Interaction between axons and specific populations of surrounding cells is indispensable for collateral formation in the mammillary system.

Background: An essential phenomenon during brain development is the extension of long collateral branches by axons. How the local cellular environment contributes to the initial sprouting of these branches in specific points of an axonal shaft remains unclear.

Methodology/principal Findings: The principal mammillary tract (pm) is a landmark axonal bundle connecting ventral diencephalon to brainstem (through the mammillotegmental tract, mtg). Late in development, the axons of the principal mammillary tract sprout collateral branches at a very specific point forming a large bundle whose target is the thalamus. Inspection of this model showed a number of distinct, identified cell populations originated in the dorsal and the ventral diencephalon and migrating during development to arrange themselves into several discrete groups around the branching point. Further analysis of this system in several mouse lines carrying mutant alleles of genes expressed in defined subpopulations (including Pax6, Foxb1, Lrp6 and Gbx2) together with the use of an unambiguous genetic marker of mammillary axons revealed: 1) a specific group of Pax6-expressing cells in close apposition with the prospective branching point is indispensable to elicit axonal branching in this system; and 2) cooperation of transcription factors Foxb1 and Pax6 to differentially regulate navigation and fasciculation of distinct branches of the principal mammillary tract.

Conclusions/significance: Our results define for the first time a model system where interaction of the axonal shaft with a specific group of surrounding cells is essential to promote branching. Additionally, we provide insight on the cooperative transcriptional regulation necessary to promote and organize an intricate axonal tree.