Various transcription factors are expressed in the developing limb bud in a region-specific manner. Therefore, it has been suggested that these transcription factors might be involved in position-dependent cell adhesiveness.
Hox genes are expressed in a region-specific manner in the limb bud, where they regulate growth and adhesion of mesodermal cells (reviewed by Pearson et al.,2005). Hoxa13 is expressed in the distal-posterior region of the limb bud at early stages. At later stages, the expression localizes to the presumptive autopod-forming region (Yokouchi et al.,1991; Nelson et al.,1996). As expected from its expression pattern, Hoxa13 is involved in autopod formation in cooperation with Hoxd13, the expression of which is partially overlapped with Hoxa13. Misexpression of Hoxa13 in the whole limb bud affects morphogenesis of proximal cartilaginous elements, such as the stylopod and zeugopod, that are normally formed from Hoxa13-negative cells (Yokouchi et al.,1995). Disruption of Hoxa13 in the limb bud causes hypoplasia of the autopod (Fromental-Ramain et al.,1996; Stadler et al.,2001). Notably, Hoxa13/d13 double knockout mice completely lack their autopods (Fromental-Ramain et al.,1996), indicating that Hoxa13, in cooperation with Hoxd13, functions in the regulation of autopod formation.
Hoxa13 regulates limb skeletal patterning by controlling cell growth and adhesiveness. Misexpression of Hoxa13 in the proximal region alters the adhesiveness of the local cells. Hoxa13-misexpressed cells are sorted out from Hoxa13-negative cells (Yokouchi et al.,1995). In addition, Hox13 paralogue genes coordinately regulate the expression of the cell surface receptor, EphA, during limb development (Stadler et al.,2001; Cobb and Duboule,2005; Salsi and Zappavigna,2006; Kawakami et al.,2009). Interactions of Eph receptors with their ephrin ligands lead to cellular recognition and subsequent cell repulsion. Therefore, it is possible that Hox13 genes regulate cell adhesiveness and migration by controlling the expression of Eph/ephrin proteins in the limb bud.
Another homeobox gene, Meis, is also involved in adhesiveness of the limb mesenchyme. In embryos of various species, both Meis1 and 2 are expressed in the proximal region of the limb bud (Mercader et al.,1999). The expression of Meis localizes to the presumptive stylopod-forming region, indicating a role for Meis in PD axis formation during limb development. Misexpression of Meis in the distal region of the limb bud proximalizes the positional identity of the limb mesenchyme. Expression of HoxA11 in the limb bud is expanded distally, whereas expression of HoxA13 is shortened along the PD axis (Mercader et al.,2000). In addition, the position of the zeugopod is shifted distally, and autopod formation is affected (Mercader et al.,1999,2009), indicating that a distal to proximal transformation occurs in the Meis-misexpressed limb bud. When Meis is misexpressed, the cellular adhesiveness of the limb mesenchyme is also proximalized (Mercader et al.,2000), suggesting that one of the roles of Meis in limb development is to specify the proximal identity of the limb mesenchyme by modulating cell adhesiveness.
Expression and function of Meis genes and their products are tightly connected with retinoic acid (RA) signaling. Recent reports have shown that RA plays a role in PD patterning. High levels of RA signaling determine proximal identity and contribute to the formation of proximal structures by activating Meis1/2, which in turn proximalize cellular identity (Mercader et al.,2000). It has been shown that CYP26 is involved in RA-mediated signaling (Yashiro et al.,2004). In addition, RA signaling affects cell adhesiveness, suggesting its involvement in the distinct adhesiveness of the limb mesenchyme. Pretreatment of the limb mesenchyme with RA interferes with segregation of cells along the PD axis (Tamura et al.,1997). This, in turn, also indicates the apparent relationship between cellular positional identity and cell adhesiveness.
Cell surface molecules.
Cell adhesiveness is primarily regulated by cell surface molecules. It is possible that the transcription factors discussed above regulate the expression of cell recognition and/or cell adhesion molecules. Ephrin–Eph interactions and cadherins are candidates for the regulation of position-specific cell adhesiveness.
Ephrin are the cell surface ligands of the Eph tyrosine kinase-type cell surface receptors. Ephrins are classified into 2 classes: type A and B. Type A ephrins are produced as a glycosylphosphatidylinositol (GPI)-anchored cell surface protein and bind preferentially to EphA receptors, while type B ephrins are membrane-spanning proteins that bind to EphB receptors, with some exceptions (Holder and Klein,1999; Wilkinson,2001). Eph–ephrin interactions induce repulsive movement between neighboring cells by altering intracellular signaling (Wilkinson,2001; Pasquale,2005). These interactions regulate cell migration and distribution in various developmental systems, such as neural crest migration, retinotectal projection, and rhombomere formation, to settle cells into the proper position in the developmental field of the systems (Kullander and Klein,2002).
Ephrin-A2 and ephrin-A5 proteins are predominantly distributed in the proximal region of the chick limb bud (Wada et al.,2003). Ephrin-A2 is strongly distributed through the presumptive stylopod and zeugpod, but weakly distributed through the autopod region, where one of the ephrin-A2 receptors, EphA4, is highly expressed (Ohta et al.,1996; Patel et al.,1996). Misexpression of ephrin-A2 in the distal region of the limb bud alters cell adhesiveness, and ephrin-A2-misexpressed cells are sorted out from normal cells in vitro. In addition, ectopic cell sorting is also observed in vivo, and induces skeletal malformations of the limb such as partial duplication or fusion of digits (Wada et al.,2003). By contrast, misexpression of ephrin-A2 has no effect on skeletogenesis in the proximal region. Therefore, it is possible that different amounts of ephrin-A2 along the PD axis influence skeletal morphogenesis in the limb bud by affecting cell adhesiveness.
In addition to EphA4, other EphA receptors, such as EphA3 and EphA7, are also expressed in the distal region of mouse limb bud (Stadler et al.,2001; Cobb and Duboule,2005; Kawakami et al.,2009). EphA7 is expressed in the distal region of the limb bud, and restricted to presumptive digits. In vitro inhibition of EphA7 function by blocking antibodies causes incomplete cartilaginous nodule formation, suggesting the involvement of EphA7 in the mesenchymal condensation that precedes cartilage differentiation. The expression of EphA7 is decreased in Hoxa13-knockout limbs (Stadler et al.,2001), and is regulated by both Hoxa13 and Hoxd13 (Salsi and Zappavigna,2006). Thus, EphA7 acts as a downstream effecter of Hox13 genes to regulate cell migration and cartilage morphogenesis during limb development.
Treatment with PI-PLC, an enzyme that removes GPI-anchor type cell surface protein from the cell membrane, interferes with sorting of cells from different PD positions, suggesting the involvement of GPI-anchored protein in distinct cell adhesiveness along the PD axis of the limb bud (Wada et al.,1998). Since ephrin-A2 is a GPI-anchored protein, GPI-anchored protein-dependent cell sorting may be partially mediated by ephrin-A2. In addition, position-dependent tissue engulfment of the regenerating blastema is also inhibited by PI-PLC treatment (see next section) (da Silva et al.,2002), suggesting that GPI-anchored proteins are generally involved in position-dependent cell–cell recognition during limb morphogenesis.
On the other hand, despite the involvement of GPI-anchored protein in cell adhesiveness, the role of GPI-protein-mediated adhesiveness in vivo remains unclear. The limbs in mice lacking PIGA, which encodes PIGA, an enzyme that is involved in the biosynthesis of the GPI-anchor, show severe chondrodysplasia phenotype, and delayed osteogenesis (Ahrens et al.,2009). Despite these skeletal defects, the skeletal pattern in the limb is barely affected, and a rough outline of skeletal pattern appears to be established without a GPI-anchored protein. Since GPI-anchored protein affects cell adhesiveness as shown above, it is possible that GPI-anchored protein-mediated adhesiveness may play a role other than initial skeletal patterning. Another possible role for GPI-anchored proteins in limb development is the formation of properly shaped cartilaginous nodules. The nodule formation of limb mesenchyme in vitro is disrupted by misexpression of ephrin-A2 (Wada et al.,2003). In this case, the expression of chondrogenic markers is maintained, suggesting the disruption of nodule formation was not a result of inhibition of chondrogenesis itself. In addition, inhibition of EphA7 function in vitro also disrupts nodule formation (Stadler et al.,2001), suggesting that interaction of EphA7 with GPI-ligand(s) regulates nodule formation. Moreover, treatment of limb mesenchyme with PI-PLC in vitro also disrupts nodule formation (Fig. 4). Therefore, GPI-anchored proteins expressed in the limb mesenchyme may regulate the proper cell–cell interaction, which is necessary for cartilaginous nodule formation. Disrupted morphogenesis of the limb cartilage observed in PIGA-deficient mice (Ahrens et al.,2009) may be due to interference of nodule formation.
Figure 4. Nodule formation in vitro is affected by PI-PLC treatment. Mesenchymal cells from the distal half of stage-24 limb buds were prepared, seeded, and incubated for 3 days. A: The control culture forms round cellular aggregates, the cartilaginous nodules (arrowheads). B: Addition of PI-PLC to the culture medium inhibits cartilaginous nodule formation, and the morphology of the cells appears uniform.
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Class-B ephrins are also expressed in the limb bud (Flenniken et al.,1996; Baker et al.,2001). The somatic cells from ephrin-B1 haploinsufficient female mice are composed of ephrin-B1-positive or ephrin-B1-negative cells after random inactivation of the X chromosome, because ephrin-B1 is mapped to the X-chromosome (Compagni et al.,2003; Davy et al.,2004). Segregation between ephrin-B1-positive and ephrin-B1-negative cells occurs in the limb bud, and skeletal morphogenesis of the limb bud is affected. This result suggests that class-B ephrins are also involved in cellular adhesiveness of the limb mesenchyme.
The involvement of the cadherin family of cell adhesion molecules in position-specific cell adhesiveness has also been suggested. Several cadherins are expressed in the limb bud in a region-specific manner (Kimura et al.,1995; Kitajima et al.,1999; Yajima et al.,1999,2002). The distribution of N-cadherin reflects the positional identity of the limb mesenchyme along the PD axis of the limb bud (Yajima et al.,1999,2002). N-cadherin is weakly distributed at the early stage of limb development. At later stages, its expression is still weak in the proximal region of the limb bud, but becomes strong in the distal region, and forms a gradient along the PD axis of the limb bud (Yajima et al.,1999,2002), suggesting that the amount of N-cadherin protein regulates position-specific adhesiveness. Position-dependent sorting of cells of the limb mesenchyme is affected by misexpression of N-cadherin or inhibition of N-cadherin protein, implying the involvement of N-cadherin in position-specific cell adhesiveness (Yajima et al.,1999,2002).
In addition to N-cadherin, other cadherins, such as cadherin-11 (Kimura et al.,1995) and PB-cadherin (Kitajima et al.,1999) are also expressed in the distal region of the limb bud, indicating that the total amount of cadherin proteins in the cells of the distal region is higher than that of the proximal region. This distribution possibly affects position-dependent cellular adhesiveness and might explain stage-dependent aggregate formation in rotation culture (Fig. 1). Cadherin-11 is involved in cell sorting of the limb mesenchyme in vitro (Kimura et al.,1995), and cadherin-dependent cell adhesion regulates chondrogenesis of the limb mesenchyme (Kim et al.,2009). The polarized distribution of cadherin proteins in the limb bud may affect position-dependent chondrogenesis and cartilage morphogenesis thorough modification of cell adhesiveness.