Chapter SummaryAxial and germ layer specification in the Xenopus embryo involves a complex set of signaling interactions and cytoplasmic rearrangements. Dorsal axial specification begins with cortical rotation, which is brought about by the dynamic nature of microtubules following fertilization. This results in the stabilization of B-catenin on the prospective dorsal side of the embryo, which then enters the nucleus leading to the transcriptional activation of the siamois gene in these cells. These cells then form the Nieuwkoop center, which leads to dorsal mesoderm specification and activation of Spemann organizer specific genes such as xgoosecoid. Furthermore, genes encoding the transcription factors vegT and veg1, which are essential for emdoderm formation, synergise with Nodal-related factors essential for mesoderm formation.
The Nieuwkoop center signals to induce the Spemann organizer which is the center of axis formation and gastrulation. The Spemann organizer expresses a distinctive set of genes such as those encoding ligands like Noggin and Chordin which have strong neuralizing influences as well as dorsalizing influences on the mesoderm. Other genes expressed by the organizer are the ones coding for activin and FGF that primarily influence the mesoderm. Thus the organizer has two kinds of activities: neuralization and dorsalization of the mesoderm. This is due to the activity of the molecules such as Noggin and Chordin, which antagonize BMP action thereby inhibiting the ectoderm inducing activity of BMP. Other molecules involved in this patterning are Xolloid and Twisted-gastrulation. Moreover, molecules such as Frizb and lunatic fringe are also present in the Xenopus. Frizb plays a role in preventing ventralization of cells in this area. Thus a complex set of molecular interactions govern pattern formation in the early embryo. In addition to the signals emanating from the organizer, the mesoderm is also under the influence of other moelcules such as Xwnt8, BMP2, and BMP4, which act as ventralizing factors. [fig 16.9]
A number of these molecules are diffusible and have different developmental consequences at different concentrations. These molecules are referred to as morphogens. For example, Noggin and Chordin, which are produced by the dorsal mesoderm, inhibit BMP activity in a concentration-dependent manner resulting in a gradient of BMP activity. This leads to higher BMP activity in ventral mesodermal regions. Another aspect of the vertebrate body plan is left-right asymmetry. A number of genes have been shown to play a role in establishing this asymmetry. For example, nodal is expressed on the left side of postgastrula embryos of the frog, chick and mouse.
In amniotes the organizer role is fulfilled by the Hensen's node. However, the molecular details of organizer function may not be identical. For example, even though noggin is expressed in Hensen's node of the mouse, a noggin null mutant still forms a notochord and a neural tube. This is perhaps because other molecules are able to satisfy the role played by Noggin in such an embryo. Candidates for such a function could be Follistatin and Chordin.
Homeobox genes have been identified in the mouse. Their linear order of organization in the DNA is the same as that seen on the Drosophila chromosome. However, the mouse has four groups of linked genes; this is thought to be a result of gene duplication during evolution. In the developing mouse embryo the anterior borders of expression of these genes are distinct; however, there is considerable overlap in their expression domains. Furthermore, the precise domain of expression of a gene is highly dynamic and may keep changing at any given time. Hox genes in vertebrates, just as those in Drosophila, function as selector genes. Thus a mutation in a Hox gene often brings about a transformation to anterior structures. However, their regulation has not been as extensively studied as in Drosophila and is only beginning to be deciphered in detail. Furthermore, a mouse homolog of Drosophila polycomb genes, bmil, plays a similar role as that in the fly. Bmil mutation results in some posterior skeletal transformations.
Vertebrate limbs arise from limb buds in the lateral plate mesoderm. Dynamic patterns of Hox gene expression along the anterior posterior axis are thought to play a role in positioning the limb fields. The system that sets up the limb field originates in the intermediate mesoderm and involves some members of the FGF family. Anterior vs. posterior identity of the limb buds is governed by the action of T-box-containing transcription factors: tbx4 is expressed in the hindlimbs and tbx5 is expressed in the forelimbs.
Dorsoventral organization of the limb bud involves a complex interplay of molecules. The homolog of the Drosophila engrailed gene, en1, is expressed in the ventral ectoderm but not dorsally in either the mesoderm or the ectoderm. The prospective dorsal ectoderm expresses a membrane protein, radical fringe (Rfng). En1 in the ventral ectoderm antagonizes Rfng expression. The Apical Ectodermal Ridge (AER) forms at the border of the dorsal Rfng-expressing and ventral En1-expressing cells. Furthermore, the dorsal ectoderm expresses xwnt7a which signals to the underlying mesoderm to express the transcription factor lmx1, which is known to be necessary for forming dorsal-pattern elements in the limb. Lmx1 is absent ventrally because En1 suppresses it.
Anterior-posterior patterning of the limb (as shown in the chick) is dependent on the proper functioning of the Zone of Polarizing Activity (ZPA), formation of which is dependent on hox gene b8. Cells making up the ZPA express Shh under the influence of the transcription factor called dHAND. Shh is in turn thought to act as a morphogen. Low concentrations induce anterior digits while high concentrations are required for posterior digit formation. Furthermore, Shh also stimulates proliferation of the mesenchyme, which is essential for limb outgrowth. There is also thought to be a positive feedback loop between the AER and the ZPA; Shh in the ZPA is thought to stimulate FGF secretion in the AER which in turn stimulates Shh. Another molecule, Gremlin (a noggin relative) is expressed by cells adjacent to the Shh-secreting cells of the ZPA; gremlin is thought to regulate the levels of BMP in the developing limb.
Proximal-distal patterning of the developing limb is thought to depend on the formation of a progresszone, a kind of stem-cell population that moves distally as the limb extends. Thus progeny of cells making up the progresszone which leave the zone early are exposed to AER influences for a shorter period of time than those leaving the zone later. Cells leaving early might attain proximal fates as opposed to those leaving later, which attain a more distal fate. Moreover, two homeobox genes, meis1 and meis2, show localized expression in proximal limb elements. Expression of these genes is dependent on retinoic acid signaling. It is thought that FGF from the AER is responsible for restricting the activity of RA that limits Meis1 and -2 expression.
In addition to the above mentioned molecules, Hox genes also regulate the limb element formed. For example, mesoderm forming the humerus expresses d9 and d10; the domain of the prospective radius and ulna express a nested array of Hox genes d9 through d13; and the prospective wrist and digit express a13 and d13. A primary role for Hox genes seems to be to regulate the rate and timing of cartilage (and hence, eventually, of bone) differentiation.
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