Primers for gender identification and genotyping.

Primers for gender identification and genotyping.</p

The effect of Hltf deletion on the fetal-to-placental-weight ratio.

<p>Fetal weight (A), placental weight (B) and fetal/placental weight ratios (C) were unaffected by <i>Hltf</i> deletion. Statistics for <i>Hltf</i> -/- animals were: fetal weight (n = 18), placental weight (n = 28) and ratio data (n = 18 paired values). Statistics for <i>Hltf</i> +/+ animals were: fetal weight (n = 19), placental weight (n = 26), and ratio data (n = 19 paired values). Values in each category were compared with Student’s <i>t</i>-test (significance, p<0.05). The histograms show data are not different (p>0.05).</p

Isoform discovery and alternative expression of Hltf.

<p>A diagram illustrating the intron retention event at exon 21 resulting in the formation of exon 21B. Cufflinks assembled the five control transcripts and estimated their abundance. Then cuffdiff tested for differential Hltf isoform expression based on exon and junction read counts. Exon 21 was joined (98 total reads) with intron B to form exon 21B; however, neither exon 21 nor intron B were joined (0 reads) to exon 22. The diagram shows the tandem stop codons (red) in exon 21B that terminate protein translation. Positive immunolabeling of the C-terminus of Hltf in secondary spermatocytes in seminiferous tubules of rat (A) and mouse (B) testis compared with the absence of immunolabeling to the C-terminus of Hltf in placenta (C; negative control D) confirms no full-length Hltf protein is expressed in any cell types of the placenta. Alternatively, immunolabeling with an antibody to amino acids (aa) 164–300 that are common to all known Hltf protein variants confirms the abundance of truncated Hltf protein in placenta (E; negative control F). 40x magnification.</p

Photomicrographs of histological sections of mouse placenta on gd18.5.

Sections from wild type (A-E) and Hltf null (F-J) placentae show widespread DBA-lectin reactivity for uNK cells at 10 X magnification (A, F). Comparisons at 40 X magnification of DBA-lectin reactivity for uNK cells (B, G) and amylase-resistant PAS positive uNK cells (C, H) show abundant cytoplasmic granules contained within the cellular membranes. Immunolabeling (20 x magnification) for perforin is negligible in uNK cells from wild type (D; blue arrows) compared with Hltf null (I). Immunolabeling (20 x magnification) for Hltf shows both nuclear and cytoplasmic staining throughout the wild type (E) placenta compared to the complete absence of Hltf protein in the null placenta (J). All insets are negative (minus primary antibody) controls.</p

Nuclear localization of protocadherin gamma in trophoblasts.

<p>Proteolytic processing by γ-secretase precedes nuclear localization of the C-terminal domains common to all γ-protocadherin adhesion proteins. Immunolabeling with an antibody to the C-terminal domain of all γ-protocadherin proteins shows extensive nuclear localization of the proteolytic fragment in wild type control (A-C) compared with staining on the inner, cytoplasmic, side of the cell membrane, diffuse cytoplasmic stain and generally negligible nuclear staining in <i>Hltf</i> null (D-F) placenta. Magnifications are A, D 20X, B, E 40X, C, F 100X oil emersion.</p

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface - Fig 5

<p><b>Gene measured composite volcano and expression bar plots:</b> A. Volcano plot highlighting increased expression of perforin 1 <i>(Prf1</i>) and granzyme B (<i>Gzmb</i>) when <i>Hltf</i> is deleted. B. All the differentially increased genes [perforin1 (<i>Prf1</i>), albumin (<i>Alb</i>), fibrinogen-like protein 2 (<i>Fgl2</i>), granzymes (<i>Gzmb-Grzmg</i>) and lysozyme 2 (<i>Lyz2</i>)] that are annotated to cytolysis are ranked based on the significance of their measured logFC. The box and whisker plot on the left summarizes the distribution of all the differentially increased genes that are annotated to this GO:0019835 term. The box represents the 1st quartile, the median and the 3rd quartile.</p

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface - Fig 2

<p><b>Volcano plots:</b> Differential expression (DE) of (A) trophoblast lineage markers [<i>Prl2c1</i>, <i>Ctsq</i>, <i>Prl4a1</i>, <i>Syna</i>, <i>Cxcr7</i> and <i>Gcm1</i>] and (B) glucose regulators [<i>Slc2a1</i> (Glut 1), <i>Slc2a3</i> (Glut 3), and <i>Slc2a12</i> (Glut 12)] is represented in terms of their measured expression change (x-axis) and the significance of the change (y-axis). <i>Prl4a1</i>, <i>Gcm1</i>, <i>Syna</i>, and <i>Cxcr7</i> are represented by a single yellow dot because they share the same change in gene expression (p = 0.004) compared to their matched controls. Significance is represented in terms of the negative log (base 10) of the p-value, so that more significant genes are plotted higher on the y-axis. The dotted lines represent the thresholds used to select the DE genes: LogFC = <b><i>0</i>.<i>6</i></b> for expression change and <b><i>0</i>.<i>05</i></b> for significance (p-value shown in terms of the negative log (base 10) value).</p

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface

<div><p><i>Hltf</i> is regulated by intron retention, and global <i>Hltf</i>-deletion causes perinatal lethality from hypoglycemia. In heart, full-length Hltf is a transcriptional regulator of <i>Hif-1α</i> that controls transport systems. Thus, we tested the hypothesis that <i>Hltf</i> deletion from placenta caused or exacerbated neonatal hypoglycemia via <i>Hif-1α</i> regulation of nutrient transporters. RNA-seq data analyses identified significant changes in transcript expression and alternative splicing (AS) in E18.5 placentome. iPathwayGuide was used for gene ontology (GO) analysis of biological processes, molecular functions and cellular components. Elim pruning algorithm identified hierarchical relationships. The methylome was interrogated by Methyl-MiniSeq Epiquest analysis. GO analysis identified gene enrichment within biological processes. Protein expression was visualized with immunohistochemistry. Although two Hltf mRNA isoforms are quantifiable in most murine tissues, only the truncated Hltf isoform is expressed in placenta. The responsible intron retention event occurs in the absence of DNA methylation. iPathwayGuide analysis identified 157 target genes of 11,538 total genes with measured expression. These were obtained using a threshold of 0.05 for statistical significance (p-value) and a long fold change of expression with absolute value of at least 0.6. <i>Hltf</i> deletion altered transcription of trophoblast lineage-specific genes, and increased transcription of the <i>Cxcr7</i> (p = 0.004) gene whose protein product is a co-receptor for human and simian immunodeficiency viruses. Concomitant increased Cxcr7 protein was identified with immunolabeling. <i>Hltf</i> deletion had no effect on transcription or site-specific methylation patterns of <i>Hif-1α</i>, the major glucose transporters, or System A amino acid transporters. There was no measureable evidence of uteroplacental dysfunction or fetal compromise. iPathGuide analysis revealed Hltf suppresses cytolysis (10/21 genes; p-value 1.900e-12; p-value correction: Elim pruning; GO:019835) including the perforin-granzyme pathway in uterine natural killer cells. Our findings 1) prove the truncated Hltf protein isoform is a transcription factor, 2) establish a functional link between AS of Hltf and immunosuppression at the feto-maternal interface, 3) correlate intron retention with the absence of DNA methylation, and 4) underscore the importance of differential splicing analysis to identify Hltf’s functional diversity.</p></div

Trilaminar trophoblast structure of the feto-maternal interface.

<p>The diagrammatic representation (A) is provided as a guide to understanding Cxcr7 protein localization on the maternal side (arrow) of Hltf null trophoblast (B) with fetal capillary (asterisk). Increased Cxcr7 protein in Hltf null (C) compared to control (D) placenta. Minus primary antibody (E) in Hltf null placenta shows negligible immunostaining. 40x magnification.</p

Exploration of the tissue architecture of IC and ID β <i>HLTF</i> KO pancreata.

(A) Two-dimensional batch-corrected t-distributed stochastic neighbor embedding (t-SNE) visualization of the UMI counts from the entire IC vs ID dataset. (B) Pancreata clusters 16 and 18 are shown in t-SNE space. (C) Pancreatic genes insulin 1 (Ins1) and insulin 2 (Ins2) illustrated differential gene expression in IC vs ID β Hltf KO mice in t-SNE plots of clusters 16 and 18. The unique expression of immune cell markers in IC vs ID tissue is elaborated in t-SNE plots of these clusters. Slamf6 and Il2rb were added to previously identified markers for NK cells (GzmA, Klrb1b). Five markers for B cells (Pax5, Blk, Fcmr, Fcrla, Tnfrasf9) and three markers for activation of innate immunity (Bpifb1, Serpinb3a, Defb36) were unique to cluster 18.</p