Comparison of overall gene expression between the three treatment groups.

<p>Mammary gland samples belong to wild-type dams (WT), Tryptophan hydroxylase (<i>Tph1</i>) deficient dams (serotonin deficient; KO), and <i>Tph1</i> deficient dams injected daily with 5-hydroxytryptophan (RC). (<b>A</b>) The bar graph shows the number of genes upregulated in each group for each of the three pairwise comparisons. The numbers represent the total number of differentially expressed genes (DEG) at 5% FDR (and at 1% FDR in parenthesis). (<b>B</b>) The Venn diagram shows the overlap between genes that showed significant differential expression at 5% FDR in each of the three pairwise comparisons.</p

Medical Subject Headings (MeSH) terms that were significantly enriched (FDR ≤ 0.01) with differentially expressed genes detected in the mammary gland on day 10 of lactation for the following pairwise comparisons: WT <i>vs</i>. KO and WT <i>vs</i>. RC.

<p>Medical Subject Headings (MeSH) terms that were significantly enriched (FDR ≤ 0.01) with differentially expressed genes detected in the mammary gland on day 10 of lactation for the following pairwise comparisons: WT <i>vs</i>. KO and WT <i>vs</i>. RC.</p

Functional characterization of the 62 most significant genes (FDR ≤ 0.01 and fold change ≥ 2) detected simultaneously in the mouse mammary gland on day 10 of lactation between WT <i>vs</i>. KO dams and WT <i>vs</i>. RC dams.

<p>WT = wild-type mice; KO = Tryptophan hydroxylase (<i>Tph1</i>) deficient mice; RC = Rescue mice (<i>Tph1</i> KO + 100 mg/kg daily injections of 5-hydroxytryptophan). Gene symbols that are underlined denote genes downregulated in the mammary gland of KO and RC dams compared to WT dams.</p><p>Functional characterization of the 62 most significant genes (FDR ≤ 0.01 and fold change ≥ 2) detected simultaneously in the mouse mammary gland on day 10 of lactation between WT <i>vs</i>. KO dams and WT <i>vs</i>. RC dams.</p

Functional characterization of the 204 most significant genes (FDR ≤ 0.01 and fold change ≥ 2) detected in the mouse mammary gland on day 10 of lactation between WT <i>vs</i>. RC dams.

<p>WT = wild-type mice; RC = Rescue mice (<i>Tph1</i> KO + 100 mg/kg daily injections of 5-hydroxytryptophan). Gene symbols that are underlined denote genes downregulated in the mammary gland of RC dams compared to WT dams.</p><p>These are functional terms that were detected exclusively in this specific comparison.</p

Functional characterization of the 97 most significant genes (FDR ≤ 0.01 and fold change ≥ 2) detected in the mouse mammary gland on day 10 of lactation between WT <i>vs</i>. KO dams.

<p>WT = wild-type mice; KO = Tryptophan hydroxylase (<i>Tph1</i>) deficient mice. Gene symbols that are underlined denote genes downregulated in the mammary gland of KO dams compared to WT dams.</p><p>These are functional terms that were detected exclusively in this specific comparison.</p

List of Gene Ontology (GO) terms that were significantly enriched (FDR ≤ 0.01) with differentially expressed genes (DEG) detected between wild-type (WT) <i>vs</i>. Knock-out mammary glands on day 10 of lactation.

<p>The graph shows the term name, the total number of genes within each term, and the number of genes up-regulated (green) and down-regulated (yellow) in the knock-out compared to the wild-type. Knock-out = Tryptophan hydroxylase (<i>Tph1</i>) deficient mice.</p

List of Gene Ontology (GO) biological process terms that were significantly enriched (FDR ≤ 0.01) with differentially expressed genes (DEG) detected simultaneously in both pairwise comparisons involving the wild-type (WT) mammary glands on day 10 of lactation: WT <i>vs</i>. Knock-out (A) and WT <i>vs</i>. Rescue (B).

<p>The graph shows the term name, the total number of genes within each term, and the number of genes up-regulated (green) and down-regulated (yellow) in the knock-out or rescue compared to the WT. Knock-out = Tryptophan hydroxylase (<i>Tph1</i>) deficient mice; Rescue = <i>Tph1</i> knock-out + 100 mg/kg daily injections of 5-hydroxytryptophan.</p

Validation of overall gene expression.

<p>Fold changes of four differentially expressed genes measured by RNA-Seq (blue) versus qRT-PCR (orange). Genes period circadian clock 2 (Per2), a novel transcript unit [Chr5:30,208,00730,212,059] (NTU), and G protein-coupled receptor 113 (Gpr113) were statistically upregulated in the mammary gland of RC dams while gene GLIS family zinc finger 3 (Glis3) was statistically upregulated in the mammary gland of WT dams.</p

List of Gene Ontology (GO) terms that were significantly enriched (FDR ≤ 0.01) with differentially expressed genes (DEG) detected between wild-type (WT) <i>vs</i>. Rescue mammary gland on day 10 of lactation.

<p>The graph shows the term name, the total number of genes within each term, and the number of genes up-regulated (green) and down-regulated (yellow) in the Rescue compared to the wild-type. Rescue = Tryptophan hydroxylase (<i>Tph1</i>) knock-out + 100 mg/kg daily injections of 5-hydroxytryptophan.</p

Knockdown of <i>CDKN1C (p57^kip2)</i> and <i>PHLDA2</i> Results in Developmental Changes in Bovine Pre-implantation Embryos

<div><p>Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (<i>MAGEL2</i>, <i>UBE3A</i>, <i>IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10</i>, <i>RTL1, IGF2, H19</i>, <i>MIM1,</i> and <i>XIST</i>) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The <i>CDKN1C</i> gene showed the highest upregulation in blastocysts whereas <i>PHLDA2</i> was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to <i>PHLDA2</i> into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of <i>CDKN1C</i>-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of <i>CDKN1C</i>-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes <i>CDKN1C</i> and <i>PHLDA2</i> are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.</p></div