PROJECT SUMMARY
The pharyngeal pouches develop into the thymus, thyroid and parathyroid glands and contribute to formation
of the ear and tonsils. They also provide signals that are essential to the morphogenesis of the craniofacial
skeleton. In this latter role, the specific morphology and position of the pouches relative to the mesenchyme of
the pharyngeal arches is critical to instruct skeletal formation. Despite these essential roles for proper
development of the vertebrate head, pharyngeal pouch development in mammals remains poorly understood.
Pharyngeal pouch formation consists of two separate morphogenetic processes, lateral out-pocketing and
proximal-distal extension. The work outlined in this proposal focuses on understanding the role of Fgf8 in the
latter process. Using an allelic series of Fgf8 mutant mice, embryos of different Fgf8 dosages can be
generated, including both a mild and severe mutant. In contrast to zebrafish, in both mouse Fgf8 mutant
genotypes, the first pharyngeal pouch (pp1) out-pockets to contact the ectoderm but fails to extend along the
proximal-distal axis. In mild mutants, pp1 is reduced in size, but does extend. In the severe mutant, pp1 does
not extend proximo-distally and the ectodermal cleft is also hypoplastic and disorganized, contributing to failure
of the first and second arches to separate distally. Severe mutants have small, round pouches in which cells
appear to stack upon each other. Together, these data suggest that Fgf8 has additional roles in pharyngeal
pouch formation beyond directing lateral out-pocketing, which may include proximal-distal extension of both the
pharyngeal endoderm and ectoderm. Previous research has suggested that Fgf8 regulates cell polarity, and
that polarity of the actin cytoskeleton is essential to pharyngeal pouch extension. The specific hypothesis to
be tested by the proposed research is that Fgf8 has a paracrine function regulating proliferation and polarity of
pharyngeal pouch epithelial cells. This hypothesis will be tested through two specific aims. In Specific Aim 1,
proliferation and polarity in pouch epithelial cells will be quantified in embryos in which Fgf8 has been reduced
globally. In Specific Aim 2, pouch shape will be evaluated in embryos in which Fgf8 is specifically ablated in the
pharyngeal mesoderm or ectoderm. The future research goals of this work are to investigate genetic and
developmental interactions underlying variation in craniofacial morphogenesis, particularly variation in the
severity and penetrance of craniofacial disorders. The Fgf8 allelic series exhibits a large range of
morphological variation, including the bilateral variation present in both mutant genotypes. Both Fgf8 mutant
genotypes exhibit directional asymmetry of the jaw, exemplified by Fgf8Neo/Neo mice in which unilateral fusion of
the jaw on the left side only is observed in 33% of neonates. Although it has yet to be shown directly,
directional asymmetry is likely due bilateral asymmetry in Fgf8 expression in the cranial mesoderm as a
consequence of heart development. Facial asymmetry and heart defects are associated in several syndromes,
notably CHARGE and DiGeorge (22q11 deletion) syndrome, which have similar phenotypes to Fgf8 mutants.