Chengbing Wang

Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors

Gli2 and Gli3 are primary transcriptional regulators that mediate hedgehog (Hh) signaling. Mechanisms that stabilize and destabilize Gli2 and Gli3 are essential for the proteins to promptly respond to Hh signaling or to be inactivated following the activation. In this study, we show that loss of suppressor of fused (Sufu; an inhibitory effector for Gli proteins) results in destabilization of Gli2 and Gli3 full-length activators but not of their C-terminally processed repressors, whereas overexpression of Sufu stabilizes them. By contrast, RNAi knockdown of Spop (a substrate-binding adaptor for the cullin3-based ubiquitin E3 ligase) in Sufu mutant mouse embryonic fibroblasts (MEFs) can restore the levels of Gli2 and Gli3 full-length proteins, but not those of their repressors, whereas introducing Sufu into the MEFs stabilizes Gli2 and Gli3 full-length proteins and rescues Gli3 processing. Consistent with these findings, forced Spop expression promotes Gli2 and Gli3 degradation and Gli3 processing. The functions of Sufu and Spop oppose each other through their competitive binding to the N- and C-terminal regions of Gli3 or the C-terminal region of Gli2. More importantly, the Gli3 repressor expressed by a Gli3 mutant allele (Gli3Δ699) can mostly rescue the ventralized neural tube phenotypes of Sufu mutant embryos, indicating that the Gli3 repressor can function independently of Sufu. Our study provides a new insight into the regulation of Gli2 and Gli3 stability and processing by Sufu and Spop, and reveals the unexpected Sufu-independent Gli3 repressor function.

Phosphorylation of Gli2 by protein kinase A is required for Gli2 processing and degradation and the Sonic Hedgehog-regulated mouse development.

In mice, Gli2 and Gli3 are the transcription factors that mediate the initial Hedgehog (Hh) signaling. In the absence of Hh signaling, the majority of the full-length Gli3 protein undergoes proteolytic processing into a repressor, while only a small fraction of the full-length Gli2 protein is processed. Gli3 processing is dependent on phosphorylation of the first four of the six protein kinase A (PKA) sites at its C-terminus. However, whether the same phosphorylation of Gli2 by PKA is required for Gli2 processing and, if so, whether such phosphorylation regulates additional Gli2 function are unknown. To address these questions, we mutated these PKA sites in the mouse Gli2 locus to create the Gli2(P1-4) allele. Gli2(P1-4) homozygous embryos die in utero and exhibit exencephaly, defects in neural tube closure, enlarged craniofacial structures, and an extra anterior digit. Analysis of spinal cord patterning shows that domains of motoneurons and V2, V1, and V0 interneurons are expanded to different degrees in both Gli2(P1-4) single and Gli2(P1-4);Shh double mutants. Furthermore, Gli2(P1-4) expression prevents massive cell death and promotes cell proliferation in Shh mutant. Analysis of Gli2(P1-4) protein in vivo reveals that the mutant protein is not processed and is twice as stable as wild type Gli2 protein. We also show that the Gli2 repressor can effectively antagonize Gli2P1-4 activity. Together, these results indicate that phosphorylation of Gli2 by PKA induces Gli2 processing and destabilization in vivo and plays an important role in the Hh-regulated mouse embryonic patterning.

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4.

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.

The decoupling of Smoothened from Gαi proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Galpha(i) proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Galpha(i) protein activity appears to significantly reduce Hh pathway activity in Ptc(-/-) mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Galpha(i) protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Galpha(i) protein, Galpha(i2)QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Galpha(i) activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.

A hypermorphic mouse Gli3 allele results in a polydactylous limb phenotype

Gli3 protein processing to generate the Gli3 repressor is mediated by proteasome and inhibited by Hedgehog signaling. The Gli3 repressor concentration is graded along the anterior–posterior axis of the developing vertebrate limb due to posteriorly restricted Sonic hedgehog expression. In this study, we created a small deletion at the Gli3 locus (Gli3Δ68), which causes a half reduction in the Gli3 repressor levels and a slightly increased activity of full‐length mutant protein in the limb. Mice homozygous for Gli3Δ68 develop one to two extra partial digits in the anterior of the limb, while mice carrying one copy of the Gli3Δ68 allele die soon after birth and display seven digits. These phenotypes are more severe than those found in mice lacking one wild‐type Gli3 allele. The expression of dHand, Hoxd12, and Hoxd13 is anteriorly expanded in the limb, even though no up‐regulation of Gli1 and Ptc RNA expression is detected. These findings suggest that a decrease in the Gli3 repressor level in combination with an increase in Gli3 full‐length activity results in more severe digit patterning abnormalities than those caused by a loss of one wild‐type Gli3 allele. Developmental Dynamics 236:769–776, 2007.

Centrosomal Protein DZIP1 Regulates Hedgehog Signaling by Promoting Cytoplasmic Retention of Transcription Factor GLI3 and Affecting Ciliogenesis

Background: All known cilia-related proteins regulate Hedgehog signaling through their role in ciliogenesis. Results: The centrosomal protein DZIP1 interacts with and sequesters GLI3 transcription factor in the cytoplasm and also regulates ciliogenesis. Conclusion: DZIP1 is the first known cilia-related protein that regulates Hedgehog signaling through a dual mechanism. Significance: Understanding how DZIP1 regulates Hedgehog signaling provides new insights into the molecular mechanism of Hedgehog signal transduction. The primary cilium is required for Hedgehog signaling. So far, all known ciliogenic proteins regulate Hedgehog signaling through their role in ciliogenesis. Here we show that the mouse DZIP1 regulates Hedgehog signaling through two mechanisms. First, DZIP1 interacts with GLI3, a transcriptional regulator for Hedgehog signaling, and prevents GLI3 from entering the nucleus. Second, DZIP1 is required for ciliogenesis. We show that DZIP1 colocalizes and interacts with CEP164, a protein localizing at appendages of the mother centrioles, and IFT88, a component of the intraflagellar transport (IFT) machinery. Functionally, both CEP164 and Ninein appendage proteins fail to localize to ciliary appendages in Dzip1 mutant cells; IFT components are not recruited to the basal body of cilia. Importantly, the accumulation of GLI3 in the nucleus is independent of loss of primary cilia in Dzip1 mutant cells. Therefore, DZIP1 is the first known ciliogenic protein that regulates Hedgehog signaling through a dual mechanism and that biochemically links IFT machinery with Hedgehog pathway components.

Mouse limbs expressing only the Gli3 repressor resemble those of Sonic hedgehog mutants.

Anterioposterior vertebrate limb patterning is controlled by opposing action between Sonic Hedgehog (Shh) and the Gli3 transcriptional repressor. Unexpectedly, Gli3(Δ699) mutant mice, which are thought to express only a Gli3 repressor and not the full-length activator, exhibit limb phenotypes inconsistent with those of Shh mutant mice. Therefore, it remains debatable whether Shh patterns the anterioposterior limb primarily by inhibiting generation of the Gli3 repressor. However, one caveat is that Gli3(Δ699) may not be as potent as the natural form of Gli3 repressor because of the nature of the mutant allele. In the present study, we created a conditional Gli3 mutant allele that exclusively expresses Gli3 repressor in the presence of Cre recombinase. Using this mutant, we show that the phenotypes of mouse limbs expressing only the Gli3 repressor exhibit no or single digit, resembling those of Shh mutant limbs. Consistent with the limb phenotypes, the expression of genes dependent on Shh signaling is also inhibited in both mutants. This inhibition by the Gli3 repressor is independent of Shh. Thus, our study clarifies the current controversy and provides important genetic evidence to support the hypothesis that Shh patterns the anterioposterior limb primarily through the inhibition of Gli3 repressor formation.

Talpid3-binding centrosomal protein Cep120 is required for centriole duplication and proliferation of cerebellar granule neuron progenitors.

Granule neuron progenitors (GNPs) are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh) secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3), another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis.

Increased proteolytic processing of full-length Gli2 transcription factor reduces the hedgehog pathway activity in vivo

The proteolytic processing of Gli2 and Gli3 full‐length transcription factors into repressors is a key step of the regulation in Hedgehog (Hh) signaling. The differential Gli2 and Gli3 processing is controlled by the processing determinant domain or PDD, but its significance is not clear. We generated a Gli2 mutant allele, Gli23PDD, in which the Gli3PDD substitutes for the Gli2PDD. As expected, Gli23PDD is processed more efficiently and at a different position as compared to Gli2, indicating that PDD also determines the extent and site of Gli2 and Gli3 processing in vivo. The increase in levels of the Gli2 repressor in Gli23PDD mutant reduces the Hh pathway activity. Gli23PDD processing is still regulated by Hh signaling. These results indicate that the proper balance between the Gli2 full‐length activator and repressor is essential for Hh signaling. Developmental Dynamics 240:766–774, 2011.

Three Tctn proteins are functionally conserved in the regulation of neural tube patterning and Gli3 processing but not ciliogenesis and Hedgehog signaling in the mouse

Tctn1, Tctn2, and Tctn3 are membrane proteins that localize at the transition zone of primary cilia. Tctn1 and Tctn2 mutations have been reported in both humans and mice, but Tctn3 mutations have been reported only in humans. It is also not clear whether the three Tctn proteins are functionally conserved with respect to ciliogenesis and Hedgehog (Hh) signaling. In the present study, we report that loss of Tctn3 gene function in mice results in a decrease in ciliogenesis and Hh signaling. Consistent with this, Tctn3 mutant mice exhibit holoprosencephaly and randomized heart looping and lack the floor plate in the neural tube, the phenotypes similar to those of Tctn1 and Tctn2 mutants. We also show that overexpression of Tctn3, but not Tctn1 or Tctn2, can rescue ciliogenesis in Tctn3 mutant cells. Similarly, replacement of Tctn3 with Tctn1 or Tctn2 in the Tctn3 gene locus results in reduced ciliogenesis and Hh signaling, holoprosencephaly, and randomized heart looping. Surprisingly, however, the neural tube patterning and the proteolytic processing of Gli3 (a transcription regulator for Hh signaling) into a repressor, both of which are usually impaired in ciliary gene mutants, are normal. These results suggest that Tctn1, Tctn2, and Tctn3 are functionally divergent with respect to their role in ciliogenesis and Hh signaling but conserved in neural tube patterning and Gli3 processing.