LMX1B and CCN4: Brains, limbs, and more in the vertebrate embryo

The first portion of my dissertation focused on the role of Lmx1b in the development of the midbrain-hindbrain region in vertebrate embryos. Cells in the caudal mesencephalon and rostral metencephalon become organized by signals emanating from the isthmus organizer (ISO). Lmx1b is expressed and functions in a distinct region of the IsO. Lmx1b expression is maintained by Fgf8. Lmx1b, in turn, maintains the expression of the secreted factor Wnt1. Ectopic Lmx1b was insufficient to alter the expression of a number of other genes expressed at the IsO, suggesting that it does not generate a new signaling center. Instead, if Lmx1b/RCAS-infected brains develop longer, changes in mesencephalic morphology are detected. Since both endogenous and ectopic Lmx1b expression occurs in regions of the isthmus undergoing morphological changes, it could play a role in this process. Finally, since a similar phenotype is not observed in Wnt1/ RCAS-infected brains, ectopic Wnt1 alone is insufficient to mediate the effect of Lmx1b/RCAS infection. Since Wnt1 function has been linked to the proper segregation of mesencephalic and metencephalic cells, we suggest that Lmx1b and Wnt1 normally function in concert to affect IsO morphogenesis. The second portion of my dissertation focused on the role of Ccn4 in early limb development. Ccn4 is likely to play roles both in the early stages of pattern formation and later stages of bone morphogenesis. Concomitant with limb out growth, Ccn4 is first expressed in the ventral ectoderm and then in the apical ectodermal ridge (AER) from its inception. Later, Ccn4 mRNA is localized in the periostium and endosteum of the forming limb bones. To address its functions, we misexpressed Ccn4 throughout the limb. The affected limbs have changes in tibia and metatarsal bone development. A molecular analysis of these limbs demonstrates that Ccn4 can regulate chondrocyte maturation and mineralization. Finally Ccn4 expression can be regulated by Bmp and Wnt signaling. Our work demonstrates that Cnn4 plays at least two roles during limb formation and is regulated by at least two signaling pathways