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Transcriptome analysis by RNA sequencing of the cortical brain in a mouse model of early life pain

ABSTRACT: Preterm children are frequently exposed to various painful stimuli in the early days of life. Early-life-pain (ELP) may affect cortical development and reduce the fitness and resilience in during youth and adult life. We developed an optogenetic Cre/loxP mouse model of "early-life-pain" (ELP) using mice with transgenic expression of an improved channelrhodopsin-2 (ChR2)/Td-Tomato fusion protein under control of the advillin promoter, that drives expression of ChR2/td-Tomato specifically in primary somatosensory neurons of the dorsal root ganglia (Avil-ChR2 mice). Floxed ChR2/td-Tomato littermates (ChR2-flfl) were used as controls. Mice were exposed to blue light in a chamber once daily from postnatal day P1-P5. Cortical gene expression was analyzed at P7 via RNA-sequencing in eight biological replicates per group. Cortices were rapidly excised and snap froze in liquid nitrogen. Total RNA was extracted from with Qiagen RNeays spin columns and quantified on a Nanodrop. Sequencing libraries were prepared using Illumina TruSeq stranded mRNA library preparation kit, and sequencing was performed on an Illumina NGS platform. The alignment was performed with SeqMan NGen 17 (Lasergene) using the reference genome mm10 provided from UCSC (GRCm38) as template, a minimum read length of 35 bp and automatic adapter trimming. Sequence reads were normalized with EdgeR and analyzed in ArrayStar 17 (Lasergene). Differential gene expression was assessed with general linear models and adjusted according to Benjamini Hochberg.

ORGANISM(S): Mus musculus

PROVIDER: GSE200140 | GEO | 2023/09/06

REPOSITORIES: GEO

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Project description:The phases of fracture healing have been well characterized. However, the exact source and genetic profile of the skeletal progenitors that participate in bone repair is somewhat unclear. Sox9 expression in skeletal elements precedes bone and cartilage formation and a Sox9+ cell type is retained in the adult periosteum. We hypothesized that Sox9+ periosteal cells are multipotent skeletal progenitors normally participating in fracture repair. To test this hypothesis we used tamoxifen (TM)-mediated lineage tracing of Sox9+ cells in Sox9CreErt2:Td-Tomato mice. TM injection indelibly labels Sox9+ cells and all their descendants with the fluorescent reporter protein Td-Tomato. Intact mouse femora were harvested 2 weeks after TM injection and analyzed by histology and immunostaining and RNA sequencing, to evaluate the skeletal distribution and gene expression profile of Td-Tomato positive cells in the adult femur. To assess the role of these cells in fracture repair, mice underwent a closed mid-diaphyseal femoral fracture and their hind limbs were harvested at 3, 9 or 56 days post-fracture to assess the contribution of Sox9+ and Td-tomato+ cells in the fracture healing process. In the intact adult mouse femur, Td-Tomato-labelled cells were observed in articular and growth plate cartilage where Sox9 is known to be expressed, but also in the primary spongiosa, periosteum, and endosteum. RNA sequencing and subsequent analysis showed that Td-Tomato positive periosteal cells were enriched in Sox9 transcripts, and transcripts for various osteogenic and chondrocyte specific genes. In a femoral fracture model, we showed that pre-labeled Td-Tomato positive descendent cells were mobilized during the fracture repair process, expanding and migrating towards the fracture site 3 days post-fracture. Here, Td-Tomato positive cells differentiate into chondrocytes and osteoblasts in the soft and hard callus, respectively, 9 days post-fracture. By 2 months post-fracture, descendants of the original Sox9 labelled cell population were prevalent in the cortex and periosteum, and amongst the differentiated osteocyte population embedded within the cortical bone. Thus, a Sox9+ progenitor population resides in the adult periosteum. Fate tracing shows that these cells likely play a substantial role in repair of the fractured femur giving rise to chondrocytes, osteoblasts and mature cortical osteocytes. To our knowledge this is the first report of this Sox9+ cell population in the periosteum of the adult long bone. Taken together with developmental studies on skeletal formation, our data suggest that Sox9+ osteochondroprogenitors play a continuous role in skeletal formation throughout life.

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Transcriptome analysis by RNA sequencing of the cortical brain in a mouse model of early life pain

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