Yad

dorsal pattern

Figure 3.21

AER formation and dorsoventral patterning of the limb bud

(a) Gene expression differs along the D/V axis of the limb bud. (b) The regulation of AER formation and dorsal patterning depends on the interactions of the BMP and Wnt signaling pathways. Downstream of BMP signaling, the En-1 gene acts to restrict Wnt-7a expression to the dorsal ectoderm, which functions to organize dorsal patterning.

Source: Part a modified from Johnson RL, Tabin CJ. Cell 1997; 90: 979-990. Part b based on Soshnikova N, Zechner D, et al. Genes Dev2003;17:1963-1968.

regulates dorsoventral polarity. The actions of Wnt-7a appear to be mediated by the Lmxlb protein, a homolog of the Drosophila apterous gene that is expressed in all dorsal mesodermal cells of the limb (Fig. 3.21b).

Patterning along the A/P axis of the limb bud depends on the function of the zone of polarizing activity (ZPA), another organizer with potent signaling activity. The ZPA is the source of the Sonic hedgehog (Shh) protein in the limb field. The ZPA and Shh influence the polarity of pattern elements along the A/P axis. In particular, digits with reversed polarity can be induced when either the ZPA is transplanted or Shh expression is induced in the anterior part of the limb.

The restriction of the ZPA and Shh expression to the posterior portion of the limb bud suggests that the limb bud contains anteroposterior positional information that predates Shh expression and function. This preexisting information may consist of rostrocaudal regulatory patterns derived from the lateral plate mesoderm. Two proteins, the basic helix-loop-helix transcription factor HAND2 (dHAND) and Gli3 , a homolog of the Drosophila Cubitus interuptus protein and a transcriptional effector of Hedgehog signaling play critical roles in anteroposterior patterning of the limb. Gli3 is expressed in the anterior limb mesoderm and HAND2 in the posterior limb mesoderm, and they each act to restrict each other's expression (Fig. 3.20c). HAND2 regulates the activation of Shh expression in the posterior distal mesoderm. Therefore, Shh expression in the ZPA appears to be regulated both by signals from the AER and inputs from the lateral plate mesodermal cells that will become incorporated into the limb bud.

The organizers in the limb field do not act independently of one another. Formation and maintenance of the ZPA depends on the presence of a functional AER, and, in turn, maintenance of the AER depends on the existence of ZPA function. Thus a regulatory circuit connects FGF signaling to the induction of Shh expression, and Shh signaling to FGF expression in the AER (Fig. 3.20d). These inductive interactions appear to operate through intermediate signals, although all of the proteins are not known as of yet.

Integration of organizing signals to form the proximodistal axis and deployment of Hox genes in the limb field

After the formation of the ZPA and AER and the proliferation of mesodermal cells, proximal mesenchymal cells begin to form cartilaginous condensations that prefigure the limb skeletal pattern. These condensations arise in a proximal to distal order, so that the humerus (the bone of the upper arm) forms first, the radius and ulna next, and the carpals (wrist bones) and digits last. The development of these individual pattern elements suggest that limb development goes through discrete temporal phases and that pattern formation is controlled by the localized expression of regulatory genes, cued by the signals emanating from the organizers, the AER and the ZPA.

The Hox genes play important roles in limb patterning, albeit in a different fashion than in appendage patterning in arthropods. Whereas individual Hox genes or pairs of Hox genes are expressed in arthropod limb fields, a much larger number of Hox genes are deployed in nested domains of vertebrate limb fields (Fig. 3.22). Detailed studies of normal and ectopic Hox gene expression and analysis of limb development in mice lacking one or more Hox gene functions have revealed a complex and dynamic spatiotemporal pattern of Hox gene patterns and gene interactions in determining the formation of limb pattern elements.

Three temporal phases of Hox gene expression appear to correlate with the temporal sequence of proximodistal pattern element formation, particularly in regard to the Hox9-13 genes of the HoxA and HoxD complexes (Fig. 3.22a-c). The first phase of Hox expression is not polarized, appears to be Shh-independent, and is associated with the development of the most proximal limb elements (upper arm/leg) (Fig. 3.22a). Subsequent phases of Hox expression arise in nested patterns whose polarity depends on AER and ZPA functions. The second phase of Hox expression occurs in the next most proximal elements (forearm or lower leg) (Fig. 3.22b). The third phase of Hox expression includes most distal elements (wrist and hand, ankle and foot) (Fig. 3.22c).

These spatial patterns of Hox gene expression reveal that many related Hox proteins are expressed in overlapping as well as adjacent domains. No simple one-to-one correspondence exists between a particular Hox gene and the growth and patterning of any particular limb pattern element. Rather, the formation and identity of particular elements appear to reflect combinations of Hox protein functions, some of which are clearly acting redundantly. For example, loss of either Hoxa-13 or Hoxd-13 function has limited effects, whereas loss of both genes dramatically affects the development of the distal limb. Similarly, Hoxd11 or Hoxa11 mutations have minor effects on the formation of the radius and ulna, whereas these long bones are almost completely lost in the double mutant.

The analysis of cz's-acting regulatory elements that control Hox gene expression in the limb bud has highlighted the complexity of Hox expression. Both gene-specific and global regulatory elements appear to control the expression of individual Hox genes and groups of Hox o

Paralog 9 10 11 12 13

Knockout phenotype Phase I expression

Phase I

Zeugopod Element

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