Project description:Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta/alpha (IL1beta/alpha) modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Interleukin-6 (IL6), also released during inflammation, affects transcriptional responses in primary chromaffin cells, and may coordinate immune and autonomic adrenomedullary responses via an autocrine mechanism, as TNFalpha itself strongly induces IL6 expression in chromaffin cells, which in turn express receptors responsive to IL6. We have examined the signaling mechanisms employed by IL6 to affect tyrosine hydroxylase (TH) enzymatic activation, and adrenomedullary gene transcription, in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and serine phosphorylation of signal transducer and activator of transcription 3 (STAT3), as do several other first messengers acting on the chromaffin cell, including histamine, nicotine and angiotensin II. IL6 uniquely activated tyrosine phosphorylation of STAT3. Consistent with a short-term ERK1/2 activation, IL6 treatment caused prompt regulation of TH phosphorylation, and up-regulation of genes encoding secreted proteins of the adrenal medulla including galanin, vasoactive intestinal peptide (VIP), gastrin releasing peptide (GRP) and parathyroid hormone-like hormone (PTHLH). We further examined the effects of IL6 treatment on the entire bovine chromaffin cell transcriptome. Of 90 genes up-regulated by IL6, only 16 of which are known targets of IL6 in the immune system. The remaining genes likely represent a combination of novel IL6/STAT3 targets, targets of ERK1/2 shared by other first messengers, and, potentially, IL6-dependent genes activated in a secondary cascade via transcription mediated by IL6-induced transcription factors, such as HIF-1alpha. Notably, genes induced by IL6 represent a cohort with a profile that includes both neuroendocrine-specific genes, including several that are activated by G-protein couple receptor (GPCR) signaling pathways initiated by histamine and pituitary adenylate cyclase-activating polypeptide (PACAP), and some transcripts also activated by cytokines including interferon-alpha (INFalpha and TNFalpha. These results suggest an integrative role for IL6 in overall fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge in the adrenal medulla.
Project description:During embryonic development, nerve-associated Schwann cell precursors (SCPs) give rise to chromaffin cells of the adrenal gland via the “bridge” transient stage, according to recent functional experiments and single cell data from humans and mice. However, currently existing data do not resolve the finest heterogeneity of developing chromaffin populations. Here we took advantage of deep SmartSeq2 transcriptomic sequencing to expand our collection of individual cells from developing murine sympatho-adrenal anlage and uncover the microheterogeneity of embryonic chromaffin cells and corresponding developmental paths. After improving our atlas of sympatho-adrenal development and performing experimental validations, we discovered that SCPs in the local nerve show high degree of microheterogeneity corresponding to early biases towards either Schwann or chromaffin terminal fates. Furthermore, we found that a post-“bridge” population of developing chromaffin cells gives rise to persisting immature chromaffin cells and the two terminal populations (adrenergic and noradrenergic) via diverging differentiation paths. Taken together, we provide a thorough identification of novel markers of adrenergic and noradrenergic populations in developing adrenal glands and report novel differentiation micro-paths leading to them.
Project description:Adrenal chromaffin cells and sympathetic neuron are derived from neural crest precursors and both cell types can give rise to childhood cancer, neuroblastoma. However only limited is known about the mechanism of their development. Better understanding of their transcriptomic profiles during development could gives an insight into the cell fates acquisition as well as the origin of neuroblastoma. Yellow fluorescent protein expressing sympathetic neuroblasts and adrenal chromaffin cells were isolated from E12.5 TH-IRES-Cre;ROSA26-EYFP mouse embryos by fluorescence-activated cell sorting. Transcriptomic profiles of sympathetic neuroblasts and adrenal chromaffin cells from embryonic age (E)12.5 TH-IRES-Cre;ROSA26-EYFP mice were generated by RNA sequencing, in four paired biological replicates.
Project description:Angiotensin II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an acute increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the upregulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species: human, bovine, and rat. Experiment Overall Design: Microarray analysis was performed using samples from control and Ang-II-(10 nM) treated (1 hour) cells from human adrenocortical tumor cell line H295-R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human HG-133 + 2 , bovine, and rat 230-2 Affymetrix chips. qPCR was performed to confirm upregulation of selected genes using mRNA. Dye Swap was not used. Human samples (H295R) include 3 replicates of Basal (controls) and 3 repeats of Angiotensin II samples. Human H295R cells include also a group of cycloheximide (protein synthesis blocker) and angiontensin II + cycloheximide in order to check if the genes were direct targets of angiotensin II.
Project description:Pheochromocytoma (PCC) and paraganglioma (PGL) are neuroendocrine tumours arising in the adrenal medulla and paraganglia of the autonomous nervous system, respectively. Malignant PCC/PGL are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Gene expression changes associated with SDHB inactivation were investigated in a two genetically defined murine cellular models. These models were generated by Cre-mediated recombination in spontaneously immortalized mouse chromaffin cells (imCCs) and adrenal fibroblasts (MAFs).
Project description:The Toll-like receptor (TLR) and peptidoglycan recognition protein 1 (PGLYRP1) genes play key roles in the innate immune systems of mammals. While the TLRs recognize a variety of invading pathogens and induce innate immune responses, PGLYRP1 is directly microbicidal. We used custom allele-specific assays to genotype and validate 220 diallelic variants, including 54 nonsynonymous SNPs in 11 bovine innate immune genes (TLR1-TLR10, PGLYRP1) for 37 cattle breeds. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and we were unable to differentiate between the specialized B. t. taurus beef and dairy breeds, despite an average polymorphism density of one locus per 219 bp. Ninety-nine tagSNPs and one tag insertion-deletion polymorphism were sufficient to predict 100% of the variation at all 11 innate immune loci in both subspecies and their hybrids, whereas 58 tagSNPs captured 100% of the variation at 172 loci in B. t. taurus. PolyPhen and SIFT analyses of nonsynonymous SNPs encoding amino acid replacements indicated that the majority of these substitutions were benign, but up to 31% were expected to potentially impact protein function. Several diversity-based tests provided support for strong purifying selection acting on TLR10 in B. t. taurus cattle. These results will broadly impact efforts related to bovine translational genomics.
Project description:BackgroundWe present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS) only assembly used for many other animal genomes including the rhesus macaque.ResultsThe assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information.Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5%) of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly.ConclusionThe biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research.