Search

Background Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Results Here we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Conclusion Our method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.

The cell differentiation of the musculoskeletal system is highly coordinated during limb development. In the distal-most region of the limb, WNT and FGF released from the apical ectodermal ridge maintain mesenchymal cells in the undifferentiated stage. Once the cells stop receiving WNT and FGF, they respond to differentiation signals. Particularly during tendon development, mesenchymal cells enter the cell differentiation program once Scleraxis (Scx) gene expression occurs. Among the signals that trigger the cell differentiation programs, TGFβ signaling has been found to be closely involved in tendon differentiation. However, whether Scx gene expression depends merely on TGFβ signaling or other signals is still not fully understood. In the present study, considering that WNT/β-catenin is an inhibitory signal of cell differentiation, we speculated possible antagonistic or additive effects between canonical Wnt/β-catenin and TGFβ/SMAD signaling pathways to control Scx gene expression. We found that the blockade of WNT/β-catenin promoted Scx gene expression. In contrast, the inhibition of TGFβ/SMAD signaling did not maintain Scx gene expression. Interestingly, the blockade of both WNT/β-catenin and TGFβ/SMAD signaling at the same time promoted Scx gene expression. Thus the inhibition of WNT/β-catenin signaling appears to be necessary and sufficient to induce Scx gene expression.

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

El desenvolupament dels membres s'inicia a partir de la formació d'unes estructures mesodèrmiques envoltades per ectoderma en les quals apareixen unes estructures, com ara la cresta ectodèrmica apical, la zona de progrés, la zona d'activitat polaritzant, ... Aquestes estructures actuen com a centres senyalitzadors per la formació dels tres eixos espacials principals: El desarrollo de los miembros se inicia a partir de la formación de unas estructuras mesodérmicas rodeadas por ectodermo en las que aparecen unas estructuras, como la cresta ectodérmica apical, la zona de progreso, la zona de actividad polarizante, ... Estas estructuras actúan como centros señalizadores por la formación de los tres ejes espaciales principales: The development of the limbs begins with the formation of mesodermal structures surrounded by ectoderm in which structures appear, such as the apical ectodermal ridge, the zone of progress, the zone of polarizing activity, ... These structures act as signaling centers for the formation of the three main spatial axes