Lineage-specific responses to reduced embryonic Pax3 expression levels

AbstractPax3 is an essential paired- and homeodomain-containing transcription factor that is necessary for closure of the neural tube, and morphogenesis of the migratory neural crest and myoblast lineages. Homozygous loss-of-function mutation results in mid-gestational lethality with defects in myogenesis, neural tube closure and neural crest-derived lineages including melanocytes, Schwann cells and insufficient mesenchymal cells to septate the cardiac outflow tract. To address the function of Pax3 in later fetal stages and in specific adult tissues, we generated a floxed Pax3 allele (Pax3flox). An intermediate allele (Pax3neo) was produced via creation of the floxed allele, in which the TK-neo(R) cassette is present between exons 5 and 6. It was deduced to be a hypomorph, as Pax3 protein expression is reduced by 80% and homozygote hypomorphs die postnatally. To assess the consequences of reduced Pax3 levels on the various Pax3-expressing lineages and to determine the underlying cause of lethality, we examined Pax3 spatiotemporal expression and the resultant defects. Defective limb and tongue musculature were observed and lethality was due to an inability to suckle. However, the heart, diaphragm, trunk musculature, as well as the various neural crest-derived lineages and neural tube were all unaffected by reduced Pax3 levels. Significantly, elevated levels of the related Pax7 protein were present in unaffected neural tube and epaxial somatic component. The limb and tongue myogenic defects were found to be due to a significant increase in apoptosis within the somites that leads to a paucity of migratory hypaxial myoblasts. These effects were attributed to the hypomorphic effect of the Pax3neo allele, as removal of the TK-neo(R) cassette completely relieves the hypomorphic effect, as 100% of the Pax3flox/flox mice were normal. These data demonstrate a lineage-specific response to ∼80% loss of Pax3 protein expression, with myogenesis of limb and tongue being most sensitive to reduced Pax3 levels. Thus, we have established that there are different minimum threshold requirements for Pax3 within different Pax3-expressing lineages