Genetic Regulation of Development and Disease


Summary: Matthew Scott is investigating how embryonic and later development is governed by proteins that control gene activity and signaling processes. He is exploring how defects in the regulators of development, or in related proteins, lead to birth defects, cancer, and neurodegenerative disease.


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.

When the Hh signal is received, a complex series of molecular steps follows inside the receiving cell. The components of this signal transduction mechanism are proteins, some of them also connected to cancer, that refine and interpret the Hh signal information. We are studying components that participate in the transduction, including proteins that form scaffolds, proteins that serve in transport processes, and proteins that modify other proteins. Among the proteins that we are studying are Rab proteins, small enzymes that regulate transfers between compartments within cells.

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.

Development, Tumorigenesis, and Neurodegeneration in the Cerebellum
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.

The Niemann-Pick type C (NPC) syndrome has a major impact on the cerebellum. People lacking either of the NPC genes, NPC1 and Npc2, 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. Curiously, the Npc1 protein is the single protein most closely related to Ptc, one of several connections between Hh signaling and cholesterol metabolism. Npc1 looks like a protein that moves small molecules across membranes; Npc2 is a mysterious secreted protein that may sense or transport cholesterol. We have used time-lapse imaging of fluorescent versions of Npc1 to study its movements, and we have determined which parts of the Npc2 protein are essential for its function. To learn how neurodegeneration occurs when Npc1 protein is not functional, we constructed chimeric mice in which only some cells lack the Npc1 gene. In this way we showed that the neurons, rather than some other cell type, require Npc1 function. Npc1 and Npc2 are ancient proteins, present even in yeast, and we are developing genetic models of NPC disease in Drosophila order to apply powerful genetics to its analysis.

The Earliest Stages of Mammalian Development
In addition to studies of the cerebellum, we are using genomics approaches to study the sequential activation of gene expression during early fly and mouse embryonic stages. In collaboration with Ron Davis (Stanford University) and Magdalena Zernicka-Goetz (University of Cambridge), we have investigated gene expression changes in preimplantation mouse embryos. The results revealed waves of signaling and gene regulation processes, but the functions of many of these activities are unknown. We are investigating selected proteins involved in gene regulation and signaling during these early stages of mammalian development; in parallel we are analyzing some of the same regulators in the context of embryonic stem cell culture and differentiation.

Our studies are also supported by grants from the National Institutes of Health, the Ludwig Foundation, and the Ara Parseghian Research Foundation.

Drosophila wing imaginal disc...

Signaling proteins traverse tissue to form morphogen gradients during animal development. Shown here is a Drosophila wing imaginal disc that will eventually give rise to adult wing blade and thorax. The wing imaginal disc is divided into anterior (A) and posterior (P) compartments. Two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), determine adult wing development. Hh protein produced by posterior disc cells (shown in red) reaches across the A-P boundary to activate Hh-responsive genes, including ptc (shown in green) in the A compartment. These target genes are essential for the proper patterning of the adult wing blade. The vertebrate neural tube is another model system used for studying morphogen gradients. Insets: immunofluorescence analyses of three mouse neural tubes at embryonic day 10.5, using antibodies directed against Sonic hedgehog (Shh) protein (green), Foxa2 (red), and Isl1/2 (blue).
�2004 Cold Spring Harbor Laboratory Press. See also Zhu, A.J., and Scott, M.P. 2004. Genes & Development 18:2985–2997.

A lobe of a developing mouse cerebellum...

lobe of a developing mouse cerebellum stained to show granule neurons (red), Purkinje neurons (blue), and granule neuron precursors (green).

Tal Raveh, in Matthew Scott's lab.

Patterns of regulatory gene expression...

Many regulatory genes serve related functions in embryos as different as flies and mice. The fly embryo (left) shows striped patterns of regulatory gene expression. The mouse embryo shows the developing hair follicles highlighted (blue) by the expression of patched1, a gene first discovered in flies.

Ljiljana Milenkovic and Matthew Scott.

A network of microtubules...

A cultured cell showing the network of microtubules that forms a cytoskeleton (orange) and organelles containing Niemann-Pick protein fused to green fluorescent protein.

Dennis Ko and Matthew Scott.

http://www.hhmi.org/research/investigators/scottm.html