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Biomedical subjects

Matthew P Scott

Publications and source records attributed to Matthew P Scott.

At least 19 recordsLinked to original sources
PubMed2006

Hedgehog modulates cell cycle regulators in stem cells to control hematopoietic regeneration.

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here, we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However, this effect is at the expense of HSC function, because continued Hh activation during regeneration represses expression of specific cell cycle regulators, leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo.

Animals↗
PubMed2006

Automated MEMS-based Drosophila embryo injection system for high-throughput RNAi screens.

We have developed an automated system based on microelectromechanical systems (MEMS) injectors for reliable mass-injection of Drosophila embryos. Targeted applications are high-throughput RNA interference (RNAi) screens. Our injection needles are made of silicon nitride. The liquid to be injected is stored in an integrated 500 nl reservoir, and an externally applied air pressure pulse precisely controls the injected volume. A steady-state water flow rate per applied pressure of 1.2 nl s(-1) bar(-1) was measured for a needle with channel width, height and length of 6.1 microm, 2.3 microm and 350 microm, respectively. A typical volume of 60 pl per embryo can be reliably and rapidly delivered within tens of milliseconds. Theoretical predictions of flow rates match measured values within +/-10%. Embryos are attached to a glass slide surface and covered with oil. Packages with the injector chip and the embryo slide are mounted on motorized xyz-stages. Two cameras allow the user to quickly align the needle tip to alignment marks on the glass slide. Our system then automatically screens the glass slide for embryos and reliably detects and injects more than 98% of all embryos. Survival rates after deionized (DI) water injection of 80% and higher were achieved. A first RNAi experiment was successfully performed with double-stranded RNA (dsRNA) corresponding to the segment polarity gene armadillo at a concentration of 0.01 microM. Almost 80% of the injected embryos expressed an expected strong loss-of-function phenotype. Our system can replace current manual injection technologies and will support systematic identification of Drosophila gene functions.

Animals↗
PubMed2006

Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells.

Sterol synthesis is required for Sonic hedgehog (Shh) signal transduction. Errors in Shh signal transduction play important roles in the formation of human tumors, including medulloblastoma (MB). It is not clear which products of sterol synthesis are necessary for Shh signal transduction or how they act. Here we show that cholesterol or specific oxysterols are the critical products of sterol synthesis required for Shh pathway signal transduction in MB cells. In MB cells, sterol synthesis inhibitors reduce Shh target gene transcription and block Shh pathway-dependent proliferation. These effects of sterol synthesis inhibitors can be reversed by exogenous cholesterol or specific oxysterols. We also show that certain oxysterols can maximally activate Shh target gene transcription through the Smoothened (Smo) protein as effectively as the known Smo full agonist, SAG. Thus, sterols are required and sufficient for Shh pathway activation. These results suggest that oxysterols may be critical regulators of Smo, and thereby Shh signal transduction. Inhibition of Shh signaling by sterol synthesis inhibitors may offer a novel approach to the treatment of MB and other Shh pathway-dependent human tumors.

Animals↗
PubMed2006

Mes2, a MADF-containing transcription factor essential for Drosophila development.

The development of the Drosophila mesoderm is initiated by the basic helix-loop-helix transcription factor twist. We identified a gene encoding a putative transcription factor, mes2, in a screen for essential mesoderm-expressed genes that function downstream of twist. Mes2 protein belongs to a family of 48 Drosophila proteins containing MADF domains. MADF domains exist in worms, flies, and fish. Mes2 is a nuclear protein first produced in trunk and head mesoderm during late gastrulation. At later embryonic stages, mes2 is expressed in glia of the central and peripheral nervous systems, and in tissues derived from the head mesoderm. We have identified a null mutation of mes2 that leads to developmental arrest in first instar larvae. Increased production of Mes2 in multiple embryonic and larval tissues almost always causes lethality. The ubiquitous or epidermal misexpression of mes2 in the embryo causes a dramatic loss of epidermal integrity resulting in the failure of dorsal closure. Our data show that the precise regulation of mes2 expression is critical for normal development in Drosophila and implicate Mes2 in the regulation of essential target genes.

Amino Acid Sequence↗
PubMed2005

A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis.

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.

Animals↗
PubMed2005

Cell-autonomous death of cerebellar purkinje neurons with autophagy in Niemann-Pick type C disease.

Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death.

Journal Article↗
PubMed2005

Communicating with Hedgehogs.

Signalling by secreted Hedgehog (Hh) proteins is important for the development of many tissues and organs. Damage to components of the Hh signal-transduction pathway can lead to birth defects and cancer. The Hh proteins are distributed in tissues in a gradient, and cells respond to different thresholds of Hh with distinct responses. The cellular machinery that is responsible for the unique molecular mechanisms of Hh signal transduction has been largely conserved during metazoan evolution.

Animals↗
PubMed2005

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused.

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. We have analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. Our results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh.

Amino Acid Substitution↗
PubMed2004

Incredible journey: how do developmental signals travel through tissue?

How developmental signaling proteins traverse tissue during animal development, through or around tightly packed cells, remains an incompletely resolved mystery. Signaling protein movement is regulated to create gradients, control amounts, impose barriers, or provide direction. Signaling can be controlled by the rate of signal production, modification, active transport, trapping along the path, or by the properties of the receptor apparatus. Signals may move by diffusion outside cells, attached to migrating cells, attached to carrier molecules, through cells by transcytosis, along cell extensions, or in released membrane packets. Recent findings about the movement of Hedgehog, Wingless (Wnt), and TGF-beta signaling proteins have helped to clarify the molecular mechanisms used to ensure that developmental signals carry only good news.

Animals↗
PubMed2004

Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain.

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [(3)H]7,7-azocholestanol ([(3)H]AC). After immunoprecipitation, (3)H-labeled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [(3)H]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [(3)H]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1.

Animals↗
PubMed2004

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo.

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.

Animals↗
PubMed2004

Drosophila Costal1 mutations are alleles of protein kinase A that modulate hedgehog signaling.

Hedgehog (Hh) signaling is crucial for the development of many tissues, and altered Hh signal transduction can result in cancer. The Drosophila Costal1 (Cos1) and costal2 (cos2) genes have been implicated in Hh signaling. cos2 encodes a kinesin-related molecule, one component of a cytoplasmic complex of Hh signal transducers. Mutations in Cos1 enhance loss-of-function cos2 mutations, but the molecular nature of Cos1 has been unknown. We found that previously identified alleles of Cos1 actually map to two separate loci. Four alleles of Cos1 appear to be dominant-negative mutations of a catalytic subunit of protein kinase A (pka-C1) and the fifth allele, Cos1(A1), is a gain-of-function allele of the PKA regulatory subunit pka-RII. PKA-RII protein levels are higher in Cos1(A1) mutants than in wild type. Overexpression of wild-type pka-RII phenocopies Cos1 mutants. PKA activity is aberrant in Cos1(A1) mutants. PKA-RII is uniformly overproduced in the wing imaginal disc in Cos1(A1) mutants, but only certain cells respond by activating the transcription factor Ci and Hh target gene transcription. This work shows that overexpression of a wild-type regulatory subunit of PKA is sufficient to activate Hh target gene transcription.

Amino Acid Sequence↗
PubMed2003

Notch and Ras signaling pathway effector genes expressed in fusion competent and founder cells during Drosophila myogenesis.

Drosophila muscles originate from the fusion of two types of myoblasts, founder cells (FCs) and fusion-competent myoblasts (FCMs). To better understand muscle diversity and morphogenesis, we performed a large-scale gene expression analysis to identify genes differentially expressed in FCs and FCMs. We employed embryos derived from Toll10b mutants to obtain primarily muscle-forming mesoderm, and expressed activated forms of Ras or Notch to induce FC or FCM fate, respectively. The transcripts present in embryos of each genotype were compared by hybridization to cDNA microarrays. Among the 83 genes differentially expressed, we found genes known to be enriched in FCs or FCMs, such as heartless or hibris, previously characterized genes with unknown roles in muscle development, and predicted genes of unknown function. Our studies of newly identified genes revealed new patterns of gene expression restricted to one of the two types of myoblasts, and also striking muscle phenotypes. Whereas genes such as phyllopod play a crucial role during specification of particular muscles, others such as tartan are necessary for normal muscle morphogenesis.

Animals↗
PubMed2003

Tgfbeta signaling acts on a Hox response element to confer specificity and diversity to Hox protein function.

Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. We show that the Dpp/Tgfbeta signal controls Abdominal A function, and that Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity.

Activating Transcription Factor 1↗
PubMed2003

Differential requirement for Gli2 and Gli3 in ventral neural cell fate specification.

Sonic hedgehog (Shh) directs the development of ventral cell fates, including floor plate and V3 interneurons, in the mouse neural tube. Here, we show that the transcription factors Gli2 and Gli3, mediators of Shh signaling, are required for the development of the ventral cell fates but make distinct contributions to controlling cell fates at different locations along the rostral-caudal axis. Mutants lacking Patched1 (Ptc1), the putative receptor of Shh, were used to analyze Gli functions. Ptc1(-/-) mutants develop floor plate, motor neuron, and V3 interneuron progenitors in lateral and dorsal regions, suggesting that the normal role of Ptc1 is to suppress ventral cell development in dorsal neural tube. The Ptc1(-/-) phenotype is rescued, with restoration of dorsal cell types, by the lack of Gli2, but only in the caudal neural tube. In triple mutants of Gli2, Gli3, and Ptc1, dorsal and lateral cell fates are restored in the entire neural tube. These observations suggest that Gli2 is essential for ventral specification in the caudal neural tube, and that in more rostral regions, only Gli3 can promote development of ventral cells if Gli2 is absent. Thus, Shh signaling is mediated by overlapping but distinct functions of Gli2 and Gli3, and their relative contributions vary along the rostral-caudal axis.

Animals↗
PubMed2003

Transcriptional profiling of the Sonic hedgehog response: a critical role for N-myc in proliferation of neuronal precursors.

Cerebellar granule cells are the most abundant neurons in the brain, and granule cell precursors (GCPs) are a common target of transformation in the pediatric brain tumor medulloblastoma. Proliferation of GCPs is regulated by the secreted signaling molecule Sonic hedgehog (Shh), but the mechanisms by which Shh controls proliferation of GCPs remain inadequately understood. We used DNA microarrays to identify targets of Shh in these cells and found that Shh activates a program of transcription that promotes cell cycle entry and DNA replication. Among the genes most robustly induced by Shh are cyclin D1 and N-myc. N-myc transcription is induced in the presence of the protein synthesis inhibitor cycloheximide, so it appears to be a direct target of Shh. Retroviral transduction of N-myc into GCPs induces expression of cyclin D1, E2F1, and E2F2, and promotes proliferation. Moreover, dominant-negative N-myc substantially reduces Shh-induced proliferation, indicating that N-myc is required for the Shh response. Finally, cyclin D1 and N-myc are overexpressed in murine medulloblastoma. These findings suggest that cyclin D1 and N-myc are important mediators of Shh-induced proliferation and tumorigenesis.

Animals↗