Hedgehog (Hh) signaling is employed for cell-cell communication in the formation of most organs and tissues. In some tissues the signal regulates differentiation, guiding stem cells to particular fates. In other cases Hh is a mitogen that spurs cell division. Defects in Hh signaling lead to birth defects and cancer. We study Hh signaling in flies, mice, and humans. We are focused on understanding how the signal is received, transduced, and interpreted.
One focus of our work has been Ptc, a Hh receptor protein encoded by a gene called patched (ptc). Hh binds to Ptc on the surfaces of receiving cells and causes them to choose a certain pathway of differentiation (e.g., motor neuron) or to divide. Since Hh and Ptc proteins act in opposition, the Ptc protein provides restraint designed to prevent excessive production of certain types of cells and to rein in growth. We showed that reduction or elimination of ptc function during mouse development leads to spina bifida, polydactyly, midbrain overgrowth, defects in the heart, and excessive body size. We found that mutations in human PATCHED (PTCH) cause a variety of birth defects, medulloblastoma of the cerebellum (the most common childhood malignant brain tumor), and basal cell carcinoma of the skin (the most common human cancer).
We have found that the primary cilium, an immotile appendage found on most cells, serves as an antenna for receiving the Sonic hedgehog (Shh) signal. The transduction machinery of the pathway is contained within such cilia. We have shown that Shh and Ptc control movements of proteins into or out of the cilia as they influence expression of genes. We are investigating the mechanisms through which cilium proteins affect Shh signaling in development and cancer.
We are using mutant mice to learn how tumors arise and to find new ways to stop them. We continue to study the mechanisms and impact of Shh signaling in the cerebellum. Much of our current research is directed at learning about genes whose transcription is controlled by Hh signaling, in normal cerebellum development and during tumorigenesis.
Among the genes that are highly expressed in tumors are some that encode components of the sterol synthesis pathway (SSP). These enzymes produce sterols, steroids, and bile acids. We found that medulloblastoma cells in culture have an exceptionally high requirement for activity of the SSP, both to grow and to activate target genes of the Hh pathway. We traced this requirement to a need for certain oxysterols, which are derivatives of cholesterol that have a hydroxyl group attached. Oxysterols are powerful activators of Hh target gene transcription, suggesting that oxysterols or a derivative of them play a role in transduction of the Hh signal. Manipulating such molecules may inhibit cancer cell growth.
A Neurodegeneration Syndrome Related to Intracellular Organelle Trafficking
Our studies of the Hh pathway led us to study a fatal neurodegenerative disease called Niemann-Pick type C (NPC) syndrome, caused by mutations in either of two genes, npc1 and npc2. The Npc1 protein is closely related to the Hh receptor Ptc and to the protein Npc1l1, the major cholesterol-uptake transporter in the mammalian gut. Npc1, Ptc, and Npc1l1 may all be transporter proteins related to the RND transporters, multidrug resistance pumps found in bacteria. NPC has a major impact on the cerebellum. People with a mutated NPC gene accumulate cholesterol deposits in their cells, and the Purkinje neurons of the cerebellum degenerate. Our hope is that by understanding the molecular and cellular basis of the disease we will find a way to lessen its severity. We are using engineered mice and biochemical approaches to investigate npc gene functions in cerebellum neurons.
Npc1 and Npc2 are ancient proteins, present even in yeast, and we developed genetic models of NPC disease in Drosophila to apply the power of genetics to its analysis. The mutant Drosophila, like mammals, accumulate sterols in aberrant intracellular compartments. This leads to deficient synthesis of the molting hormone ecdysone, because steroids like ecdysone are normally made in the mitochondria by enzymes that convert sterols to steroids.
Dramatic movements of proteins and organelles have become apparent in our studies of Hh signal transduction and of Niemann-Pick disease. Many trafficking events depend on a family of remarkable, small GTP-binding proteins called Rab proteins. To investigate the ~30 DrosophilaRab genes systematically, we have collaborated with Hugo Bellen (HHMI, Baylor College of Medicine) to construct transgenic fly lines that allow all DrosophilaRab activities to be reduced or increased in specific tissues and organs. We are using the transgenic flies to investigate how Rab proteins contribute to developmental signal transduction and to cell morphogenesis. Presently we are investigating a Rab protein that controls the assembly of the actin cytoskeleton.
Neural Control of Growth
Growth, a fundamental aspect of development, is highly regulated in evolution to give species their distinctive sizes. The timing and rate of growth must be controlled in response to food supply, energy requirements, stage of life, and season; such control often involves neural inputs. We are investigating the neural control of growth. The growth of tissues and organs is coordinated in Drosophila and mammals by insulin-like signals. In flies, the signals are released from specific neurons, while in mammals they come mostly from the liver. By analyzing mutations in a nucleostemin gene, we found that Drosophila insulin-producing neurons are regulated by serotonergic neurons. Drosophilanucleostemin mutants are much smaller than normal because of defective serotonergic signaling and consequent failure to release insulin. Nucleostemin proteins, originally identified in mammals, are nucleolar proteins with roles in stem cells and ribonucleoprotein complex assembly. Our genetic screens are identifying additional genes that act in identified neurons to control growth. To test hypotheses about neuron functions, we are using light-activated channel proteins that can stimulate or inhibit neural activity to manipulate neurons.