Veterinarians in the UK on the use of non-steroidal anti-inflammatory drugs (NSAIDs) for post-disbudding analgesia of calves

<p>Top 20 down-regulated genes after DAPT treatment in P0 Lfng-GFP⁺ cells.</p

Isolation of neonatal supporting cells and analysis by RNA-seq.

<p>(A) Sections through postnatal day 1 and 6 <i>LFng-GFP</i> transgenic mice. At both ages, the GFP transgene is expressed in border cells, inner phalangeal cells, outer pillar cells and all three rows of Deiters’ cells, but not inner pillar cells (arrowheads). Sections are counterstained with antibodies to MYOSIN VI to show hair cells and SOX2, PROX1, GLAST or NGFR to show distinct types of supporting cells (magenta). Scale bar 20 μm. (B) Representative FACS profile for sorting of P1 <i>LFng-GFP</i> transgenic cochleas. GFP intensity is shown on the x-axis, with four fractions (F1-F4) identified on the sorting profile. (C) QPCR analysis of the different fractions for expression of a hair cell marker (<i>Myo6</i>) and a supporting cell marker (<i>Prox1</i>). Cells falling in F3, which contained the highest <i>Prox1</i> signal and lowest <i>Myo6</i> signal were used in subsequent experiments. (D) Identification of transcripts enriched in the GFP⁺ fractions at P1 (left) and P6 (center) using the intersection of differential gene expression between GFP⁺ and GFP^- cells analyzed with DESeq and Cufflinks. 586 consensus GFP⁺ transcripts were identified at P1 and 508 at P6. The right Venn diagram shows the intersection of enriched GFP+ transcripts differentially expressed between P1 and P6 analyzed with DESeq and Cufflinks. 79 transcripts were enriched in P1 GFP⁺ cells compared to P6 GFP⁺ cells, whereas 259 transcripts were enriched in P6 GFP⁺ cells compared to P1 GFP⁺ cells. <i>p</i> < 0.01, q < 0.01 and log2(fold change) > 2.</p

Top 20 genes enriched in Lfng-GFP⁺ cells at P1.

<p>Top 20 genes enriched in Lfng-GFP⁺ cells at P1.</p

Notch and Wnt pathway genes expressed in Lfng-GFP⁺ cells at P1 and P6.

<p>Notch and Wnt pathway genes expressed in Lfng-GFP⁺ cells at P1 and P6.</p

In situ validation of supporting cell-specific transcripts.

<p>Examples of supporting cell genes enriched in <b>(A)</b> P1 supporting cells with basal P6 sections to show negative expression <b>(B)</b> P6 supporting cells with basal P1 sections to show negative expression <b>(C)</b> Both P1 and P6 supporting cells, with sections to show both apical (less mature) and basal (more mature) regions at each age. Brackets: Deiters’ cells; Arrowhead: pillar cell region; horizontal line, greater epithelial ridge region.</p

Gene expression changes in P0 supporting cells in response to Notch inhibition.

<p>Demonstration of rapid down-regulation by <i>in</i> situ hybridization of six supporting cell-specific genes in P0 cochleas after 24 hours of culture in DAPT versus DMSO controls. Scale Bar: 500 μm. Star: apex tip.</p

Biological processes (BP) GO terms represented in the genes enriched in P6 Lfng-GFP⁺ cells.

<p>Biological processes (BP) GO terms represented in the genes enriched in P6 Lfng-GFP⁺ cells.</p

Top 20 differentially expressed genes enriched in P1 Lfng-GFP⁺ cells versus P6 Lfng-GFP⁺ cells.

<p>Top 20 differentially expressed genes enriched in P1 Lfng-GFP⁺ cells versus P6 Lfng-GFP⁺ cells.</p

All significantly up- or down-regulated genes after DAPT treatment in P5 Lfng-GFP⁺ cells.

<p>All significantly up- or down-regulated genes after DAPT treatment in P5 Lfng-GFP⁺ cells.</p

Biological processes (BP) GO terms represented in the list of transcripts down-regulated after DAPT treatment of P0 Lfng-GFP⁺cells that were also enriched in P1 Lfng-GFP⁺cells.

<p>Biological processes (BP) GO terms represented in the list of transcripts down-regulated after DAPT treatment of P0 Lfng-GFP⁺cells that were also enriched in P1 Lfng-GFP⁺cells.</p