Faculty Bibliography

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1-induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA-binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus nondifferentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1-bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that polycomb repressive complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1-repressed genes. These data provide insights into GATA-1-mediated gene regulation in vivo.

Although mature neurotrophins are well described trophic factors that elicit retrograde survival signaling, the precursor forms of neurotrophins (i.e., proneurotrophins) can function as high-affinity apoptotic ligands for selected neural populations. An outstanding question is whether target-derived proneurotrophins might affect neuronal survival/death decisions through a retrograde transport mechanism. Since neurotrophin-3 (NT-3) is highly expressed in non-neural tissues that receive peripheral innervation, we investigated the localized actions of its precursor (proNT-3) on sympathetic neurons in the present study. Pharmacological inhibition of intracellular furin proteinase activity in 293T cells resulted in proNT-3 release instead of mature NT-3, whereas membrane depolarization in cerebellar granule neurons stimulated endogenous proNT-3 secretion, suggesting that proNT-3 is an inducible bona fide ligand in the nervous system. Our data also indicate that recombinant proNT-3 induced sympathetic neuron death that is p75(NTR)- and sortilin-dependent, with hallmark features of apoptosis including JNK (c-Jun N-terminal kinase) activation and nuclear fragmentation. Using compartmentalized culture systems that segregate neuronal cell bodies from axons, proNT-3, acting within the distal axon compartment, elicited sympathetic neuron death and overrode the survival-promoting actions of NGF. Together, these results raise the intriguing possibility that dysregulation of proneurotrophin processing/release by innervated targets can be deleterious to the neurons projecting to these sites

Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors

Neurogenesis and cell differentiation in the brain continues throughout life. In the subventricular zone and rostral migratory stream, precursor cells contact each other. Cell-cell interactions mediated via adhesion molecules are no doubt involved in establishing and maintaining the neurogenic ability of these cells. Here, we demonstrate that N-cadherin plays important roles in forming cell clusters and in regulating cell differentiation. N-cadherin is abundantly expressed in chain migrating cells in the subventricular zone and rostral migratory stream but is down-regulated after cells exit these regions. We also show that neurosphere formation is inhibited via suppression of N-cadherin function and that N-cadherin expression is decreased after induction of neurosphere differentiation. Furthermore, we demonstrate that functional blockade of N-cadherin can enhance glial cell differentiation in explant cultures of precursors from the subventricular zone.

BACKGROUND:This study is a comparative epigenetic evaluation of the methylation status of the DLC1 tumor suppressor gene in naturally-occurring canine lymphoma. Canine non-Hodgkin's lymphoma (NHL) has been proposed to be a relevant preclinical model that occurs spontaneously and may share causative factors with human NHL due to a shared home environment. The canine DLC1 mRNA sequence was derived from normal tissue. Using lymphoid samples from 21 dogs with NHL and 7 normal dogs, the methylation status of the promoter CpG island of the gene was defined for each sample using combined bisulfite restriction analysis (COBRA), methylation-specific PCR (MSP), and bisulfite sequencing methods. Relative gene expression was determined using real-time PCR. RESULTS:The mRNA sequence of canine DLC1 is highly similar to the human orthologue and contains all protein functional groups, with 97% or greater similarity in functional regions. Hypermethylation of the 5' and 3' flanking regions of the promoter was statistically significantly associated with the NHL phenotype, but was not associated with silencing of expression or differences in survival. CONCLUSION/CONCLUSIONS:The canine DLC1 is constructed highly similarly to the human gene, which has been shown to be an important tumor suppressor in many forms of cancer. As in human NHL, the promoter CpG island of DLC1 in canine NHL samples is abnormally hypermethylated, relative to normal lymphoid tissue. This study confirms that hypermethylation occurs in canine cancers, further supporting the use of companion dogs as comparative models of disease for evaluation of carcinogenesis, biomarker diagnosis, and therapy.

The C protein of human parainfluenza virus type 3 (HPIV3) is a multifunctional accessory protein that inhibits viral transcription and interferon (IFN) signaling. In the present study, we found that removal of N-terminal 25 or 50 amino acid residues from the C protein (CNDelta25 or CNDelta50) totally abolished viral RNA synthesis in the HPIV3 minigenome system. Further N-terminal or C-terminal deletion impaired the inhibitory ability of CNDelta25 and CNDelta50. Subsequent mutagenesis analysis suggested that the N-terminal-charged amino acid residues (K3, K6, K12, E16, and R24) contribute to the higher inhibition caused by CNDelta25 than the C protein. Consistent with viral RNA synthesis inhibition, the growth of HPIV3 was significantly decreased by 5 logs in HeLa-derived cell line expressing CNDelta25. Interestingly, replication of respiratory syncytial virus (RSV), another important respiratory tract pathogen, was also strongly inhibited in the presence of CNDelta25. These findings provide a promising potential to use CNDelta25 as an antiviral agent against the clinically important respiratory tract diseases caused by HPIV3 and RSV.

Nrf2:INrf2 (Keap1) are cellular sensors of chemical- and radiation-induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor, INrf2 which functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2 cysteine 151 and/or protein kinase C phosphorylation of Nrf2 serine 40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds the antioxidant response element, which leads to coordinated activation of gene expression. It takes less than 15 min from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls the switching off of Nrf2 activation of gene expression. GSK3beta phosphorylates Fyn at an unknown threonine residue(s), leading to the nuclear localization of Fyn. Fyn phosphorylates Nrf2 tyrosine 568, resulting in the nuclear export of Nrf2, binding with INrf2, and degradation of Nrf2. The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis, and promotes cell survival.

The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, we first conducted a molecular screen for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. We also find that the genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with our findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination.

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.