Faculty Bibliography

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Delta14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Delta14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone.

BACKGROUND: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors that modulate the pathway. RESULTS: During a forward genetic screen for novel regulators of lymphangiogenesis, we isolated a mutant with reduced lymphatic vessel development. This mutant also exhibited hyperbranching arteries, reminiscent of Notch pathway mutants. Positional cloning identified a missense mutation in the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) gene. Cad is essential for UDP biosynthesis, which is necessary for protein glycosylation and de novo biosynthesis of pyrimidine-based nucleotides. Using a transgenic reporter of Notch activity, we demonstrate that Notch signaling is significantly reduced in cad(hu10125) mutants. In this context, genetic epistasis showed that increased endothelial cell responsiveness to Vegfc/Vegfr3 signaling drives excessive artery branching. CONCLUSIONS: These findings suggest important posttranslational modifications requiring Cad as an unappreciated mechanism that regulates Notch/Vegf signaling during angiogenesis.

The atrial and ventricular chambers of the heart behave as distinct subunits with unique morphological, electrophysiological, and contractile properties. Proper maintenance of atrial and ventricular features is therefore essential for the differentiation and homeostasis of a functional heart. However, chamber fate assignments seem relatively plastic: for example, work in zebrafish has demonstrated that cells retain the potential to alter their chamber-specific traits, even after initiating differentiation. Our recent studies of zebrafish nkx2.5 and nkx2.7 mutants has shown that these transcription factors play key roles in enforcing chamber-specific gene expression programs, but the signaling pathways that function upstream of these factors are less well understood. Here, we show that the FGF signaling pathway, which facilitates ventricular specification, is required to preserve ventricular identity. We find that inhibition of FGF signaling with the FGFR antagonist SU5402 can generate ectopic atrial cardiomyocytes within the already differentiated ventricle; similar phenotypes are evident in fgf8 mutants and in embryos overexpressing a dominant-negative FGF receptor. Additionally, temporally restricted manipulation of the FGF pathway reveals a specific time window during which ventricular chamber identity is vulnerable to the loss of FGF signaling. Analysis using photoconvertible reporter transgenes indicates that the ectopic cells in SU5402-treated embryos are not derived from the atrium. Instead, examination of the dynamic localization of atrial and ventricular proteins suggests that ectopic atrial cells are produced through transformation of ventricular cardiomyocytes. Furthermore, we find that FGF signaling is required for normal maintenance of nkx2.5 and nkx2.7 expression. Overexpression of nkx2.5 can improve the preservation of ventricular character in SU5402-treated embryos, suggesting that FGF signaling functions upstream of Nkx factors to promote retention of ventricular identity. Together, our data suggest a model in which continuous FGF signaling acts to suppress the plasticity of differentiated cardiomyocytes and to preserve the integrity of the ventricular chamber

Background: GWAS studies in schizophrenia have not yielded targets for person-specific interventions. Alternatively, studies can focus on genes that were initially identified as harboring disruptive de novo mutations in sporadic cases. We examined the impact of four such genes on illness phenotypes. Methods: Structured interviews (DIGS), cognition (WAIS III), symptoms (PANSS) were examined in 48 genotyped cases finding that over 30% of the sample carried a rare/ missense mutations in any of 4 genes. Gene carrier groups were compared to cases without any of these mutations and healthy controls. Results: Carriers of disrupted genes showed significant differences, as follows: SLC39A13 (zinc transporter) (n=4) had the greatest psychopathology and severe cognitive deficits; TGM5 (n=4) had fewer symptoms but slower processing speed; PTPRG (n=5) had prematurity, childhood psychosis and good cognition except poor working memory; ARMS/KIDINS220 (n=5) had comparable severe pathology in all symptom factors and cognitive scores, though degeneration is suggested in light of their early accomplishments. Individual case vignettes highlighted familial psychosis, learning disorders, substance abuse, traumatic brain injuries and medical comorbidity in all 4 subgroups. Conclusions: The results suggest that genes prone to de novo mutations in sporadic cases may provide missing leverage to resolve the complexity of schizophrenia. A differential focus on working memory, processing speed, neuroprotection and zinc treatment should be pursued for these newly identified conditions. Other findings are that ethnicity may not limit genetic research when the focus is on gene function rather than particular sequence variations, and that premorbid exposures may sometimes reflect pleiotrophic effects of psychosis vulnerability genes rather than exposures producing nongenetic phenocopies. This novel approach may be applicable to other complex disorders

The yeast homologs of the ATM and ATR DNA damage response kinases play key roles in telomerase-mediated telomere maintenance, but the role of ATM/ATR in the mammalian telomerase pathway has been less clear. Here, we demonstrate the requirement for ATM and ATR in the localization of telomerase to telomeres and telomere elongation in immortal human cells. Stalled replication forks increased telomerase recruitment in an ATR-dependent manner. Furthermore, increased telomerase recruitment was observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site (S367). This phosphorylation leads to loss of TRF1 from telomeres and may therefore increase replication fork stalling. ATM and ATR depletion reduced assembly of the telomerase complex, and ATM was required for telomere elongation in cells expressing POT1DeltaOB, an allele of POT1 that disrupts telomere-length homeostasis. These data establish that human telomerase recruitment and telomere elongation are modulated by DNA-damage-transducing kinases.

ALS is a devastating disease, progressing from detachment of motor nerve terminals to paralytic, lethal respiratory failure within several years of diagnosis. The mechanisms responsible for axon withdrawal are poorly understood, but the loss of neuromuscular synapses is sufficient to cause muscle paralysis and therefore central to the disease. Although the subsequent loss of motor neurons has received more attention, preventing or delaying motor neuron cell death without preserving neuromuscular synapses cannot stop disease progression. Skeletal muscles provide retrograde signals that promote the differentiation and stabilization of motor nerve terminals (1, 2). The production of retrograde signals depends upon a synaptic receptor tyrosine kinase, termed MuSK, and Lrp4, a receptor for Agrin that forms a complex with MuSK (3). Because a failure to maintain neuromuscular synapses is central to all forms of ALS, we tested whether increasing retrograde signalling in SOD1^G93A transgenic mice would stabilize neuromuscular synapses, delay axon withdrawal and ameliorate disease symptoms (4). We found that a modest increase in MuSK expression is sufficient to maintain neuromuscular synapses in SOD1^G93A mice, delaying muscle denervation and improving muscle function for over one month (4). Thus, the loss of motor nerve terminals can be delayed by co-opting a retrograde signalling pathway that normally functions to stimulate the differentiation of these terminals (4). These findings suggest a novel therapeutic approach to slow the steady decline in motor function in ALS. Moreover, because motor axon withdrawal is an early, characteristic and critical feature of disease in all forms of ALS, increasing MuSK activity might provide benefit in both familial and sporadic forms of ALS. We sought a more practical therapeutic approach to activate MuSK in vivo. A previous study reported that two human single chain variable region antibodies (ScFv) to MuSK, as well as IgG molecules reconstituted from these ScFv antibodies, stimulate MuSK in cultured myotubes (5). Thus, these antibodies provide an attractive means to activate MuSK in vivo. We found that a single injection of a humanized agonist antibody to MuSK substantially reduced denervation and increased innervation for one month. Thus, increasing MuSK activity, after denervation and disease symptoms were evident, slows synaptic loss. We are currently studying whether the agonist antibody improves motor function and whether chronic dosing with a murinized agonist antibody preserves synapses, improves motor performance, reduces motor neuron cell death and prolongs longevity of SOD1^G93A mice

Inhibitors of apoptosis (IAPs) family of genes encode baculovirus IAP-repeat domain-containing proteins with antiapoptotic function. These proteins also contain RING or UBC domains and act by binding to major proapoptotic factors and ubiquitylating them. High levels of IAPs inhibit caspase-mediated apoptosis. For these cells to undergo apoptosis, IAP function must be neutralized by IAP-antagonists. Mammalian IAP knockouts do not exhibit obvious developmental phenotypes, but the cells are more sensitized to apoptosis in response to injury. Loss of the mammalian IAP-antagonist ARTS results in reduced stem cell apoptosis. In addition to the antiapoptotic properties, IAPs regulate the innate immune response, and the loss of IAP function in humans is associated with immunodeficiency. The roles of IAPs in Drosophila apoptosis regulation are more apparent, where the loss of IAP1, or the expression of IAP-antagonists in Drosophila cells, is sufficient to trigger apoptosis. In this organism, apoptosis as a fate is conferred by the transcriptional induction of the IAP-antagonists. Many signaling pathways often converge on shared enhancer regions of IAP-antagonists. Cell death sensitivity is further regulated by posttranscriptional mechanisms, including those regulated by kinases, miRs, and ubiquitin ligases. These mechanisms are employed to eliminate damaged or virus-infected cells, limit neuroblast (neural stem cell) numbers, generate neuronal diversity, and sculpt tissue morphogenesis.

BACKGROUND: Food intake of the adult fruit fly Drosophila melanogaster, an intermittent feeder, is attributed to several behavioral elements including foraging, feeding initiation and termination, and food ingestion. Despite the development of various feeding assays in fruit flies, how each of these behavioral elements, particularly food ingestion, is regulated remains largely uncharacterized. RESULTS: To this end, we have developed a manual feeding (MAFE) assay that specifically measures food ingestion of an individual fly completely independent of the other behavioral elements. This assay reliably recapitulates the effects of known feeding modulators, and offers temporal resolution in the scale of seconds. Using this assay, we find that fruit flies can rapidly assess the nutritional value of sugars within 20-30 s, and increase the ingestion of nutritive sugars after prolonged periods of starvation. Two candidate nutrient sensors, SLC5A11 and Gr43a, are required for discriminating the nutritive sugars, D-glucose and D-fructose, from their non-nutritive enantiomers, respectively. This suggests that differential sensing mechanisms play a key role in determining food nutritional value. CONCLUSIONS: Taken together, our MAFE assay offers a platform to specifically examine the regulation of food ingestion with excellent temporal resolution, and identifies a fast-acting neural mechanism that assesses food nutritional value and modulates food intake.