Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and-independent mechanisms

The axial midline mesoderm and the ventral midline of the neural tube, the floor plate, share the property of being a source of the secreted protein, Sonic hedgehog (Shh), which has the capacity to induce a variety of ventral cell types along the length of the mouse CNS, To gain insight into the mec hanisms by which Shh transcription is initiated in these tissues, we set ou t to identify the cis-acting sequences regulating Shh gene expression. As a n approach, we have tested genomic clones encompassing 35 kb of the Shh loc us for their ability to direct a lacZ reporter gene to the temporally and s patially restricted confines of the Shh expression domains in transgenic mi ce. Three enhancers were identified that directed lacZ expression to distin ct regions along the anteroposterior axis including the ventral midline of the spinal cord, hindbrain, rostral midbrain and caudal diencephalon, sugge sting that multiple transcriptional regulators are required to initiate Shh gene expression within the CNS, In addition, regulatory sequences expressi on identified notochord, that directed reporter expression to the notochord , albeit, under limited circumstances. Sequence analysis of the genomic clo nes responsible for enhancer activity from a variety of organisms, includin g mouse, chicken and human, have identified highly conserved binding sites for the hepatocyte nuclear factor 3 (Hnf3) family of transcriptional regula tors in some, but not all, of the enhancers, Moreover, the generation of mu tations in the Hnf3-binding sites showed their requirement in certain, but not all, aspects of Shh reporter expression. Taken together, our results su pport the existence of Hnf3-dependent and -independent mechanisms in the di rect activation of Shh transcription within the CNS and axial mesoderm.