Scott Lab In The Department of Developmental Biology

Scott Lab Research Areas

Patterns of regulatory gene expression...

Early embryonic development is governed by an exquisite interplay of genes that organizes cells as they proliferate. Signals flow between cells to control their fates; information inherited by the cells influences their responses to the signals. Much of the genetic machinery that builds the embryo is ancient. Transcription factors necessary for forming particular parts of the body—such as head-to-tail differences, heart, eyes, or nervous system—have remained dedicated to those tasks through evolution. Similarly, the genes and proteins that code for signals, signal receptors, and information transfer within the cell have been preserved, as have many of the relationships among them. We study evolutionarily conserved regulators in flies and in mice to learn how the embryo is constructed and how pattern-organizing genetic programs arose, function, and change. Genetic damage to developmental regulators can lead to cancer, birth defects, and neurodegeneration; we study all of these processes in the mammalian cerebellum. By examining the roles of particular genes in normal development and in disease, we learn about fundamental molecular and cellular mechanisms relevant to both.

The Hedgehog Signaling System in Development and Cancer

The Hedgehog (Hh) signaling system was discovered in fruit flies and named after the bristly appearance of mutants where the signal is not working.  Hh signaling is used in most animals to control the embryonic development of numerous tissues, such as brain and spinal cord, limbs, skeleton, and skin.  The Hh signal, a protein, is emitted by specific cells during development. Nearby cells are influenced if they have the appropriate receptors and transducers to sense and interpret the signal. Receipt of the signal changes which genes are active within the receiving cells, altering cell growth rates or causing cells to form particular types of tissue. 

We have studied Hh signaling in flies, mice, and humans. In general we have asked three questions: (1) Where does the signal go from and to? (2) What information does it carry? (3) How is the signal received, transduced, and interpreted? For these experiments we are studying fly embryonic development and metamorphosis, the mouse spinal cord, and the mouse cerebellum. In each case we are investigating which genes are activated or repressed in cells that receive the Hh signal.

One particular focus of our work has been the Hh receptor protein, which is encoded by a gene called patched (ptc). Hh binds to the Ptc protein 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, excessive body size, and certain types of cancer.

Fruit fly wing imaginal disc

A fruit fly wing imaginal disc, stained with antibodies to show the locations of three key regulatory proteins involved in controlling wing development. The disc, which is taken from a fly larva, is composed of about 50,000 cells that will, during metamorphosis, form a wing. The labeled proteins are Engrailed (En), a transcription factor made in the posterior half of the disc; Ci, a transcription factor that is most abundant in the central disc; and Costal-2 (Cos-2), a "motor" protein that moves cell components and is made in the anterior (left) of the disc. All three proteins are involved in signaling events that use the Hedgehog signaling protein.

We found that mutations in human PATCHED (PTCH) are inherited in families with the basal cell nevus syndrome. These individuals exhibit a variety of birth defects and often develop medulloblastoma of the cerebellum, the most common childhood malignant brain tumor, and basal cell carcinoma of the skin, the most common human cancer. We confirmed the involvement of PTCH in these tumors by looking for mutations in sporadic cases of the disease and by constructing mouse models of both cancers, using knowledge of the signaling pathway derived from studies in flies. We are using these mice to investigate gene function changes that occur during the conversion of normal cells to tumor cells, with the goals of understanding how tumors arise and finding new ways to stop them.

The role of PTCH in medulloblastoma suggested a role for Hh signaling in normal cerebellar development. We found that Shh, one of the vertebrate Hh proteins, is a powerful stimulant of cell division for the major type of cerebellar neuron. We are continuing to study the mechanisms and impact of Shh signaling in the cerebellum. We have broadened the work to other regulators that control the formation and differentiation of cell fates in the cerebellum. Using genomics approaches, we have identified many genes active during key early steps of cerebellar growth; we are comparing these gene regulation events to those in early tumorigenesis steps.  Our general goal is to understand how cells are born, shaped, polarized, and connected. To obtain information about gene function more quickly than is possible using mouse mutants, we have begun to study zebrafish cerebellum development in collaboration with Dr. Will Talbot in our department.  In zebrafish we test gene functions by injecting inactivating molecules specific for a particular gene. 

Our genomics analyses revealed that when normal cells transform into tumor cells, several genes that control sterol synthesis become more highly active. We found that sterols and certain related molecules are required for the growth of tumor cells in culture, and that sterol synthesis inhibitors can reduce cancer cell growth.  Inhibition of Shh signaling by sterol synthesis inhibitors may offer a useful approach to the treatment of medulloblastoma and other Shh pathway-dependent human tumors.

Molecular and genetic analysis of the Niemann-Pick type C syndrome, a neurodegenerative disorder

Children mutant in either of the two NPC genes undergo neurodegeneration and usually die by the teenage years.  At the cellular level, this terrible disease is marked by accumulation of masses of sterols in aberrant organelles due to defective intracellular trafficking.  The movements of organelles within cells do not occur properly without both genes.  We were led to an interest in NPC when the gene damaged by most of the inherited NPC mutations was isolated at the National Institutes of Health by E. Carstea and colleagues in 1997.  The encoded NPC1 protein has striking sequence similarity to Patched, the Hedgehog receptor.   Both proteins appear to be 12-13 transmembrane domain proteins that may be transporters.  Later a second NPC gene, NPC2, was isolated; it encodes a small, secreted cholesterol-binding protein. 

We have studied the movements of the late endosomes that contain NPC1 using fluorescent protein tags and time-lapse imaging.  We found that the organelles move rapidly on microtubule tracks, and when NPC1 protein is not functional the organelles coalesce into sterol-rich blobs near the nucleus.  Our studies of NPC2 showed which parts of the protein are needed for its function, that it binds strongly to cholesterol, and the location of a cavity on the surface that is likely to be the binding site for sterol.  Much remains to be learned about the roles of both proteins.

In humans and mice, NPC disease causes the death of the Purkinje neurons of the cerebellum, the neurons that we have studied for their roles in sending Hedgehog signal to the granule neuron precursors.  The NPC genes are active in all cells, but the Purkinje cells are most sensitive to their loss.  We have built genetically chimeric mice composed partly of wild-type cells and partly of NPC mutant cells in order to find out where the gene is needed to prevent Purkinje cell death.  The answer might have been, for example, that the NPC gene is needed in cells that provide a crucial hormone to the Purkinje cells.  Instead we found that NPC1 is needed in the Purkinje cells themselves, presumably for the proper movements of organelles within the cells.  We are now studying what the NPC proteins do within Purkinje neurons.

To gain further insight into how NPC proteins work, we are employing the powerful genetics of the fly Drosophila.  Like mammals, flies have both npc1 and npc2 genes.  We have made mutants in both and found an accumulation of sterols highly similar to the change seen in mutant npc mammalian cells.  By studying the fly mutants we were able to determine that npc has a particularly crucial role in a part of the brain called the ring gland.  The ring gland produces the steroid molting hormone, ecdysone, that allows larva to metamorphose into flies.  Without npc1, larvae die without molting due to a failure to produce molting hormone.  Analyses in other labs show that a failure to produce steroids may underlie aspects of NPC disease in worms and mice as well.  The next job is to find out why the ring gland cells are unable to produce steroid. 

Rab proteins in development and cell biology

Small proteins called Rabs are used to control movements of organelles and assembly of subcellular compartments and skeletal elements.  With some 80 Rab proteins or so, with overlapping functions, genetic analyses that would reveal Rab functions are challenging.  Flies have 31 Rab genes, most of which correspond to specific classes of mammalian proteins.  We have built a large set of modified Rab genes and introduced them into transgenic flies to analyze their functions.  For each gene, versions were made that are uncontrollably active, normal, or will interfere with the function of the normal gene.  Each is engineered so that it can be produced in any of many tissues during the growth of the embryo.  By activating production of a Rab protein at specific times and places, we are investigating the capabilities of each protein.  Some are required for developmental processes in specific tissues; some for apparently universal cell functions.  The goal is to assemble a broad view of Rab functions in the context of development and then investigate in detail the roles of a few chosen ones. 

Chromatin factors in embryonic stem cells

We have applied genomics approaches to early embryogenesis, first in Drosophila and more recently in the mouse.  The Drosophila studies were done in collaboration with the labs of Bruce Baker, Ron Davis, and Mark Krasnow at Stanford; the mouse work in collaboration with Ron Davis and with Magdalena Zernicka-Goetz at the Gurdon Institute in Cambridge, England.  By monitoring gene expression in carefully staged embryos we were able to discover changes in pattern of transcription during key events in embryonic pattern formation.  In Drosophila we also used mutants and an embryo-sorting instrument we designed to identify genes involved in muscle development.

Massive changes occur in transcription in very early mouse embryos, prior to implantation and during the period when the embryo develops from a single cell into 32 cells.  Among the genes changing are some that encode components of chromatin.  Chromatin proteins can alter the structure and accessibility and chemical modifications of genes.  We have begun to investigate the roles of chromatin proteins in pre-implantation mouse embryos and in cultured mouse embryonic stem cells.  This work is in collaboration with Drs. Ron Davis and Wing Wong at Stanford.  The goal is to understand how chromatin proteins, particularly those in the chromatin-remodeling class, contribute to the deployment of the genome in very early mammalian embryos.

Vertebrate functions of Planar Cell Polarity (PCP) genes

PCP genes were discovered in Drosophila as regulators of the orientation of epidermal bristles and the ommatidia of the fly eye.  PCP proteins form a signaling system that coordinates the polarity of cells within epithelial sheets.  We are working with Prof. Jeff Axelrod in the Department of Pathology at Stanford to examine roles of vertebrate planar cell polarity (PCP) genes. Vertebrate PCP proteins are involved in orienting inner ear hair cells and in cell movements during the sliding and folding processes that create tissue layers in early embryos.  PCP genes are also active in many other tissues, including the cerebellum, though their functions are unknown.  We are studying the roles of the genes using antibodies to track the proteins and engineered mice to determine functions.

The work described here was also supported by a grant from the National Institutes of Health, a grant from the Parseghian Medical Research Foundation, and a grant from the Ludwig Foundation.

This text comes from Prof. Scott's web page on the HHMI site.

Stanford Medicine Resources:

Footer Links: