The timing of gastrulation
During gastrulation, the cell cycle expands from 55 minutes to 4 hours, a lengthening that is essential for further development (Fig. 1). The control of cytostasis is not understood, although TGFß signaling is likely to be involved as it is known to limit re-entry into the cell cycle (Siegel and Massague, 2003) and is necessary downstream of VegT for gastrulation to occur (Zhang et al., 1998). WEE1, an antagonist of M-phase re-entry, is clearly required because its depletion causes an increased mitotic index from 10% to 25% during gastrulation and results in abnormalities in gastrulation movements. Zygotic gene expression continues, although the positioning of Xbra and chordin expression is disrupted, which may be crucial for correct cell movements (see below) (Murakami et al., 2004). WEE1 may regulate the mitotic activity of bottle cells, the shape changes of which in response to Xnr/Vg1/activin are responsible for the first invagination movements of gastrulation. These cells are the earliest non-mitotic population at gastrulation, and promoting mitosis arrests bottle cell formation (Kurth, 2005). WEE1 is a maternal protein, which may explain a long-standing observation that the timing of MBT and gastrulation onset are not linked (Smith and Howard, 1992). The timing of gastrulation is also dependent on several other maternal inputs. Abrogating either the maternal Wnt or Vg1 pathway delays formation of the dorsal lip (Birsoy et al., 2006; Heasman et al., 1994), and maternal CREB depletion slows ventral lip formation (Sundaram et al., 2003).
Events in the embryonic dorsal mid-line
Blastopore invagination resulting from bottle cell formation is the first external sign that gastrulation is under way. Internally, gastrulation movements are also beginning. Initial movement occurs not in the marginal zone but in the vegetal mass, which undergoes an active inward surging in the animal direction, causing an increase in the blastocoel floor area (Winklbauer and Schurfeld, 1999), and driving firstly the involution of the prechordal (head) mesoderm, followed by chordamesoderm (presumptive notochord) (Fig. 6). The fact that these two domains can be physically separated suggests that AP patterning in the dorsal mesoderm is established by this time. After involution, prechordal mesoderm cells become actively migratory and move animally (Shook et al., 2004). This behavior is regulated by multiple factors but the definitive endogenous combination is not yet known.
One secreted protein known to be involved in chordamesoderm cell behavior is platelet-derived growth factor, PDGFA, which is secreted by blastocoel roof cells. PDGFA depletion causes random protrusive activity of prechordal mesoderm and loss of head structures (Nagel et al., 2004). A second regulator of prechordal mesoderm is the Wnt antagonist dickkopf (Kazanskaya et al., 2000). Inhibition of its activity results in microcephaly, while overexpression of dickkopf expands the size of the prechordal plate at the neurula stage, without affecting chordamesoderm formation. An important issue here is which Wnt signal is being antagonized by dickkopf. Third, the migrating prechordal mesoderm zone is an area of active repression of Xbra expression. Repression is achieved by several inputs, including the binding of the transcriptional repressor goosecoid to the Xbra promoter (Yao and Kessler, 2001). Xbra overexpression in the prechordal mesoderm prevents cell adhesion to the extracellular matrix protein fibronectin (Kwan and Kirschner, 2003). As with PDGFA depletion, abrogation of goosecoid activity or of its transcriptional activator siamois blocks prechordal migration, presumably by increasing Xbra expression. Last, the size, shape and correct placement of the entire involuted region is regulated by nodal, BMP and Wnt antagonists, particularly by the TGFß family member antivin/lefty (Branford and Yost, 2002). Antivin-depleted embryos have increased and expanded Gsc/Xnr3 and Xbra expression, exogastrulate at the mid-gastrula stage and fail to form heads (Branford and Yost, 2002).
The period of prechordal mesoderm migration is brief and is limited by the direct adhesion of the prechordal mesoderm to the head neuroectoderm (Koide et al., 2002) (Fig. 2), so the major force in extending the AP axis in gastrulation is the involution and convergence extension of the chordamesoderm (Figs 2 and 6). Many factors regulate this convergence extension, as described in Box 2. In summary, many essential early zygotic patterning genes are not tissue differentiation genes, but are involved in regulating cell behavior. For example, loss of dickkopf, siamois or goosecoid activity abrogates prechordal mesoderm migration and prevents the formation of head structures, while Xbra is required for convergence extension of the chordamesoderm.
Events in the non-organizer marginal zone
Meanwhile, away from the organizer region, convergence extension movements are delayed. Several pathways are involved in establishing muscle precursor fates. First, FGF signaling and Xbra expression are required and maintain each others expression, as described above (Fig. 7). The expression pattern of Xbra mRNA, which forms a ring around the blastopore of the gastrula, is evidence that its role is not confined to dorsal convergence extension activity. Promoter studies show that dorsal and ventrolateral Xbra expression are differently regulated (Latinkic and Smith, 1999; Lerchner et al., 2000; Papin et al., 2002). By mid-gastrulation the myogenic markers Mespo, Myf5 and MyoD mRNA are all expressed in an equatorial ring similar to Xbra and FGF, and depletion of FGF4, FGFR1 or Xbra causes severe reduction in their expression (Conlon et al., 1996; Fisher et al., 2002; Yokota et al., 2003).
How does Xbra function in both myogenic and convergence extension regulation? The expression of one target gene, the cytoplasmic regulator sprouty, may be particularly important in this regard. Sprouty is a cytoplasmic antagonist of FGF signaling that blocks convergence extension movements by interfering with protein kinase C (PKC) function, without blocking activation of myogenic gene expression. It can thus separate the convergence extension and muscle specification functions of Xbra (Sivak et al., 2005). A second receptor tyrosine kinase regulator, Spred, has the opposite effect; it is required for somite specification but not for convergence extension movements (Sivak et al., 2005). Muscle specification during gastrulation also depends on the repression of BMP signaling, as triple depletions of noggin, chordin and follistatin eliminate muscle precursor gene expression (Khokha et al., 2005).
An essential aspect of mesoderm patterning is the interaction of several pathways in the initiation of Hox gene expression during gastrulation and neurulation. The AP patterning in the definitive trunk region results from the expression of nine co-linear Hox genes, which are expressed in a specific temporal order in the non-organizer mesoderm, beginning at the early gastrula stage with HoxD1
expression (Wacker et al., 2004a
). The early Hox expression determines the length of the trunk region, and depletion of HoxA1, HoxB1 and HoxD1 results in reduced MyoD
expression (Wacker et al., 2004a
; Wacker et al., 2004b
). Hox expression depends on the FGF/Xbra pathway (Fig. 7
), and is limited by BMP signaling (Wacker et al., 2004b
). Another important mesodermal regulator is retinoic acid (RA). Depletion of retinoic acid receptor Xrar
2 causes both microcephaly and tailless embryos, and reduces expression of both FGF receptors FGFR1 and FGFR4, and posterior Hox gene expression (HoxB9) (Shiotsugu et al., 2004
). The Xenopus
caudal family member Xcad3
is essential for tail somite formation, and its transcriptional regulation is complex and involves maternal CREB, FGF signaling and RA activity (Isaacs et al., 1998
; Shiotsugu et al., 2004
; Sundaram et al., 2003
Unexpectedly, a key BMP antagonist, the frizzled-related protein sizzled, is expressed not dorsally, but in the ventral lip of the blastopore. Sizzled acts as a competitive inhibitor of the chordin metalloproteinases, Xlr1 and BMP1 (Lee et al., 2006). Its expression is activated by BMP signaling and is repressed by VegT/Vg1 via the BMP antagonists (Birsoy et al., 2006; Lee et al., 2006; Salic et al., 1997). Depletion of sizzled causes an expansion of ventral blood islands and does not affect the expression of MyoD or of organizer genes. It has an essential role in regulating epidermal versus neural cell fates (Lee et al., 2006).
Events in the animal cap
While these complex events are occurring in mesodermal precursors, cells in the animal cap remain set to become epidermis, as dictated by continuing BMP signaling, providing that they are not underlain by involuted prechordal mesoderm or chordamesoderm. Switching from epidermal to proneural fate absolutely requires BMP suppression, as described previously (Khokha et al., 2005); the entire ectoderm becomes neural when all BMP signaling is depleted (Reversade et al., 2005; Reversade and De Robertis, 2005). The epidermis is a bilayer at the gastrula stage, and both layers overlying the chordameosderm express proneural genes, although only the inner layer undergoes primary neuronal differentiation at the neurula stage (Chalmers et al., 2002) (Fig. 5).
It is clear that neural specification, like that of muscle, is an active process, involving a complex network of transcription factors (Fig. 5). Transcription factors of the Sox class are expressed in ectoderm from the late blastula stage, and dominant-negative Sox2 inhibits neural induction when it is expressed specifically during the gastrula stage (Kishi et al., 2000). SoxD and Smad interacting protein 1, SIP1, a member of the EF1/ZFH family, maintain expression of each other after the gastrula stage. Loss-of-function experiments show that they are necessary for neural induction, with SIP1 preventing the activation of pro-epidermal genes (Nitta et al., 2004). The three paralogous Hox group 1 genes, HoxA1, HoxB1 and HoxD1, are essential for neural patterning and begin to be expressed at the gastrula stage; their simultaneous depletion has severe effects on hindbrain and neural crest patterning (McNulty et al., 2005). FGF8 and RA are the most likely candidate activators of Hox gene expression (Christen and Slack, 1997; Shiotsugu et al., 2004). Cell cycle withdrawal is also important for further neural differentiation. Depletion of the maternally expressed SWI/SNF remodeling protein BRG1 causes the proliferation and expansion of proneural gene expression (Sox2), but reduces neural differentiation. BRG1 also directly binds to and co-activates several bHLH transcription factors in the differentiation pathway (Seo et al., 2005).
As gastrulation proceeds, anterior- and posterior-specific neural genes begin to be expressed in the neural anlagen, a process that depends on the anterior repression of Wnt signaling via both intrinsic and extrinsic pathways. An important regulator of neural AP patterning is the zinc-finger protein Xsalf, which is activated in the anterior neural ectoderm in the late gastrula. Xsalf both activates the expression of anterior neural genes (Otx1) and represses posteriorizing Wnt signals by activating the expression of Wnt repressors Xtcf3 and GSK3ß, so that Xsalf-depleted embryos have reduced heads and lack forebrain gene expression (Onai et al., 2004). The picture that emerges is different from the original activation/transformation model of neural induction, which suggested that the activation step turns the entire neural anlagen into a pro-anterior neural state, which is later transformed by a posteriorizing gradient. The activation/transformation model predicts that Xsalf would be expressed throughout the neural ectoderm, which it is not. This lends support to an alternative, regional activation model, as described further elsewhere (Onai et al., 2004).
Events in the vegetal mass
The vegetal mass, the cells of which are determined towards endodermal fates by the early gastrula stage (Heasman et al., 1984), is the least studied region of the gastrula. Immunostaining of the vegetal mass identifies the major signaling activities as those continuing from the blastula stages: TGFß proteins that activate Smad2 and Wnt signaling (Faure et al., 2000; Schohl and Fagotto, 2002). It remains to be shown whether the nuclear ß-catenin present at this time is the result of maternal Wnt11 signaling or of new zygotic Wnt pathways. FGF signaling is very low in this region, consistent with the fact that overexpressed FGF causes reduced endoderm formation, and blocking FGF signaling expands the expression of endodermal genes into the equatorial zone (Cha et al., 2004). BMP signaling activity is almost completely excluded from the dorsal vegetal area, suggesting its antagonism is necessary for endoderm specification. In agreement with this, chordin and noggin treatment of animal caps causes ectopic endodermal gene expression (Sasai et al., 1996).
Gene expression in the gastrula stage is dictated by the four pathways that are activated in the blastula vegetal mass. These pathways can be placed into three groups; those involved in boundary formation between mesoderm and endoderm [such as the Bix and Mix homebox transcription factors (Casey et al., 1999; Kofron et al., 2004b), which are expressed during gastrulation only]; a dorsally localized group whose expression domain includes the dorsal prechordal mesoderm (often described as anterior mesendodermal genes), including cerberus, Xlim1, dickkopf and Xhex, the main function of which may be in establishing correct gastrulation movements; and those that are distributed throughout the vegetal mass (Xsox17, Gata4, Gata5 and Gata6). This last group of transcription factors continues to establish regulatory networks required for endoderm specification and maintenance after gastrulation (Afouda et al., 2005; Xanthos et al., 2002; Xanthos et al., 2001). A new player on the signaling scene in the presumptive endoderm is the short-range signal receptor Notch. Notch suppression leads to the expansion of mesodermal molecular markers and to the loss of endodermal markers, endodermin and the HMG box transcription factor Xsox17 (Contakos et al., 2005).
The timing and order of patterning events in the endoderm, from the gastrula stage onwards, is not clear. Although early explant studies showed that AP patterning is autonomous to the endoderm (Gamer and Wright, 1995), this has not been supported by more recent work, which suggests that mesodermal signals as late as the tailbud stage are necessary to specify foregut versus hindgut fates (Horb and Slack, 2001). It is nevertheless the case that blocking the establishment of the embryonic dorsal axis by UV treatment of the fertilized egg causes a loss of expression of the late anterior gut marker Pdx1, as well as of early gastrula dorsal endoderm markers, indicating that the early regionalization of the endoderm foreshadows later events (Henry et al., 1996). Future studies will determine the extent to which endoderm patterning and shape changes are regulated by the same signaling networks and their antagonists that operate in the mesoderm and ectoderm.