טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKeren Aviad
SubjectThe p38 MAPK/CREB Pathway Regulates Mesoderm Patterning
during Early Xenopus laevis Development
DepartmentDepartment of Medicine
Supervisor Professor Eyal Bengal
Full Thesis textFull thesis text - English Version


Abstract

The Spemann’s organizer is a dorsal signaling center patterning neighboring mesoderm tissues during Xenopus gastrulation.  We find that p38 MAPK and CREB are part of an organizer signaling pathway functioning in mesoderm dorsal-ventral patterning. Our results indicate that the phosphorylated active form of CREB is localized in the dorsal marginal zone. Antisense- morpholino-mediated knockdown of CREB prevents typical mesoderm patterning activity of the Spemann organizer. Different criteria indicate the involvement of the p38 MAPK pathway upstream of CREB. Expression of activated MKK6 (MKK6E) leads to the phosphorylation of CREB and mesoderm dorsalization. Moreover, ventral mesoderm explants expressing MKK6E or CREB-VP16, function non-autonomously to dorsalize cells of adjacent ventral mesoderm explants. Knockdown of CREB prevents the patterning activity of MKK6. Interestingly, knockdown of either CREB or p38 MAPK enables the expansion of the ventral homeobox gene Vent1 into the dorsal marginal region, preventing XMyf5 expression. Simultaneous knockdown of CREB and Vents rescued dorsal lateral Myf5 expression. Therefore, the antagonizing activities of p38-CREB in the dorsal domain and Vents in the ventral domain are necessary for the fine-tuned patterning of mesoderm.

Our results indicate that knockdown of p38 MAPK affected the organizer activity. Still, inhibition of p38 MAPK was not catastrophic to early development; embryos developed past gastrulation and later somites were formed. Therefore we investigated later aspects of p38 MAPK affecting myogenesis. The p38 MAPK signaling pathway is essential for skeletal muscle differentiation in tissue culture models. We demonstrate a novel role for p38 MAPK in myogenesis during early Xenopus laevis development. Interfering with p38 MAPK causes distinct defects in myogenesis. The initial expression of XMyf5 was selectively blocked, while expression of XMyoD is unaffected. Convergent extension movements are prevented and segmentation of the paraxial mesoderm is delayed, probably due to the failure of cells to withdraw from the cell cycle. Myotubes are properly formed, however, at later stages, they begin to degenerate, and the boundaries between somites disappear. The ventral body wall muscle derived from migratory progenitor cells of the ventral somite region is poorly formed. Our data indicate that the developmental defects caused by p38α-knockdown were mediated by the loss of XMyf5 expression. Thus, this study identifies a specific intracellular pathway in which p38 MAPK and Myf5 proteins regulate a distinct myogenic program. Overall, the p38 MAPK-CREB pathway is involved in early aspects of mesoderm patterning and in later effects on muscle specification through the expression of Myf5.