GROSS ANALYSIS OF CRANIOFACIAL PHENOTYPES

As a first step in the evaluation of morphological phenotypes, we generally carry out
alizarin red S and Alcian blue stains of whole skulls (11). Nontransgenic littermates are
used as controls. This technique can be easily used in conjunction with animal necropsy
to identify changes in the whole animal. For example, we knew that Msx2 was
expressed in abundant amounts in a wide variety of tissues outside the skull. Consequently,
overexpressing the Msx2 protein under the control of either the CMV promoter
or the Msx promoter might alter the developmental fate of noncalvarial tissues in
transgenic animals. To identify such changes, we undertook complete necropsies on
animals from each transgenic line from several selected stages of development. These
necropsy studies of visceral organs could be easily combined with other approaches,
such as preserving the bodies for alizarin red staining of the skeleton. In this manner,
alterations in the visceral organs or the skeletal body plan could be identified and an
approach developed to explore the role of the expression of the transgene in the development
of the affected tissue.