A simple in vitro culture system for tracheal cartilage development.

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal­lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.

Regulation of Sox9 by Sonic Hedgehog (Shh) is essential for patterning and formation of tracheal cartilage.

We report that Sonic Hedgehog (Shh) regulates both formation and patterning of tracheal cartilage by controlling the expression pattern and level of the chondrogenic gene, Sox9. In Shh(-/-) tracheo-esophageal tubes, Sox9 expression is transient and not restricted ventrally to the site of chondrogenesis, and is absent at the time of chondrogenesis, resulting in the failure of tracheal cartilage formation. Inhibition of Hedgehog signalling with cyclopamine in tracheal cultures prevents tracheal cartilage formation, while treatment of Shh(-/-) tracheal explant with exogenous Shh peptide rescues cartilage formation. Both exogenous Bmp4 and Noggin rescue cartilage phenotype in Shh(-/-) tracheal culture, while promoting excessive cartilage development in wild-type trachea through induction of Sox9 expression. The ventral and segmented expression of Sox9 in tracheal primordia under Shh modulated by Bmp4 and Noggin thus determine where and when tracheal cartilage develops. These results indicate that Shh signalling is a critical determinant in tracheal cartilage development.

The role of Shh transcription activator Gli2 in chick cloacal development.

Patterning and differentiation along the dorsal-ventral (D-V) axis lead to cloacal partitioning into ventral urinary and dorsal alimentary tracts in most mammals, but not birds and fish. We previously reported that the major activator of Sonic hedgehog (Shh) signaling transcription factor Gli2 plays an essential role in cloacal partitioning along the D-V axis in a mouse model. Here, we report that chick cloacal patterning and differentiation is along the anterior-posterior axis. During chick cloacal formation, Shh is expressed strongly in hindgut endoderm; Gli2 is very weakly detected in the surrounding hindgut mesoderm. In the mesoderm of the cloacal region, the over-expression of the constitutively active form of mouse Gli2 has been shown to: not induce cloacal partitioning along the D-V axis; induce expression of Ptch1, Gli2, bmp4, wnt5a, and hoxd-13, which have been previously shown to play a role in hindgut patterning; increase cell proliferation; and reduce apoptosis. Interestingly, p63 expression in the cloacal endoderm is also up-regulated, suggesting an interaction between the Shh and p63 pathways. In conclusion, Gli2 alone is insufficient to induce partitioning along the D-V axis in the chick embryo. However, Gli2 regulates both epithelial and mesenchymal cell proliferation and apoptosis during cloacal development.

DeltaNp63 plays an anti-apoptotic role in ventral bladder development.

The bladder, the largest smooth-muscle organ in the human body, is responsible for urine storage and micturition. P63, a homolog of the p53 tumor-suppressor gene, is essential for the development of all stratified epithelia, including the bladder urothelium. The N-terminal truncated isoform of p63, DeltaNp63, is known to have anti-apoptotic characteristics. We have established that DeltaNp63 is not only the predominant isoform expressed throughout the bladder, but is also preferentially expressed in the ventral bladder urothelium during early development. We observed a host of ventral defects in p63-/- embryos, including the absence of the abdominal and ventral bladder walls. This number of ventral defects is identical to bladder exstrophy, a congenital anomaly exhibited in human neonates. In the absence of p63, the ventral urothelium was neither committed nor differentiated, whereas the dorsal urothelium was both committed and differentiated. Furthermore, in p63-/- bladders, apoptosis in the ventral urothelium was significantly increased. This was accompanied by the upregulation of mitochondrial apoptotic mediators Bax and Apaf1, and concurrent upregulation of p53. Overexpression of DeltaNp63gamma and DeltaNp63beta in p63-/- bladder primary cell cultures resulted in a rescue, evidenced by significantly reduced expressions of Bax and Apaf1. We conclude that DeltaNp63 plays a crucial anti-apoptotic role in normal bladder development.