GSE173284
Transcriptional dysregulation in developing trigeminal sensory neurons in the LgDel mouse model of DiGeorge 22q11.2 deletion syndrome.
Purpose: The cellular composition of the trigeminal ganglion is altered in the mouse model of 22q11.2 deletion syndrome (the LgDel mouse). The goal of this study is to use RNA-seq to identify transcriptional differences in the embryonic day 10.5 trigeminal ganglion that prefigure changes in mature cell identity in this cranial nerve ganglion. Methods: Trigeminal ganglion mRNA profiles of E10.5 trigeminal ganglia from WT and LgDel mice were generated by deep sequencing.  Ganglia samples were prepared as pools of at least 6 ganglia each, from at least 3 embryos.  5 biological replicates of LgDel and WT pools were sequenced.  Paired-end libraries were constructed according to the Illumina protocol for the HiSeq2000 platform. Each pooled CNgV RNA sample, defined as a single biological replicate, was fragmented prior to cDNA conversion to ensure transcript coverage. WT (n?=?5 replicates) and LgDel (n?=?5 replicates) were subjected to sequencing (~100 million paired-end reads/replicate, 100 bp read length) on the Illumina HiSeq2000 platform. Quality-filtered reads were aligned against the mouse reference genome (GRCm38/mm10) using HISAT2 (v.2.1.0). These alignments were indexed using SAMtools (v.1.4) and aligned reads assembled into transcripts using Cufflinks (v.2.2.1) (58). Differential expression analysis was performed using Cufflinks.  Validation of key transcriptional differences was performed by SYBR Green qPCR. Results: For WT RNA-Seq, 111–123 million paired-end reads were generated after QC filtering, and 97.3–97.9% of the reads aligned to the mouse genome reference sequence. For LgDel RNA-Seq, 77–137 million paired-end reads were generated after QC filtering, and 94.3–97.8% of the reads aligned to the mouse genome reference sequence. LgDel versus wild-type (WT) CNgV transcriptomes differ significantly at E10.5 just after the ganglion has coalesced. Some changes parallel altered proportions of cranial placode versus cranial neural crest-derived CNgV cells. Others are consistent with a shift in anterior–posterior patterning associated with divergent LgDel cranial nerve differentiation. The most robust quantitative distinction, however, is statistically verifiable increased variability of expression levels for most of the over 17?000 genes expressed in common in LgDel versus WT CNgV. Conclusions: Quantitative expression changes of functionally relevant genes and increased stochastic variation across the entire CNgV transcriptome at the onset of CN V differentiation prefigure subsequent disruption of cranial nerve differentiation and oropharyngeal function in LgDel mice. Overall design: RNA-seq analysis of trigeminal nucleus in LgDel mouse