Hedgehog Signal Transduction Pathway Research Articles

Genetic and molecular control of folate-homocysteine metabolism in mutant mice.

Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.

Skinny hedgehog Puts on Fat

The Hedgehog (Hh) signal transduction pathway plays a key role in both mammalian and insect development. In addition, when the pathway is inappropriately activated by mutation, it contributes to the development of a common form of human skin cancer. Chamoun et al. show that the secreted Hh protein in the fruit fly Drosophila is palmitoylated at its amino terminus and that this modification is essential for the activity of Hh in embryonic and larval patterning. Genetic experiments indicate that the acyltransferase that catalyzes this modification is encoded by the Skinny hedgehog ( ski ) gene. The ski gene is related in sequence to a gene required for the Wingless signaling pathway, and its function raises the possibility that acylation of secreted proteins may play a widespread role in development (see the Perspective by Van den Heuvel ). Z. Chamoun, R. K. Mann, D. Nellen, D. P. von Kessler, M. Bellotto, P. A. Beachy, K. Basler, Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal. Science 293 , 2080-2084 (2001). [Abstract] [Full Text] M. van den Heuvel, Fat hedgehogs, slower or richer? Science's STKE (2001), http://www.stke.org/cgi/content/full/OC_sigtrans;2001/100/pe31. [Abstract] [Full Text]

Skinny hedgehog Puts on Fat

The Hedgehog (Hh) signal transduction pathway plays a key role in both mammalian and insect development. In addition, when the pathway is inappropriately activated by mutation, it contributes to the development of a common form of human skin cancer. Chamoun et al. show that the secreted Hh protein in the fruit fly Drosophila is palmitoylated at its amino terminus and that this modification is essential for the activity of Hh in embryonic and larval patterning. Genetic experiments indicate that the acyltransferase that catalyzes this modification is encoded by the Skinny hedgehog (ski) gene. The ski gene is related in sequence to a gene required for the Wingless signaling pathway, and its function raises the possibility that acylation of secreted proteins may play a widespread role in development (see the Perspective by Van den Heuvel).Z. Chamoun, R. K. Mann, D. Nellen, D. P. von Kessler, M. Bellotto, P. A. Beachy, K. Basler, Skinny Hedgehog, an acyltransferase required for palmitoylation and activity of the Hedgehog signal. Science 293, 2080-2084 (2001). [Abstract] [Full Text]M. van den Heuvel, Fat hedgehogs, slower or richer? Science's STKE (2001), http://www.stke.org/cgi/content/full/OC_sigtrans;2001/100/pe31. [Abstract] [Full Text]

Sonic hedgehogRegulates Proliferation and Inhibits Differentiation of CNS Precursor Cells

Activation of the Sonic hedgehog (Shh) signal transduction pathway is essential for normal pattern formation and cellular differentiation in the developing CNS. However, it is also thought to be etiological in primitive neuroectodermal tumors. We adapted GAL4/UAS methodology to ectopically express full-length Shh in the dorsal neural tube of transgenic mouse embryos commencing at 10 d postcoitum (dpc), beyond the period of primary dorsal-ventral pattern formation and floorplate induction. Expression of Shh was maintained until birth, permitting us to investigate effects of ongoing exposure to Shh on CNS precursors in vivo. Proliferative rates of spinal cord precursors were twice that of wild-type littermates at 12.5 dpc. In contrast, at late fetal stages (18.5 dpc), cells that were Shh-responsive but postmitotic were present in persistent structures reminiscent of the ventricular zone germinal matrix. This tissue remained blocked in an undifferentiated state. These results indicate that cellular competence restricts the proliferative response to Shh in vivo and provide evidence that proliferation and differentiation can be regulated separately in precursor cells of the spinal cord. Thus, Hedgehog signaling may contribute to CNS tumorigenesis by directly enhancing proliferation and preventing neural differentiation in selected precursor cells.

Proteolysis of Cubitus interruptus in Drosophila requires phosphorylation by Protein Kinase A

The Hedgehog signal transduction pathway is involved in diverse patterning events in many organisms. In Drosophila, Hedgehog signaling regulates transcription of target genes by modifying the activity of the DNA-binding protein Cubitus interruptus (Ci). Hedgehog signaling inhibits proteolytic cleavage of full-length Ci (Ci-155) to Ci-75, a form that represses some target genes, and also converts the full-length form to a potent transcriptional activator. Reduction of protein kinase A (PKA) activity also leads to accumulation of full-length Ci and to ectopic expression of Hedgehog target genes, prompting the hypothesis that PKA might normally promote cleavage to Ci-75 by directly phosphorylating Ci-155. Here we show that a mutant form of Ci lacking five potential PKA phosphorylation sites (Ci5m) is not detectably cleaved to Ci-75 in Drosophila embryos. Moreover, changes in PKA activity dramatically altered levels of full-length wild-type Ci in embryos and imaginal discs, but did not significantly alter full-length Ci5m levels. We corroborate these results by showing that Ci5m is more active than wild-type Ci at inducing ectopic transcription of the Hh target gene wingless in embryos and that inhibition of PKA enhances induction of wingless by wild-type Ci but not by Ci5m. We therefore propose that PKA phosphorylation of Ci is required for the proteolysis of Ci-155 to Ci-75 in vivo. We also show that the activity of Ci5m remains Hedgehog responsive if expressed at low levels, providing further evidence that the full-length form of Ci undergoes a Hedgehog-dependent activation step.

A frequent activated smoothened mutation in sporadic basal cell carcinomas.

Basal-cell carcinomas (BCCs) are the most common cancer in Caucasians. It has been reported that the patched gene is inactivated in 30-40% sporadic BCCs and 20% sporadic medulloblastomas via loss of heterozygosity and nonsense mutations. Recently, two activating smoothened mutations have been found in the sporadic basal cell carcinomas. One, at base pair 1604 (G-to-T transversion) of exon 9, changes codon 535 from tryptophan to leucine, and the other, at base pair 1685 (G-to-A transition) of exon 10, changes codon 562 from arginine to glutamine (Xie et al., 1998). In our study, 1604G-->T was found in 20 out of 97 (20.6%) sporadic BCCs. The high prevalence indicates that 1604G is the mutation hot spot in our tumor samples. This mutation was detected in all three histological subtypes of BCCs, suggesting that smoothened mutation is an early event during the development of the tumor. Our finding of a high smoothened mutation rate, together with high frequent patched gene mutations reported recently, indicates that activation of the hedgehog signal transduction pathway is the most common and early event in the development of sporadic BCCs. Additionally, to determine whether smoothened, like patched, is also involved in the carcinogenesis of medulloblastomas, we screened medulloblastoma samples for these two mutations by restriction analysis. We have found the 1604G-->T mutation in 1 out of 21 medulloblastomas. This result confirmed smoothened gene involvement in the carcinogenesis of medulloblastoma.

Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice

The three mouse Gli genes are putative transcription factors which are the homologs of cubitus interruptus ( ci) in Drosophila. Along with the gene patched ( Ptc), ci has been implicated in the hedgehog ( Hh) signal transduction pathway. To assess the role of Gli in embryogenesis, we compared its expression with that of Ptc and Hh family members in mouse. We found that Gli and Ptc are expressed in similar domains in diverse regions of the developing mouse embryo and these regions are adjacent to Hh signals. We also show that Gli is expressed ectopically along with Ptc and Shh in Strong's luxoid mutant mice. These results are consistent with conservation of the Hh signal transduction pathway in mice with Gli potentially mediating Hh signaling in multiple regions of the developing embryo.

Drosophila cubitus interruptus forms a negative feedback loop with patched and regulates expression of Hedgehog target genes.

The Drosophila segment polarity gene cubitus interruptus (ci) encodes a zinc finger protein that is required for the proper patterning of segments and imaginal discs. Epistasis analysis indicates that ci functions in the Hedgehog (Hh) signal transduction pathway and is required to maintain wingless expression in the embryo. In this paper, the role of the Ci protein in the Hh signaling pathway is examined in more detail. Our results show that ectopic expression of ci in imaginal discs and the embryo activates the expression of Hh target genes. One of these target genes, patched, forms a negative feedback loop with ci that is regulated by Hh signal transduction. Activation is also achieved using the Ci zinc finger domain fused to a heterologous transactivation domain. Conversely, repression of Hh target genes occurs in animals expressing the Ci zinc finger domain fused to a repression domain. To examine Ci function in more detail, regions of the Ci protein that are responsible for its ability to transactivate and its subcellular distribution have been identified.

The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways

The segment polarity gene cubitus interruptus (ci) is required to maintain expression of the wingless gene and to specify naked cuticle within each epidermal segment. Antibodies were generated against the ci protein and used to analyze its pattern of expression. By stage 11, post-transcriptional regulation of ci is observed. ci transcript levels are uniform across the anterior compartment, but protein levels are higher next to the compartment boundaries. The distribution of the ci protein is altered in fused, hedgehog and wingless mutants suggesting cell-cell signaling may regulate ci protein levels. The role of ci in cell-cell signaling and pattern formation was examined in double mutants of ci with patched and zeste-white3/shaggy.