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Keywords:

  • definitive endoderm;
  • mouse embryo;
  • morphogenesis;
  • tissue patterning;
  • cell fate

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

The endoderm is one of the primary germ layers but, in comparison to ectoderm and mesoderm, has received less attention. The definitive endoderm forms during gastrulation and replaces the extraembryonic visceral endoderm. It participates in the complex morphogenesis of the gut tube and contributes to the associated visceral organs. This review highlights the role of the definitive endoderm as a source of patterning cues for the morphogenesis of other germ-layer tissues, such as the anterior neurectoderm and the pharyngeal region, and also emphasizes the intricate patterning that the endoderm itself undergoes enabling the acquisition of regionalized cell fates. Developmental Dynamics 235:2315–2329, 2006. © 2006 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Gastrulation results in formation of the three primary germ layers and establishment of the embryonic body plan. Of the three germ layers, attention has been focused predominantly on the derivatives of the ectoderm and the mesoderm, which undergo distinctive morphogenetic processes during organogenesis. For example, the ectoderm during neurulation forms the neural tube and generates the neural crest cells, which contribute to the patterning of craniofacial structures, and the mesoderm involves with the morphogenesis of the heart and the segmental organization of the body by means of somitogenesis. By contrast, morphogenesis of the endoderm is less overt and has been considered subservient to the morphogenetic activity of the ectoderm and the mesoderm. However, recent embryological and genetic studies in the mouse reveal that the endoderm serves more than a structural role and is emerging as a potential source of morphogenetic activity. This review emphasizes the role of the definitive endoderm as a driver of morphogenesis by acting as a source of patterning cues to other germ-layer tissues while it undergoes intricate patterning accompanied by the acquisition of regionalized cell fates.

THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Endoderm is traditionally regarded as the innermost germ layer of the embryo. However, the terminology is confusing for the mouse, where first, the layer of extraembryonic tissues covering the epiblast is also termed endoderm, and second, the endoderm is initially the outermost germ layer of the embryo due to an “inside-out” arrangement of germ layers. The extraembryonic “endoderm” surrounding the early epiblast is the visceral endoderm and it is derived from the inner cell mass of the blastocyst (Weber et al.,1999), presumably by sorting out from other cells that are fated to become the blastocyst, and subsequently forms an epithelium (Yang et al.,2002). The visceral endoderm forms the epithelial lining of the yolk sac (Kadokawa et al.,1987) and can contribute during neurulation to the embryonic gut, albeit in a minor way (Tam and Beddington,1992). The definitive endoderm that forms the internal (mucosal) lining of the embryonic gut and its associated organs such as liver and pancreas is formed by the recruitment of epiblast cells through the primitive streak. Of interest, both the visceral and the definitive endoderm function as adsorptive epithelial structures (Enders et al.,1978) and they share some common genetic activity, such as that of Hnf4α (Duncan et al.,1994), Sox17 (Kanai-Azuma et al.,2002), and Gata4 (Kuo et al.,1997).

The epiblast of the mouse embryo, which contains the precursors of the definitive endoderm (Fig. 1A: early-streak embryo), is a cup-shaped epithelial layer covered on its basal surface by the visceral endoderm. Gastrulation commences with the formation of the primitive streak, which is the conduit through which cells ingress to become the mesoderm and the endoderm. The definitive endoderm emerges 8 to 10 hr after the onset of gastrulation from the anterior segment of the primitive streak (Fig. 1A: mid-streak embryo; Lawson et al.,1991; Tam and Beddington,1992) After egression from the primitive streak, definitive endoderm cells are incorporated into the pre-existing epithelium, the visceral endoderm. Lineage tracing study has shown that there may be reversed trafficking of cells from the endoderm to the mesoderm and/or ectoderm of the early-streak embryo (Lawson and Pedersen,1987). The significance and extent of this two-way traffic to lineage allocation and endoderm development is not known. As more definitive endoderm is recruited into the endodermal layer during gastrulation, the incoming population expands anteriorly and laterally from the site of integration. The definitive endoderm thereby progressively displaces the visceral endoderm anteriorly and proximally over to the extraembryonic region of the conceptus (Tam et al.,1993; Lin et al.,1994). The recruitment of the definitive endoderm is predominately through the primitive streak; however, labeling of epiblast cells suggests there may be a small contribution from cells that delaminate from the epiblast and directly integrate into the endodermal layer (Tam and Beddington,1992; Weber et al.,1999). Displacement of the visceral endoderm is probably not completed by the late-streak to early-bud stage (Fig. 1C). Fate mapping studies show that the endodermal cells in the vicinity of the embryonic–extraembryonic border may contribute to endoderm of the yolk sac and the lateral region of the embryonic gut (Tam et al.,2004) and some descendants of visceral endoderm remain in the prospective fore- and hindgut (Tam and Beddington,1992).

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Figure 1. A: Fate maps showing the regionalization of the progenitors of the definitive endoderm in the epiblast of the early- and midstreak embryo (Lawson et al.,1991) and the localization of the precursors of the endoderm of the embryonic fore-, mid-, and hindgut in the endoderm of the midstreak embryo (Lawson et al.,1991), which is shown as an exploded view of the epiblast and the endoderm with the mesoderm omitted. Mouse embryos are staged according to Theiler staging criteria (Web site: http://genex.hgu.mrc.ac.uk/Atlas/intro.html). B: Formation of the definitive endoderm at the midstreak stage visualized by molecular markers. Sox17 is expressed in the anterior visceral endoderm (asterisk) and extraembryonic visceral endoderm (bracket) of the embryonic day (E) 6.0 prestreak embryo and in the definitive endoderm (arrow) of the midstreak embryo. Cerl is also expressed in the definitive endoderm (arrow) and anterior visceral endoderm (AVE, asterisk). C: Formation of the gut tube showing the location of the endodermal precursors of the foregut (orange), midgut (pink), and hindgut (yellow) at the early-bud, early-somite, and forelimb-bud stages. At the early somite stage, the foregut pocket (FP) is extending deeply into the anterior region of the embryo and closing, but the hindgut pocket (asterisk) is just beginning to form. The embryonic gut is shown schematically in a ventral view to illustrate the morphogenetic movement (arrows) associated with the closure of the foregut and hindgut pockets and the ventral folding of the lateral body wall in the formation of the embryonic gut. At the organogenesis stage, the foregut pocket, hindgut pocket, and the lateral endodermal walls have met at the yolk stalk, thus closing the gut tube.

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Morphogenesis of the Embryonic Gut

At the completion of gastrulation, the definitive endoderm forms a squamous epithelium on the outside of the conceptus covering the ectoderm and mesoderm. Morphogenesis of the primitive gut begins with the formation of two intestinal pockets, one each in the anterior and the posterior region of the embryo (Fig. 1C). The foregut pocket initially develops as a crescent-shaped depression beneath the neural folds. It then deepens to form a pocket that extends rostrally. The opening moves posteriorly and closes up as the foregut develops.

In the posterior region, the hindgut pocket forms in a similar manner 6–8 hr later (equivalent to the time taken for the segmentation of six to eight somites) and its opening closes anteriorly (Fig. 1C). As the two gut pockets elongate and close, the lateral walls of the embryonic gut between the invaginations folds ventrally, culminating in the meeting of all the tissue folds at the yolk stalk and the formation of a gut tube by the 20–25 somite (organogenesis; Theiler Stage 15) stage (Fig. 1C).

Three major morphogenetic movements occur during the development of the foregut invagination (Tremblay and Zaret,2005). First, the mid-line cells of the anterior-most definitive endoderm are displaced ventrally then posteriorly to become the floor of the foregut invagination: these cells constitute the ventral midline of the gut rostral to the yolk stalk. Second, cells at the same axial level in the lateral regions of the definitive endoderm do not extend rostrocaudally in situ as in the chick embryo, but instead, the lateral endoderm moves toward the ventral midline. These tissue movements may be instrumental in bringing together the progenitor tissue of the liver from the lateral and anterior domains of the anterior definitive endoderm to the ventral midline. Third, cells in more posterior regions of the anterior definitive endoderm extend along the rostrocaudal axis to occupy the dorsal wall of the embryonic foregut.

The closure of the gut tube is intimately linked to change in the posture of the embryo from a lordose to a fetal position. The internalization of the endoderm is accomplished by the inversion of the arrangement of the germ-layer derivatives so that the ectoderm is rotated to the outside and the endoderm rolls into the inside of the embryo. Mutant embryos, such as those lacking Gata4 (Molkentin et al.,1994; Kuo et al.,1997), Sox17 (Kanai-Azuma et al.,2002), and Furin/SPC1 function (Roebroek et al.,1998; Constam and Robertson,2000), which fail to undergo proper body rotation, invariably display ventral body wall defects with an open gut tube. The severity of the defects is correlated with the loss of Furin function in the definitive endoderm (Constam and Robertson,2000) and could be ameliorated by the presence of wild-type cells in the embryonic gut (Narita et al.,1997). After gut tube formation, visceral organs are generated from specific regions of the gut by local swelling, budding or coiling of endodermal tissues at sites that are delineated by restricted domains of gene expression and inductive interactions with other germ layer derivatives (Bort et al.,2004; Molotkov et al.,2005; Serls et al.,2005; Tremblay and Zaret,2005).

SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Concept of a Mesendodermal Progenitor Population

It is unclear when the mesoderm and endoderm lineages of the mouse segregate and whether there is an epiblast-derived mesendodermal progenitor that is competent to differentiate into either endoderm or mesoderm but not ectoderm. In Caenorhabditis elegans and sea urchins, a mesendodermal progenitor can be identified at the 4-cell and 16-cell stage, respectively. Activity of signaling cascades leads to allocation of the mesendodermal progenitors and then their differentiation into mesoderm and endoderm (Angerer and Angerer,2000; Davidson et al.,2002; Maduro and Rothman,2002). Single-cell fate mapping in zebrafish at the mid-blastula stage localizes a mesendoderm population distinct from that which solely produces mesoderm (Warga and Nusslein-Volhard,1999). Molecular findings reveal the coexpression of the mesodermal gene Brachyury (T) and the endodermal gene Gata5 in cells that are fated to be mesendoderm, whereas cells expressing Brachyury only are localized to the region that forms mesoderm exclusively (Rodaway et al.,1999).

Evidence for a mesendodermal progenitor in amniotes, and specifically in the mouse, is more limited. Recently, several studies on embryonic stem (ES) cell differentiation in vitro point to the existence of a common mesendodermal precursor. Overexpression of Nodal in mouse embryonic stem cells leads to an up-regulation of both endodermal (Hnf4α, Ihh, Afp, Alb) and mesodermal genes (T, Bmp5, Meox1, Flk1) and a down-regulation of neurectodermal (Sox2, Otx2, Ncam) genes (Pfendler et al.,2005). The differentiation of endoderm (Foxa2, Sox17) occurs in cells derived from the T-positive population, which can also differentiate into mesoderm, but not from the T-negative population, implicating the former as bipotential mesendodermal precursors (Kubo et al.,2004). Of interest, the ability to form endoderm and mesoderm can be influenced by the level of either Nodal or activin, which share a common downstream signaling cascade, with higher levels favoring the formation of endoderm. However, only specific types of mesoderm are induced together with the endoderm (Kubo et al.,2004), raising the possibility that not all mesoderm is formed by means of the mesendoderm route. When induced, Goosecoid–green fluorescent protein (GFP; Gsc-GFP) -positive ES cells express organizer markers (Foxa2, Lhx1, and Chrd) and preferentially form mesoderm (that expresses Vegfr2 and Pdgfrb) and endoderm (expressing Foxa2). Endodermal cells derived from Gsc-GFP+ cells are Gsc+/Sox17+ and do not express Sox7, Hnf4, or Pthr1, suggesting they are unlikely to be visceral endoderm. Under conditions that generate the definitive endoderm, a Gsc+/Sox17 mesodermal population is also generated, further supporting the presence of a mesendodermal precursor in vitro (Yasunaga et al.,2005). However, no Gsc-GFP positive cells have been shown to form endodermal cells only, indicating that the transition through the mesendodermal phase is a prerequisite for endoderm formation in vitro (Tada et al.,2005).

In vivo, lineage analysis of the epiblast cells of the early- and midstreak embryo demonstrates that cell fate is regionalized in the epiblast such that precursors of definitive endoderm are found adjacent to the anterior segment of the primitive streak (Figs. 1A, 2A). Fate maps of the early streak epiblast generated from these experiments show this region to be in the posterior–distal region of the epiblast, and this area lies within the mesoderm forming region (Lawson et al.,1991). However, the clonal descendants of these epiblast cells are not restricted to one germ layer (Lawson et al.,1991). Transplantation studies have confirmed that this population contributes to both anterior mesoderm and endoderm (Kinder et al.,2001) and is characterized by the expression of T, Gsc (Fig. 2B), Foxa2, Eomes, Wnt3, and Mixl1 (Robb and Tam,2004). Taken together, the gene expression and cell fate data highlight the anterior region of the primitive streak as being the location of the putative mesendodermal progenitor in the mouse embryo.

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Figure 2. A: Fate map of the midstreak epiblast showing the endoderm forming region (green) and its overlap with the regions forming mesoderm (pink) and ectoderm (blue; Modified from Lawson, 1999). B: Expression of Gsc in the midgastrula organizer and Brachyury in the primitive streak at the midstreak stage. Their expression overlaps at the anterior end of the primitive streak, where the endodermal precursors lie. C: Schematic of Nodal mutants and their impact on the formation of the mesoderm (red) and endoderm (green) showing the correlation of the varying levels of nodal signaling and specification of the endodermal tissues. PrChP, prechordal plate.

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Molecular evidence for a mesendodermal progenitor population in the mouse is found in mouse mutants displaying defects in germ layer formation. In the mouse, Nodal signaling is required generally for gastrulation, making it difficult to establish a specific role in mesendoderm formation. Mutants with dramatically curtailed Nodal signaling activity (Zhou et al.,1993; Ding et al.,1998; Weinstein et al.,1998) fail to form a primitive streak and, consequently, lack both mesoderm and endoderm. Hypomorphic mutants with moderate levels of Nodal signaling activity can form some endoderm, whereas a further reduced level leads to a loss of endoderm, although not mesoderm. Of interest, the Nodal dosage influences the type of endoderm and mesoderm that may be formed, with anterior endoderm requiring stronger signaling than posterior endoderm (Lowe et al.,2001; Liu et al.,2004). Elevated Nodal signaling caused by loss of the repressor DRAP1 results in an expanded primitive streak and production of excessive mesoderm (Iratni et al.,2002). These data implicate the level of Nodal signaling in regulating the selective allocation of mesoderm and endoderm (Fig. 2C) and are consistent with, but not sufficient to prove, the presence of a common progenitor for mesoderm and endoderm.

The signaling pathway that generates definitive endoderm in zebrafish and Xenopus involves key factors, including Nodal, Mix-type, Sox, and Gata factors (Stainier,2002; Tam et al.,2003). Although the interactions are much less well defined in the mouse, the phenotypes of mouse mutants suggest that the molecular pathways may be broadly conserved. One mix-like gene, Mixl1, has been identified in mouse, whereas there are three in zebrafish and seven in Xenopus (Pearce and Evans,1999; Robb et al.,2000). Mouse embryos lacking Mixl1 are deficient of definitive endoderm but contain excess T- and Nodal-expressing tissues. Consistent with an impaired endodermal potency, Mixl1-null ES cells are less able to colonize the embryonic gut of chimeric embryos (Hart et al.,2002). Furthermore, promoter analysis suggests that Mixl1 is a downstream target of Nodal signaling (Hart et al.,2005), which may act to curtail Nodal and T activity, thus facilitating endoderm differentiation.

Evidence for the role of wingless-type MMTV integration site (WNT) signaling in endoderm formation in the mouse embryo is less substantial. In embryos that lack β-catenin activity specifically in the endodermal derivatives, cells that are reminiscent of cardiac mesoderm are found among the definitive endoderm. Chimera analysis suggests that β-catenin–deficient cells lose the ability to form endoderm and adopt a mesodermal fate instead (Lickert et al.,2002). This phenotype may represent a WNT-mediated lineage choice by a common mesendodermal progenitor, but the possibility of a fate switch of endodermal cells to mesoderm cannot be excluded. A potential downstream target of β-catenin is Sox17, which plays an essential role in endoderm formation in Xenopus and interacts with β-catenin to regulate the transcription of other endodermal genes (Sinner et al.,2004).

A role for Sox17 as a downstream transcription factor in endoderm formation is partially conserved in vertebrates with the mouse mutant deficient of definitive endoderm and Sox17−/− ES cells excluded from the gut of the chimera (Kanai-Azuma et al.,2002). However, the details differ or remain unclear, for example, there is no mouse ortholog of the sox factor Casanova, which is essential for endoderm formation in zebrafish (Alexander et al.,1999). Similarly, although GATA factors are required for endoderm differentiation in zebrafish and Xenopus, a role in the mouse has not been definitively established, which may be because first, these genes are expressed and required in tissues such as the visceral endoderm and heart as well as the definitive endoderm, and second, they are likely to function redundantly (Molkentin,2000; Stainier,2002; Watt et al.,2004; Zhao et al.,2005).

In the final analysis, although the precursors of the mesoderm and endoderm are colocalized in the epiblast and cells of the two germ layers emerge together during gastrulation and their formation is regulated by common molecular activities, the evidence for a mesendodermal progenitor in the mouse remains inconclusive. Although studies in stem cells are suggestive of a mesendodermal precursor, in vitro studies are artificial and do not replicate the interactions that occur among heterogeneous cell types in the embryo. The spectrum of defects in Nodal-related mutants highlights the temporal influence and gene dosage effects on lineage choices. Thus, if there is a mesendodermal progenitor, its lineage potency might not remain constant but could vary depending on the time of lineage allocation during gastrulation.

Regionalization of Cell Fates and Organ-Forming Potency in the Embryonic Gut

The initial regionalization of the definitive endoderm occurs concurrently with the formation of the definitive endoderm as it exits the primitive streak. The timing of cell migration from the primitive streak influences the final destination in the anterior–posterior body axis, such that cells recruited earlier into the primitive streak will form the foregut, and those recruited later will contribute to gut in progressively more posterior regions (Lawson et al.,1986; Lawson and Pedersen,1987; Tam and Beddington,1992). There is a paucity of pan-tissue markers for the definitive endoderm, and this finding, to some degree, reflects the regionalization of the definitive endoderm from the time of its allocation and/or its functional similarity to the visceral endoderm. Two of the earliest markers are Cerl and Sox17, although neither is restricted to the definitive endoderm nor are they uniformly expressed by the definitive endoderm (Fig. 1B). Cerl expression in the definitive endoderm is transient and restricted to the anterior definitive endoderm (Belo et al.,1997). Sox17 expression is not restricted permanently to any segment of the gut: expression begins in the anterior definitive endoderm (prospective foregut endoderm) and is progressively up-regulated successively in the mid- and hindgut and down-regulated in anterior segments (Kanai-Azuma et al.,2002). The early restriction of gene expression in the definitive endoderm suggests an early regionalization of cell fate along the anterior–posterior length of the embryonic gut.

Further evidence of segmental specialization of cell fates arises from the study of chimeric mice in which Sox17-null cells are incapable of colonizing the mid- and hindgut but do contribute to the foregut, but at a reduced frequency. Similar results were observed for Mixl1 (Hart et al.,2002) and Smad2 (Tremblay et al.,2000). In contrast, Foxa2 (Hnf3β) null cells were found to colonize the hindgut but not the fore- or midgut (Dufort et al.,1998). Again, this finding demonstrates molecular heterogeneity of the definitive endoderm along its anterior–posterior axis. Nodal signaling impacts on cell fate decisions, but the mouse mutants suggest that graded Nodal signaling governs endodermal fate decision as well as segmental contribution to the gut, with higher Nodal signaling specifying more anterior fates and vice versa (Fig. 2C). As a result, the foregut endoderm is more sensitive to a reduction in Nodal signaling than the mid- or hindgut. This finding suggests that initial regionalization of cell fates is mediated by Nodal signaling and occurs concurrently with endoderm specification.

During the morphogenesis of the embryonic gut, the expression of early genes such as Cerberus-like (Cerl) and Lhx1 is down-regulated, whereas other genes such as Hhex and Dickkopf-1 (Dkk1) become restricted to specific regions where organ primordia are formed (Table 1). The definitive endoderm is regionalized through reciprocal interactions with surrounding tissues that delineate sites of organ formation. Many of the genes that display regionalized expression are members of the Hox and ParaHox families and are reputed to confer anterior–posterior identity. However, many Hox genes are expressed in both the endoderm and the adjacent mesoderm and expression in the endoderm is more limited than in the mesoderm, so their specific role in the endoderm is unclear (Beck et al.,2000). Whereas the pattern of gene expression may reflect the underlying regionalization of the embryonic gut, it offers no clues as to the mechanism that establishes this tissue's pattern or the degree of determination of tissue fate. In vitro culture studies show that the molecular identity of the endoderm at the late-streak stage may be influenced by the presence of ectoderm/mesoderm from specific regions along the anterior–posterior axis, which release soluble factors such as fibroblast growth factor-4 (FGF4) to pattern the endoderm (Wells and Melton,2000); nevertheless, the early stages of regionalization are poorly defined. An interaction with surrounding tissues continues to be critical for endoderm differentiation, as fate becomes progressively more restricted, and such interactions govern the emergence of organ primordia (Wells and Melton,1999). By E9.0, several genes are expressed in well-defined restricted domains of the endoderm. Some such as Hhex, Pax1, and Pdx1, which are expressed initially in a regionalized pattern remain actively transcribed during the development of specific visceral organs and, thus, may have later organ-specific roles in addition to a role in the establishment of the organ primordium (Grapin-Botton and Melton,2000).

Table 1. Selection of Genes Expressed in the Endoderm at the Late-Streak and Early-Somite Stage
 GeneEndoderm expressionOther tissuesReference
  1. aExpression of most genes is regionalized within the endoderm, and many are expressed in other germ layers. AVE, anterior visceral endoderm.

Late-streakDkk1Anterior-most foregutAnterior-most AVEMukhopadhyay et al.,2001
 CerlAnterior endodermAVEBelo et al.,1997
 Sox17Anterior endodermExtraembryonic and anterior visceral endodermKanai-Azuma et al.,2002
Early SomiteIrx3Anterior foregutMidbrain, hindbrain, and anterior spinal cordBosse et al.,1997
 Irx1ForegutMidbrainBosse et al.,1997
 Pax1Mediolateral anterior endodermSomitesWallin et al.,1996
 Dkk1Anterior and ventral foregut and posterior hindgutPrimitive streak and pre-somitic mesenchyme, midbrain neuroepitheliumMukhopadhyay et al.,2001
 HexAIP (liver forming region)Extraembryonic blood islandsMartinez-Barbera and Beddington,2001
 Hoxb1Gut caudal to headfoldsParaxial mesoderm and somites caudal to headfolds and rhombomere fourHuang et al.,1998
 Sox17Mid- and hindgut endodermNoneKanai-Azuma et al.,2002
 Cdx2HindgutPosterior mesoderm, tail bud, caudal neural tubeBeck et al.,1995

ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

Anterior Definitive Endoderm Versus Anterior Axial Mesendoderm

The anterior definitive endoderm (ADE), which is localized in the region of the embryo anterior to the node and underneath the headfolds (Fig. 3A), is fated to become the endoderm of the embryonic foregut (Tam et al.,2004; Tremblay and Zaret,2005). In the gastrula embryo, the expression domain of Cerl and Sox17 encompasses most of the ADE, whereas in the early-somite stage embryo, only part of it is covered by the expression domain of Dkk1 and Foxa2 (Fig. 3B,E). In the midline region, the ADE is congruent with the axial mesendoderm (AME), which could be recognized by the expression of Foxa2 (Fig. 3E). Morphologically, the AME is composed of two regions. In the rostral most region, the endoderm and the mesoderm merge into a single tissue layer, which is juxtaposed directly to the underside of the neural plate to form the prechordal plate, the fate of which is not well understood in the mouse (Sulik et al.,1994). More posteriorly, the endoderm is separated from the anterior notochord, which lies between the neural plate and the endoderm. However, the morphogenesis of the anterior notochord involves a transition through the notochordal plate where a mixed population of endoderm and the notochordal precursors are organized into an axial epithelial domain, which is histologically a continuum of the endodermal layer. The notochordal cells later segregate from the endoderm in the dorsal wall of the foregut pocket to form the notochord. The prechordal plate expresses Gsc (Fig. 3C), molecularly distinguishing it from the more caudal axial mesendoderm, which expresses Noggin (Nog) and Chordin (Chrd; Fig. 3D), whose anterior limit of expression demarcates the caudal border of the prechordal plate (Anderson et al.,2002). Dkk1 is expressed in the prechordal plate but is also expressed more broadly across the anterior-most definitive endoderm (Mukhopadhyay et al.,2001) (Fig. 3). The midline tissues are a critical source of patterning information and are considered here because they have been part of the endodermal layer or still remain congruent with the ADE. However, the intersection in the midline of ADE and AME makes it more difficult to discern a specific role for the ADE in anterior morphogenesis of the mouse.

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Figure 3. A: Transverse histological section (shown in false colors) through a headfold stage embryo showing the germ layers: neurectoderm (blue), mesoderm (pink), endoderm (black). The axial mesendoderm is in green and is indicated with an asterisk. B–E: Regionalized pattern of gene expression in the anterior endoderm at the headfold stage. B: Dkk1 expression in the anterior endoderm. C: Gsc expression in the prechordal plate. D: Chrd expression in the axial mesendoderm up to the caudal border of the prechordal plate. E: Foxa2 expression in the axial mesoderm and the anterior-most region of the anterior definitive endoderm.

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Loss of Inductive Activity and Abnormal Head Morphogenesis

Tissue recombination and ablation experiments have provided evidence supporting a morphogenetic role for ADE and AME in anterior development. Explant cultures of anterior epiblast with and without the mesoderm and endoderm that are destined to the head region demonstrate a requirement for inductive signals from adjacent germ layers to induce En1 and to maintain Otx2 (Ang and Rossant,1993,1994). However, the relative contribution of endoderm vs. mesoderm has yet to be tested. Microsurgical ablation of the rostral AME leads to the truncation of anterior structures, particularly the forebrain, demonstrating a requirement for this tissue in head morphogenesis (Camus et al.,2000). These experiments provide strong evidence that inductive signals for anterior neural specification may be provided by the ADE and the AME, but it is not known if the provision of the morphogenetic activity is tissue-specific and/or stage-dependent.

Mouse mutants lacking the function of Hhex, Lhx1, Otx2, or Foxa2 display varying degrees of anterior truncation. Lhx1, Otx2, and Foxa2 mutants display more severe anterior truncations than Hhex mutants. All these genes are expressed in the AVE, and the defects initially were pinned on this tissue as evidence that the AVE is an essential inducer of anterior neural specification. However, the presence of wild-type extraembryonic tissue in chimeras only partially ameliorates the defects (Dufort et al.,1998; Rhinn et al.,1998; Shawlot et al.,1999; Martinez Barbera et al.,2000). Rescuing the defects of AVE development by expressing Dkk1 from the Otx2 locus allows gastrulation to proceed in the Otx2-null embryo but they still develop abnormal anterior patterning (Kimura-Yoshida et al.,2005). These findings suggest that the function of these genes is required in epiblast-derived tissue for head morphogenesis, in addition to their role in the visceral endoderm.

Hhex null embryos exhibit moderate anterior truncations (Martinez Barbera et al.,2000; Martinez-Barbera and Beddington,2001). Failure of anterior patterning is attributed to a requirement for Hhex function in the definitive endoderm based on chimera data and the loss of Cerl expression in the ADE but not the AVE. Hhex is a transcription factor acting downstream of bone morphogenetic protein (BMP) signaling through the SMAD effectors (Zhang et al.,2002). The loss of Hhex appears to impact the differentiation of the definitive endoderm such that it is no longer competent to maintain signaling to the overlying tissue layers, resulting in a failure to correctly pattern the anterior most neurectoderm.

Some mouse mutants associated with BMP, WNT, and Nodal signaling pathways display similar phenotypes and defects in anterior patterning to the Hhex mutants. Twisted Gastrulation (TWSG1) is a secreted cysteine-rich protein that can enhance or inhibit BMP signaling, depending on the context. Mutant embryos have anterior truncations and other craniofacial defects and have reduced Hhex expression in the ADE and a reduction in the extent of Foxa2 expression in the foregut region (Petryk et al.,2004). The phenotypic similarity of Twsg1 to the Hhex mutant and its involvement in the same molecular pathway, plus the morphological and molecular defects in the ADE, suggest that the loss of Twsg1 impacts on the patterning function of the foregut by affecting its differentiation and viability (Petryk et al.,2004).

The anterior truncation phenotype of mouse mutants for Dkk1 (a WNT inhibitor), Nog/Chrd (BMP inhibitor), and Bmpr1a (BMP receptor) highlights the requirement for the corepression of WNT and BMP activity by the ADE and AME in patterning the neurectoderm (Glinka et al.,1997). The Dkk1-null embryo and Nog/Chrd compound mutants display moderate anterior truncations (Mukhopadhyay et al.,2001; Anderson et al.,2002), whereas Dkk1/Nog compound mutants display more severe truncations (del Barco Barrantes et al.,2003). Like Hhex and Twsg1 mutants, there is likely to be an inability to maintain patterning of the neurectoderm by the mesendodermal tissues. In contrast, Bmpr1a conditional mutants with loss of activity restricted to embryonic tissues exhibit a broader anterior domain comprising a broad, convoluted, and enlarged neurectoderm at the expense of surface ectoderm. The expression domain of molecular markers of prechordal plate and ADE is expanded in the Bmpr1a mutant, suggesting that the phenotype may be the result of excessive morphogenetic activity of the ADE and AME (Davis et al.,2004).

In the Foxa2 mutant, there is stronger evidence of failure to differentiate definitive endoderm. Morphogenesis of the foregut is compromised, but the hindgut forms relatively normally (Ang and Rossant,1994; Weinstein et al.,1994). In Foxa2 chimeras, the failure of foregut morphogenesis results from a failure to displace the visceral endoderm from the gut-forming region due to a deficiency in ADE formation (Dufort et al.,1998). Foxa2 is strongly implicated as a downstream effector of Nodal signaling (Vincent et al.,2003) and a specific loss of anterior structures fits a paradigm of nodal dependent induction of cell fate. In Xenopus, Otx2 and Lhx1 have also been linked to this pathway with Lhx1 and Otx2 part of the machinery, which activates Cerberus in response to nodal signaling (Yamamoto et al.,2003). It is likely, therefore, that in mouse, Otx2, Lhx1, and Foxa2 are required for the specification of cell fates. If this is the case, failure to properly specify cell fate would result in a failure of ADE and/or AME formation and a subsequent failure of anterior patterning.

A milder phenotype is observed in Foxa2 conditional mutants and in Smad2/Smad3 compound mutants, which are known downstream components of Nodal signaling. Smad2/Smad3 compound heterozygotes display moderate rostral truncations (Liu et al.,2004). The analysis of Smad2/Smad3 mutant reveals a consistent reduction in endodermal expression of Foxa2, Hhex, and Alb1. The loss of endodermal gene activity occurs before the onset of anterior patterning defects that may be associated with the loss of Fgf8 and Shh later in development, pointing to a primary defect of the ADE. Although expression of endoderm specific genes is reduced, the foregut pocket is formed normally. The presence of Afp-expressing cells in the foregut pocket suggests that the visceral endoderm may not have been fully replaced by the definitive endoderm. Unlike Hhex and Twsg1 mutants, in which anterior endoderm loses competence in inductive activity, the Smad2/3 compound mutant seems to have a deficiency of ADE itself due to failure of specification.

Defective Movement of the Endoderm Leads to Head Truncation

It is also likely that failure of morphogenetic movement of the endoderm to the anterior region of the embryo precludes the occurrence of inductive interactions between germ layer derivatives to initiate head morphogenesis. Lhx1 is required for the migration of mesoderm during gastrulation (Hukriede et al.,2003), the longitudinal extension of the organizer tissue and the movement of the definitive endoderm (Tam et al.,2004). Because Lhx1 is not expressed in the definitive endoderm, this raises the possibility that the failure of its movement could be secondary to the defective migration of other cell populations. Angiomotin (amot) acts downstream of Lhx1, and the loss of Amot activity impairs the movement of the visceral endoderm from the anterior region of the embryo to the extraembryonic sites (Shimono and Behringer,2003). The stagnation of the visceral endoderm, therefore, may impede anterior displacement of the definitive endoderm in the Lhx1 mutant embryo. The Otx2 null phenotype is due, in part, to failure of β-catenin- and Dkk1-dependent VE migration (Kimura-Yoshida et al.,2005), and a similar migratory mechanism may be required in the ADE. Therefore, the failure of VE migration subsequently may inhibit the anterior movement of ADE and/or AME which in turn disrupts the process of inductive interaction required for anterior morphogenesis.

Interdependence of ADE and AME Function

Foxa2 conditional mutants, in which Foxa2 is specifically inactivated in the embryonic tissues by Cre-mediated recombination, also develop anterior truncations (Hallonet et al.,2002). The initial defect is failure of the axial mesendoderm to form, with no identifiable axial structures morphologically and loss of Gsc from the prechordal plate and Shh in the midline. The definitive endoderm forms as Cerl is present initially, but its expression is lost at the headfold stage. Foxa2 driven LacZ is still expressed in the definitive endoderm, again demonstrating that the definitive endoderm is formed, but there is a failure to maintain its specification and a subsequent loss of patterning ability. This shows that the ADE requires signals from the AME to maintain its own specification and that in turn, both the AME and the ADE are required to maintain correct anterior patterning. It is likely that a similar failure occurs in Nog/Chrd mutants whose initial defect is in the AME. Single-stranded DNA-binding protein 1 (ssdp1) mutants (Nishioka et al.,2005) have a primary failure in the prechordal plate, there is a loss of Dkk1 expression from the prechordal plate and anterior most endoderm, which could suggest a subsequent impact on the entire AME and/or ADE, but this conclusion remains to be determined.

Interdependence between the ADE and AME is not unexpected given their close morphological association, the axial mesendoderm forming the midline of the anterior endoderm, their close history, both originating from the anterior primitive streak, and their close molecular association with both requiring high levels of Nodal signaling for their generation. The occurrence of specific midline defects, along with anterior truncations, in many of the mutants discussed supports this interdependence. It is plausible that the midline defects result from disruption of the axial mesendoderm, whereas anterior truncations result from disruption of anterior endoderm; therefore, their frequent concurrence is due to the mutual interdependence of these tissues at the early stages of anterior morphogenesis.

ADE and Heart Morphogenies

The formation of the heart occurs in close association with the lateral and then ventral foregut endoderm, and evidence from a variety of species suggests that the induction of cardiac cells requires an inductive signal from the endoderm. In the mouse, such an interaction is not well defined. There is some evidence from explant cultures that the differentiation of cardiac cells from mesoderm requires both the primitive streak and the visceral and/or definitive endoderm (Arai et al.,1997). Hex mutant mice have cardiac and vascular defects but it has not been demonstrated whether these stem from a requirement for Hex activity in the foregut endoderm or the endothelium (Hallaq et al.,2004). In Xenopus, an inductive pathway has been defined whereby the Wnt antagonists Dkk1 and crescent induce Hex expression in the endoderm, which then initiates cardiogenesis in the mesoderm through the production of a diffusible factor (Foley and Mercola,2005), and it is possible that a similar mechanism exists in the mouse.

FOREGUT ENDODERM PATTERNS THE PHARYNX

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

In the embryonic foregut, lateral pockets of endoderm called branchial (pharyngeal) pouches are formed in between the branchial arches (Fig. 4). The branchial arches encase a core of mesenchyme derived from the paraxial mesoderm and surrounded by neural crest mesenchyme. The branchial arches and pouches are integral components of the pharyngeal apparatus that forms the craniofacial structures, the pharynx, and associated glandular organs.

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Figure 4. A: The pharyngeal apparatus of an embryonic day (E) 9.5 embryo, showing branchial arches 1 to 3 (BA; blue) and the endoderm of the branchial pouches (BP, outlined in yellow). HT, heart. B: Schematic section through the plane of the embryo shown in A. The pharyngeal endoderm (yellow) lines the branchial pouches up to the level of branchial pouch 1 where the lining of the oral cavity is derived from the ectoderm (blue). The branchial pouches are composed of mesenchyme (red), which is derived from both mesoderm and neural crest. The pharyngeal arches are defined by the presence of a pharyngeal artery (red ovals). C,D: Dorsal view (C) and lateral view (D) of the head region of an E8.5 embryo, showing expression of Pax1 in the medial–lateral foregut endoderm. E: Pax1 expression in the branchial pouch endoderm of an E9.5 embryo.

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Four branchial pouches form in mouse from endoderm of the foregut which is marked by Pax1 and Pax9 (Fig. 4; Wallin et al.,1996; Peters et al.,1998). In the chick embryo, the expression of Pax1 and Pax9 in the pharyngeal endoderm does not require signals from the midline tissues nor the cranial mesoderm, suggesting that patterning is intrinsic to the foregut endoderm (Muller et al.,1996). Gene expression within the pharyngeal endoderm is highly regionalized: Pax1 is expressed in a more dorsal domain than Pax9, the rostral half of each pouch expresses Bmp7 whereas the caudal half expresses Fgf8, and Shh is expressed in the posterior endoderm of pouch two (Veitch et al.,1999). The pharyngeal endoderm expresses Hox genes with, for example, Hoxa3 expressed in pharyngeal endoderm in a domain caudal to the level of the second pouch (Manley and Capecchi,1995). Therefore, branchial pouches are patterned along dorsoventral and rostrocaudal axes and, furthermore, have individual identities. Each pouch contributes to specific organ primordia such as the thyroid from the ventral foregut at the level of the second arch, the thymus and parathyroid from the third pouch, and the ultimobranchial bodies from the fourth pouch (Manley and Capecchi,1995).

The patterning of the pharyngeal apparatus to generate distinctive craniofacial structures involves multiple tissue interactions. The neural crest cells have long been regarded as the primary source of information for patterning the brachial arches. However, experimental evidence from a range of vertebrate species now points to a critical role of the pharyngeal endoderm in patterning the pharynx.

Several mouse mutants display specific malformations of the posterior pharyngeal apparatus and their derivatives that point to a defect in the pharyngeal endoderm. Retinoic acid (RA) is a potent teratogen and an essential regulator of early development. Treatment of headfold stage mouse embryos with a pan-retinoic acid receptor (RAR) antagonist results in loss of the 3rd and 4th pharyngeal pouches and a loss of the mesenchyme, arteries, and nerves associated with their corresponding arches (Wendling et al.,2000). A strikingly similar phenotype is observed with the loss of the retinoic acid synthesis gene Raldh2 (Niederreither et al.,2003). Alterations specifically in the endodermal expression of the genes, Pax1, Pax9, Fgf3, and Fgf8, demonstrate that specification of the pharyngeal endoderm is disrupted in these mutants. Critically, the temporal window of RAR agonist efficacy does not correlate with the time of neural crest migration (Wendling et al.,2000), so a failure to pattern the pharyngeal endoderm, rather than a defect of the cranial neural crest, is the more likely cause of the pharyngeal defect. Hox genes (e.g., Hoxa1, Hoxb1) provide positional information during development, and their misexpression results in homeotic transformations. Hoxb1 is expressed in the pharyngeal endoderm, rhombomere 4, and posterior regions of the embryo. The endodermal expression of Hoxb1 is dependent on a specific RA response element and is expanded anteriorly in response to excess RA (Huang et al.,1998), and markedly decreased, specifically in the endoderm but not in rhombomere 4, in RA-deficient models (Niederreither et al.,2003). In these mouse models, only the caudal pouches and arches are affected, which corresponds to the region of foregut endoderm expressing RARβ and Hoxb1/Hoxa1. Treatment with a RARβ agonist results in defects in pharyngeal arches 1 and 2 attributable to ectopic expression of Hoxb1/Hoxa1 in the anterior pharyngeal endoderm (Matt et al.,2003). Together, these studies show that RA regulation of Hox gene expression in the pharyngeal endoderm is essential for patterning of the surrounding pharyngeal apparatus.

Tbx1 has been strongly implicated in the 22q11 deletion syndrome, as it is located within the commonly deleted region; mouse mutants for Tbx1 have a loss of pharyngeal pouches 3 and 4 and defects in their derivatives. Tbx1 expression is restricted predominately to the endoderm; furthermore, a conditional mouse mutant in which Tbx1 is specifically inactivated in the pharyngeal endoderm has identical defects to those of the Tbx1 null mouse, so the root of the defects is in the pharyngeal endoderm, which results in a loss of pouch outgrowth and of guidance cues for the neural crest (Vitelli et al.,2002; Arnold et al.,2006) The van gogh mutant in zebrafish disrupts Tbx1, causing pharyngeal defects that are rescued by transplantation of wild-type endoderm so Tbx1 has an autonomous role in the pharyngeal endoderm, which is essential for patterning the pharyngeal derivatives (Piotrowski and Nusslein-Volhard,2000).

Mouse mutants with reduced levels of FGF8, phenocopy the 22q11 deletion syndrome (Frank et al.,2002), and although Fgf8 is expressed in both the ectoderm and endoderm of the pharyngeal region, using conditional mutants the loss of endodermal and not ectodermal Fgf8 was demonstrated to be the cause of both glandular and cardiac defects (Macatee et al.,2003). A common molecular link between RA signaling deficient mice and Tbx1 mutants is reduction of Fgf8 expression specifically in the endoderm. A pathway establishing pharyngeal pouch patterning is beginning to be elucidated. Tbx1 and Crk1 double heterozygotes also phenocopy the 22q11 deletion syndrome, and there is an ectopic activation of RA due to changes in the expression of RA synthesis genes. Furthermore, thymic defects can be rescued by a reduction in RA (Guris et al.,2006). Crk1 is an adaptor protein and has been shown to act downstream of Fgf8 during morphogenesis of the pharyngeal apparatus (Moon et al.,2006). Therefore, correct Fgf8 expression in the pharyngeal pouch endoderm is essential for patterning of the surrounding pharyngeal arches and migrating neural crest, and key regulators of this expression are Tbx1 and RA regulated Hox genes.

POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

The evidence for a patterning role of the definitive endoderm thus far has focused on the anterior definitive endoderm; much less is understood about the endoderm of the other segments of the gut and any role they may have in patterning surrounding tissues. The endoderm of the mid- and hindgut is derived from definitive endoderm localized in the distal to posterior regions of the gastrula. This tissue undergoes substantial expansion such that cells from a small region of definitive endoderm can populate a substantial length of the embryonic gut (Tam et al.,2004; Tanaka et al.,2005). The morphogenesis of the posterior gut involves the lateral and anterior movement of the most posterior cells and the posterior and paraxial extension of the medial and more distal cell population during the formation of posterior gut.

Sox17 null embryos are deficient in definitive endoderm, especially that of the mid- and hindgut rather than the foregut (Kanai-Azuma et al.,2002). These embryos appear morphologically normal until early somite stage, when they become distinguished by lack of axis rotation and deteriorating growth and organization of the trunk. This is accompanied by loss of Shh and Ihh activity in the endoderm of the posterior gut, and a corresponding loss of Patched activity in the adjacent lateral plate mesoderm. Several studies have identified a role for Hedgehog signaling in endoderm development in both the pharyngeal regions (Moore-Scott and Manley,2005), the midgut (Apelqvist et al.,1997), and the hindgut (Ramalho-Santos et al.,2000). Shh is widely expressed in the gut, although at the level of the pancreatic bud, it is absent from the dorsal and ventral regions where the pancreatic buds form. Ectopic expression in these regions causes intestinal mesoderm to develop in place of pancreatic mesoderm (Apelqvist et al.,1997). Shh and Ihh mutant mice both display multiple defects of the gastrointestinal tract, including intestinal transformation of the stomach, suggestive of a critical role in endoderm development; however, the expression of Hox and para-Hox genes was not altered (Ramalho-Santos et al.,2000).

Some Hox genes are expressed in spatially distinct regions along the anterior–posterior axis of the endoderm. One such gene is Hoxa13, which is specifically expressed in the posterior endoderm and mesoderm. A specific role for Hoxa13 in the endoderm has been demonstrated in the chick (de Santa Barbara and Roberts,2002). During embryonic development, the hindgut pocket initiates the development of the hindgut and tail and the cloaca. Overexpression of mutant Hoxa13 specifically in the chick hindgut results in severe defects of the hindgut, tail, and cloaca but not the neural tube. The defects are not observed upon overexpression in the mesoderm, demonstrating a specific role for the endoderm in posterior patterning. Ablation of the posterior endoderm causes similar phenotypes, which can be rescued by replacement with posterior endoderm but not with anterior endoderm, demonstrating that signals from the posterior endoderm are required for normal tail and hindgut development in the chick. In the mouse, Hoxd genes regulate the formation of gut sphincters (Kondo et al.,1996; Zakany and Duboule,1999), but the relative importance of their activity in the endoderm versus the mesoderm has not been dissected.

The para-Hox gene Cdx2 is also expressed in the posterior endoderm and mesoderm. Null mice do not complete gastrulation due to an earlier requirement for Cdx2 in the extraembryonic tissues, but Cdx2 heterozygotes display lesions in the colon, which resemble ectopic stomach and small intestine, a partial homeotic transformation which shows that Cdx2 is required to direct endoderm toward a caudal phenotype (Beck et al.,1999) but to what extent this phenotype depends on Cdx2 expression in the mesoderm or endoderm is unclear. WNT signaling also may be essential for hindgut development as mice mutant for the downstream transcription factors Tcf4−/−/Tcf1−/− exhibit severe caudal truncations in which the earliest molecular defects are loss of Sox17, Foxa1, Foxa2, and Shh from the caudal endoderm and a resultant failure of hindgut morphogenesis, suggesting that the loss of posterior development stems from failure of posterior endoderm specification (Gregorieff et al.,2004).

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

A major hurdle in analysis of early development of the definitive endoderm is the lack of both pan-endodermal and region-specific genetic markers. A majority of definitive endoderm markers are also expressed in the visceral endoderm; although this likely reflects functional overlap, it hampers the interpretation of experimental outcome regarding the specification and differentiation of the embryonic versus extraembryonic endoderm. Additionally, some genes are expressed in multiple germ layers (e.g., Hox and ParaHox genes). More markers would greatly aid our ability to specifically analyze the role of the definitive endoderm in developmental processes and to this end, screens have been performed (for example, Sousa-Nunes et al.,2003) with some success, although new technologies will facilitate further experiments to fully characterize the transcriptome of the definitive endoderm. The failure thus far to identify pan-definitive endoderm genes may reflect the heterogeneity of the endoderm.

The definitive endoderm has been overlooked as a source of morphogenetic activity. The AME is clearly essential for organizing anterior development, but its lineage relationship to the ADE is ambiguous. The timing and mechanism of allocation of the AME and ADE at gastrulation is unclear and the fate of the prechordal plate is unknown. Furthermore, the morphogenetic role of the ADE versus the AME is unresolved. Despite a handful of detailed studies, the morphogenetic movement of the definitive endoderm, including the means by which it intercalates into the pre-existing visceral endoderm and replaces the visceral endoderm and the pattern and driving forces of morphogenetic movement of the definitive endoderm particularly that of the posterior embryonic gut is far from known. Other aspects of endoderm development, such as the existence of the mesendodermal progenitor, the molecular mechanism of lineage choice, the timing and duration of recruitment of definitive endoderm, the population dynamics of the gut endoderm, and the precise nature of the morphogenetic cues to other germ-layer derivatives are not fully understood. Embryological experiments that address these issues are technically difficult in the mouse. For example, the identification of a mesendodermal progenitor at the early gastrulation stage will require the ability to accurately map single cells and their derivatives, preferably in real time, and to combine this with molecular markers of cell fate. Data from genome-wide characterization and large-scale functional assays of endodermal genetic activity would be immensely useful to focus the embryological investigation to the relevant molecular pathways and the analysis of mutant phenotypes. Answering such questions would provide a better understanding of the how lineage separation occurs at gastrulation, which could in turn inform stem cell research and shed light on the myriad other cell fate decisions that occur during development and are central to the developmental process.

An understanding of the genetic network of endoderm specification and differentiation in mouse lags behind that of other species, and reflects some of the difficulties of the mouse as an experimental model. Although there are clearly conserved network components across species, there are also distinct differences. The effort to better understand the specification and differentiation of definitive endoderm in the mouse would benefit not only from discovering the peculiarity of this species but to draw on the common features of different models. The advances in stem cell studies will facilitate embryological investigation by identifying markers and pathways of differentiation. However, it cannot fully reveal the complexity and interactive nature of the developmental processes in vivo, the study of which in turn will inform and enable the understanding of the biology of the stem cells.

Table 2. Mouse Mutants Displaying Anterior Defects that Are Reputed to Be Associated with Morphogenetic Deficiency of the Endoderm*
GeneMutationExpressionPhenotypeMorphogenetic processes affectedPutative defect of the endodermMolecular PathwayType of moleculeReference
AVEADEAMEMGOW/S
  • *

    Phenotypes are as follows: A, anterior truncation; B, midline defects; C, pharyngeal arch 1 defects; and D, enlarged rostral structure.

  • Putative defects of the endoderm are as follows: 1, impact on the formation of the definitive endoderm (specification and/or allocation of the endodermal precursor); 2, lack of morphogenetic movement of the definitive endoderm; 3, failure to proliferate or maintain viability leading to reduction and/or depletion of the definitive endoderm; 4, impact on the differentiation of the definitive endoderm, failure to acquire the competence to pattern overlying germ layers; and 5, impairment of the inductive or patterning activity despite the formation of the proper types of definitive endoderm. PrChP, prechordal plate; TF, transcription factor; MGO, midgastrula organizer; W/S, widespread.

Otx2Null×A+• Loss of A-P axis rotation and AVE migration1 and 2Wnt? Nodal?TFAng et al.,1996; Kinder et al.,2001
• Possible loss/reduction of ADE/AME formation and/or migration
Foxa2Null×A+• Loss of A-P axis rotation and AVE migration1 and 2downstream of NodalTFAng and Rossant,1994; Weinstein et al.,1994; Kinder et al.,2001
• Possible loss/reduction of ADE/AME formation and/or migration
Lhx1Null×××A+• Loss of A-P axis rotation and AVE migration1 and 2Wnt? Nodal?TF complex with Ldb1/Ssdp1Shawlot and Behringer,1995; Kinder et al.,2001
• Possible loss/reduction of ADE/AME formation and/or migration
Ldb1NullA+• Loss of Dkk1 and Cerl in AVE/ADE/PrChP1 and/or 2Wnt? Nodal?TF complex with Lhx1/Ssdp1Mukhopadhyay et al.,2003
• Failure to extend AME
Smad 2/3Double HeterozygoteA, B• Loss of FGF8 from ANR and Shh from rostral ventral midline1 and/or 2BMP NodaleffectorsLiu et al.,2004
• Reduction of endoderm (Foxa2, Hhex, Albumin) and ectopic VE (AFP)
Twsg1NullA,B,C• Reduction of Hhex expression and of Foxa1 in foregut3 or 4BMPagonist and/or antagonistPetryk et al.,2004
• Foregut narrowed and separated from notochord
HhexNull×××A, C• AVE normal4Downstream of BMPTFMartinez-Barbera et al.,2000; Martinez-Barbera and Beddington,2001
• Absence of cerl expression in DE
• Loss of Lhx1 and Foxa2 in axial mesendoderm
Foxa2conditional AME only×A• Failure of PrChP to differentiate5 secondary to AMEDownstream of NodalTFHallonet et al.,2002
• ADE formed but not maintained
Ssdp1insertion××A• Normal AVE5 secondary to PrChPWnt? Nodal?TF complex with Lhx1/Ldb1Nishioka et al.,2005
• Early ADE normal (Hhex and Cerl)
• Loss of PrChP (Cerl, Gsc, Dkk1, Foxa2)
Bmp1aconditional embryonic onlyD• Enlarged VE and DE domains and PrChP platenaBMPreceptorDavis et al.,2004

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES

We thank Poh-Lynn Khoo, Nicole Wong, Joshua Studdert and Kirsten Steiner for assistance with figure preparation and Peter Rowe, David Loebel, and Sabine Pfister for comments on the manuscript. Our work is supported by Mr. James Fairfax. P.P.L.T. is an NHMRC Senior Principal Research Fellow. Nomenclature of embryonic structures and genes follows the Anatomical Dictionary and Gene Symbols/Name of Mouse Genome Informatics 3.43 (http://www.informatics.jax.org/).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. THE ANATOMY OF ENDODERM FORMATION DURING GASTRULATION
  5. SPECIFICATION AND DIFFERENTIATION OF TISSUE LINEAGES OF THE DEFINITIVE ENDODERM
  6. ROLE OF ANTERIOR DEFINITIVE ENDODERM IN HEAD MORPHOGENESIS
  7. FOREGUT ENDODERM PATTERNS THE PHARYNX
  8. POSTERIOR DEFINITIVE ENDODERM AND GUT FORMATION
  9. PERSPECTIVES
  10. Acknowledgements
  11. REFERENCES
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