Transcriptomic analysis indicates a role for oxidative phosphorylation in postnatal development of rat carotid body hypoxic sensitivity

The carotid body (CB), located bilaterally at the carotid artery bifurcations, is the primary sensory organ for monitoring arterial blood O2 levels. Carotid bodies are immature at birth, exhibiting low sensitivity to hypoxia and become more sensitive with maturation during the first few weeks of neonatal life. To understand the molecular basis for the postnatal developmental hypoxic responses of CB, we isolated CBs from 5-day and 21-day-old Sprague Dawley rats, and performed RNA-sequencing, which allows comprehensive analysis of gene expression. Differentially expressed genes (DEGs) were generated using Edge R, while functional enrichment analysis was performed using GSEA. Analysis of RNA seq data showed 1769 DEGs of the total 12,696 genes shared between neonates and adults. Of the 1769 DEGs, 924 genes were upregulated, and 1680 genes were downregulated. Further analysis showed that genes related to oxidative phosphorylation (Ox/phos) and hypoxia-signaling pathways were significantly upregulated in neonatal compared to adult CBs, suggesting a possible link to differential developmental hypoxic responses seen in CB. Genes related to cytokine signaling (INF? and TNFa) and transcription factors (CREB and NF?B) mediated pathways were enriched in adult CBs suggesting expression of these pathways may be linked to developmental regulation. The RNA-seq results were verified by analyzing mRNA changes in selected genes by qRT-PCR. Our results of enrichment analysis of biological pathways offer a valuable insight into CB hypoxic sensing responses and development process. Overall design: Sprague Dawley pregnant rats purchased from Charles River Laboratories were used for the experiments. Mother was housed on a 12h light dark cycle and fed ad libitum a standard pelleted rodent diet with free access to the water. Pups aged 5 days were sacrificed by IP urethane injection and bifurcations were harvested. One third of the litter was allowed to grow to 21 days after which they were sacrificed, and CB bifurcations were harvested. CBs were isolated from the bifurcations and immediately transferred to RNA Later at 40C. Total RNAs were extracted using the Direct-ZOL RNA micro prep kit (Zymo Research; #R2060) according to the manufacturer's instructions. For each experiment, CBs from 6 pups (post-natal day 5) or 3 adult rats (post-natal day 21) were pooled per sample. We then performed gene expression profiling analysis using data obtained from RNA seq of CBs at two time points (5day and 21 day old) run in duplicates