Sonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. Here, we show that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, we show that the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein.

The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.

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Regulatory factor X (Rfx) homologs regulate the transcription of genes necessary for ciliogenesis in invertebrates and vertebrates. Primary cilia are necessary for Hedgehog signaling and regulation of the activity of the transcriptional regulators known as Gli proteins, which are targets of Hedgehog signaling. Here, we describe an Rfx4(L298P) mouse mutant with distinct dorsoventral patterning defects in the ventral spinal cord and telencephalon due to aberrant Sonic hedgehog (Shh) signaling and Gli3 activity. We find that Ift172, which encodes an intraflagellar transport protein necessary for ciliogenesis, is a direct transcriptional target of Rfx4, and the decrease in its expression in the developing telencephalon and spinal cord of Rfx4(L298P) mutants correlates with defects in patterning and cilia formation. Our data indicate that Rfx4 is a regionally specific transcriptional regulator of ciliogenesis and thus is also a regionally specific modulator of Shh signaling during development of the central nervous system.

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