Differential Cell Affinity and Sorting of Anterior and Posterior Cells during Outgrowth of Recombinant Avian Limb Buds

Abstract

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.

Limb regeneration in higher vertebrates: Developing a roadmap

“…FGF4 and FGF8 are known to be mitogenic for limb bud cells (Niswander and Martin, 1993;Kawakami et al, 2003), in addition to their role as cell survival factors (Dudley et al, 2002;Sun et al, 2002). FGF4 produced by the AER acts as a chemoattractant for limb-and digit-forming cells (Li and Muneoka, 1999;NgoMuller and Muneoka, 2000), and the data suggest that organized cell migration is critical for limb bud morphogenesis (Omi et al, 2002;NgoMuller et al, 2004). While removal of the AER causes limb truncation, the presence of an ectopic AER redirects outgrowth and stimulates the formation of additional limb structures.…”

Digit regeneration is regulated byMsx1andBMP4in fetal mice

“…For in situ hybridization, digoxigenin-labeled riboprobes complementary to Msx1 (Ngo- Muller and Muneoka, 2000b), Msx2 (Ngo- Muller and Muneoka, 2000b), Bmp4 (Jones et al, 1991), Hoxc13 (Godwin and Capecchi, 1998) and Ihh (St-Jacques et al, 1999) were used in whole-mount or paraffin-sectioned preparations as described (Schaller and Muneoka, 2001;Omi et al, 2002). Fetal digit tissues were fixed by immersion in 4% paraformaldehyde and post-natal digits were fixed by injection of fixative into the digits followed by immersion in 4% paraformaldehyde.…”

Budding behaviors: Growth of the limb as a model of morphogenesis

“…There is also a substantial body of evidence that region-specific adhesion differences exist between limb bud cells (Ide et al, 1998;Barna and Niswander, 2007). These differences cause cell sorting and provide a potential mechanism for maintaining positional identity along the anteroposterior (Omi et al, 2002) and the proximodistal axes (Ide et al, 1994;Tamura et al, 1997). The cell adhesion hypothesis links outgrowth with pattern formation (Yokouchi et al, 1995;Omi et al, 2002), however a concrete hypothesis of how the spatial distribution of differential adhesion could itself lead to mechanical elongation of the limb bud has not been proposed.…”

“…These differences cause cell sorting and provide a potential mechanism for maintaining positional identity along the anteroposterior (Omi et al, 2002) and the proximodistal axes (Ide et al, 1994;Tamura et al, 1997). The cell adhesion hypothesis links outgrowth with pattern formation (Yokouchi et al, 1995;Omi et al, 2002), however a concrete hypothesis of how the spatial distribution of differential adhesion could itself lead to mechanical elongation of the limb bud has not been proposed. In particular, tissues driven by strong cell adhesion effects tend to display behaviors dictated by their surface tension, which results in increasingly rounded or spherical shapes over time, while by contrast the distal elongation of the limb bud is accompanied by a flat dorsoventral axis which appears to argue against a possible cell-adhesion driven elongation effect.…”