Gap junctions are comprised of membrane channels that mediate the diffusion of ions and small molecules such as calcium, cAMP and other second messengers. Given these interesting membrane permeability properties, it has been suggested that gap that play a role in cell signaling. Consistent with gap playing a role in development is the finding that in mouse embryos, gap junctional communication (gjc) and expression of gap junction genes are modulated in a developmentally significant manner. Thus, in the limb bud and central nervous system (CNS), expression of the gap junction gene Cx43 is spatially restricted in a pattern which is coincident with or complementary to those of wnt genes-a family of growth factors that mediate cell signaling in development. Particularly striking is a band of Cx43 and wnt-expression spanning the midbrain/hindbrain junction. That wnt-1 plays an important role in mouse development is indicated by the finding of midbrain/hindbrain ectopically expressing wnt-1 in the limb. Our hypothesis is that gap junctions may mediate the transmission of second messengers derived from signal transduction cascades triggered by wnt-1, and in this manner, play an integral role in wnt-1 mediated events in mammalian development.
In Aim 1, we will determine if Cx43 expression and function may be subject to regulation via wnt-1 signaling. Thus, we will examine if the absence or ectopic expression of wnt-1 may perturb gjc or Cx43 expression.
In Aim 2, we will examine the developmental effects of inhibiting Cx43 function in the wnt-1 cell signaling, then the loss of Cx43 function may result in developmental defects similar to those found in wnt-1 null mutants.
In Aim 3, we will use transgenic approaches to determine if the assembly and gating of Cx43 encoded gap junctions may be subjects to regulation by signal transduction pathways triggered by wnt-1. We will focus these analyses on Cx43 constructs in which mutations have been introduced into the cytoplasmic tail, a region of the protein which undergoes phosphorylation.
In Aim 4, we will generate lacZ reporter constructs driven by genomic DNA from the Cx43 gene, and determine if there are regulatory elements that may respond to wnt-1 expression. In addition, using luciferase reporter constructs, we will search for retinoic acid response elements (RARE), as retinoic acid is known to affect gjc, Cx43 expression, as well as patterning in the limb and neural tube.
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