in vivo (in
contrast to Pdx1). Issues of redundancy remain to be resolved
regarding the function of these proteins at the insulin promoter;
Mafa, for example, is co-expressed with the related cMaf
(v-maf oncogene homolog) gene, which also activates the insulin
promoter in vitro (Nishimura et al.,
2006). Finally, global and tissue-specific knockouts in mice
reveals that the Foxa1 and Foxa2 transcription factors are
required for glucose-stimulated insulin secretion by ß-cells, although
not for insulin mRNA expression
(Lantz et al., 2004;
Vatamaniuk et al., 2006).
Importantly, prenatal ß-cell development occurs normally in the absence
of Mafa, Foxa1 or Foxa2, emphasizing the distinction between
genes that regulate specification and differentiation and those that regulate
mature cellular function. It will be of considerable interest to determine
whether Maf-family or Foxa-family mutations underlie any diabetes-related
conditions in humans.
With respect to ß-cell numbers, mice that lack the cyclindependent
kinase Cdk4 have a normal complement of ß-cells at birth, but the failure
of these cells to subsequently proliferate produces progressive hyperglycemia
(Rane et al., 1999). A
similar phenomenon occurs in mice deficient for cyclin D1 and/or cyclin D2
(Georgia and Bhushan, 2004;
Kushner et al., 2005), and in
mice with a pancreas-specific deletion of the Foxm1 (forkhead box M1)
transcription factor (Zhang et al., 1999). Essentially opposite results are
obtained in mice heterozygous for the tumor-suppressor gene Men1
(multiple endocrine neoplasia 1), or in those carrying a gain-of-function
cdk4 allele: normal pancreas development occurs until birth, followed
by progressive ß-cell hyperplasia
(Crabtree et al., 2001;
Rane et al., 1999). These
results imply that, whereas ß-cell production in utero depends on
progenitor cell differentiation, their postnatal maintenance and expansion
occurs primarily through proliferation. This has been confirmed by a
genetic-lineage-tracing study, in which differentiated ß-cells were
labeled in adult mice and followed over several months. Despite an increase in
total ß-cell numbers during this time, the proportion of labeling did not
decrease, indicating that the new cells arose from pre-existing ß-cells
(Dor et al., 2004).
The signals controlling ß-cell proliferation are becoming increasingly
well-understood. Mice lacking the insulin receptor in ß-cells exhibit a
severe reduction in adult ß-cell mass
(Otani et al., 2004), whereas
ß-cells deleted for Pten, a negative regulator of
insulin-receptor signaling, undergo hyperplasia
(Stiles et al., 2006). If
this and other pathways could be manipulated in vitro, clinicians might be
able to grow unlimited numbers of ß-cells from scarce donor islets,
providing transplantable material without even having to worry about
developmental biology. Indeed, recent studies of diabetic mice suggest that
blocking autoimmunity at early stages of the human disease could restore
normoglycemia through the expansion of residual ß-cells
(Chong et al., 2006;
Nishio et al., 2006;
Suri et al., 2006).
Does ß-cell neogenesis ever occur in the adult (e.g. through a
pancreatic stem cell)? In uninjured mice, as summarized above, the weight of
evidence is negative. Islet cell mass can regenerate following partial
pancreatectomy, however, and strong correlative evidence from rodent studies
suggests that at least part of this regenerative response reflects neogenesis
from pre-existing duct cells or acini (reviewed by
Bonner-Weir and Weir, 2005;
Lardon and Bouwens, 2005). It
should be noticed that the lineage-tracing study described above failed to
find evidence for ß-cell neogenesis following partial pancreatectomy
(Dor et al., 2004), and an
independent study found that Ngn3 expression, which marks islet
precursors in utero, could not be detected in pancreata recovering from
partial pancreatectomy (Lee et al.,
2006). Nonetheless, neither of these studies focused on the
precise regions of the pancreas damaged during surgery, in which neogenesis
might be most pronounced (Sharma et al.,
1999). Past and present studies may also have differed in the
severity or type of injury models that were applied. It now seems imperative
to replicate past correlative studies with genetic-lineage-tracing techniques
to answer this controversial and fundamental question.
Even if ß-cell neogenesis does not occur endogenously, it may be
experimentally inducible ex vivo. For example, transducing cultured human duct
cells with Ngn3 activates many islet-specific genes, including
insulin, albeit at low levels
(Gasa et al., 2004;
Heremans et al., 2002). A
recent report indicates that non-endocrine epithelial cells of the adult human
pancreas (presumably ductal or acinar) can be induced to adopt a ß-cell
fate by signals from fetal pancreas (Hao
et al., 2006). Similar findings in the rat
(Dudek et al., 1991) suggest
that an embryonic differentiation program can be reactivated in adult
pancreatic ducts, consistent with the overlap in gene expression that is seen
between mature ducts and embryonic progenitors
(Pierreux et al., 2006;
Piper et al., 2004).
Moreover, a recent study that combined in vitro culture with genetic-lineage
tracing suggests that adult acinar cells can be coaxed to a ß-cell fate
(Minami et al., 2005), a
result consistent with independent work documenting the plasticity of
differentiation in mature acini (Means et
al., 2005). As ducts and acini are currently discarded during
human islet isolation, the prospect of spinning this dross into gold is highly
attractive.
Conclusion
The past decade has seen remarkable progress in understanding the molecular
program of islet specification and differentiation. Nevertheless, we still
cannot answer a number of questions raised in the premolecular era. Why, for
instance, do -cells begin to differentiate days earlier than
ß-cells? Why, in turn, does ß-cell differentiation occur
simultaneously with that of acinar cells, yet in a spatially distinct region
of the developing organ? What is the anatomical relationship between the
duct-like progenitors of the embryo and the mature ducts of the adult, and can
the functional program of the former be re-established in the latter? The
application of molecular genetics to the study of ß-cell development has
itself raised almost as many questions as it has answered. When, for example,
do Ngn3+ cells become terminally specified, and to how
many different cells and cell types can a single Ngn3+
cell contribute? Although novel technical approaches are clearly required, for
example a method of easily tracing the progeny of single progenitor cells, the
history of the field encourages optimism that those approaches will be found
and exploited.
Another set of open questions in pancreas development concerns the role of extracellular signaling pathways in cell fate specification. To date, the only strong genetic evidence for such a role comes from studies of the Notch, and to a lesser extent TGFß, pathways. Testing the in vivo role of other signals will require that we carefully consider the different stages of ß-cell specification, across which a single molecule might have multiple roles. The field will also benefit from the development of better in vitro culture techniques. It is easy enough to buy a growth factor and add it to a cultured pancreatic bud, yet relatively few consistent results have been reported from such studies, probably owing to minor technical differences.
One of the strengths of pancreas development research is its diversity of
approaches, and maintaining that diversity is essential for the field to
progress towards the controlled generation of human ß-cells. Although the
mouse offers numerous experimental advantages, important contributions have
been made by studies in the embryos of the chick, fish and frog (e.g.
Afelik et al., 2006;
Dessimoz et al., 2006;
Zecchin et al., 2006), and
work on human tissue remains essential as well. Each stage of ß-cell
development, as summarized in Figs
1,
2, offers a unique starting
point for potential therapeutic ß-cell neogenesis: re-specification of
hepatocytes, for example, via Pdx1-VP16 expression, or ß-cell
induction from pancreatic duct cells by Ngn3. It may also be possible
to fully recapitulate the process of pancreas and ß-cell development in
ES cells, and the recent efforts of D'Amour et al.
(D'Amour et al., 2006) along
these lines suggest that such an approach will hinge on a robust understanding
of normal developmental mechanisms. I must emphasize that, even with an
unlimited supply of perfect ß-cells, we will have to clear numerous
hurdles before curing diabetes, not least of which is the autoimmune reaction
that causes the disease in the first place. Nonetheless, the future study of
pancreas development will not only enrich our basic understanding of
organogenesis, but should also advance translational efforts towards that
ultimate end.
ACKNOWLEDGMENTS
I thank Sheldon Rowan, Gabrielle Kardon, Kristen Kwan and my anonymous reviewers for helpful comments on the manuscript, and I am indebted to Daniel Kopinke for the staining depicted in Fig. 3. Work in my lab on pancreas development is supported by the Searle Scholars Foundation.
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