| Neural patterning within the mammalian spinal cord and brain relies upon signaling cues from surrounding tissues, and the mouse neural tube serves as a good model to uncover genes involved in neural patterning. Through analysis of recessive mutations causing developmental and neural tube dorso-ventral patterning defects, novel Sonic hedgehog (Shh) signaling components can be identified and characterized based on changes in Shh-dependent cell identities. This work characterizes Tubby-like Protein 3 (Tulp3 ) as a novel negative regulator of Shh signaling in the mouse embryo. Tulp3 is required for proper embryonic development in mice; genetic disruption of mouse Tulp3 generates morphological defects in the embryonic craniofacial regions, spinal cord, and limbs. In Tulp3 mutants, ventral cell types in the neural tube are ectopically specified at the expense of dorsal cell types. These ventral cell types acquire their identity primarily from Shh signaling, suggesting that Tulp3 mutants de-repress Shh signaling.;From genetic epistasis experiments, we show that the Tulp3 ventralized phenotype occurs independently of Shh, the transmembrane protein Smoothened, and transcription factor Gli3. Tulp3 is, however, dependent upon the transcription factor Gli2 and kinesin II subunit Kif3A, which regulates intraflagellar transport and formation of the primary cilium. Tulp3 also localizes to the cilium, a site where other Shh components Smoothened, Gli transcription factors, and Suppressor of Fused are found. Although trafficking or processing events occurring in the cilium are not well understood, biochemical data from other organisms suggest that members of the Tubby family of proteins function in vesicular trafficking and/or signaling mediated through the cilium. Importantly, Tulp3 mutants do assemble primary cilia of normal length, but demonstrate loss of another Shh regulator Arl13b in neural tube cilia, indicating that Tulp3 may regulate trafficking of Arl13b. Further genetic epistasis suggests that in addition to Arl13b, Tulp3 may regulate other unknown components for ventral neural patterning. This work lastly identifies a Tulp3 protein interaction with CaMKII, a kinase involved in Ca2+-dependent responses, as one of these potential components. Through these genetic and biochemical analyses of Tulp3 function, we have gained further insight to the complexities and regulation unique to mammalian Shh signal transduction. |
