Deletion of <i>Snai2</i> and <i>Snai3</i> Results in Impaired Physical Development Compounded by Lymphocyte Deficiency

<div><p>The Snail family of transcriptional regulators consists of three highly conserved members. These proteins regulate (repress) transcription via the recruitment of histone deacetylases to target gene promoters that possess the appropriate E-box binding sequences. Murine <i>Snai1</i> is required for mouse development while <i>Snai2</i> deficient animals survive with some anomalies. Less is known about the third member of the family, <i>Snai3</i>. To investigate the function of <i>Snai3</i>, we generated a conditional knockin mouse. Utilizing <i>Cre</i>-mediated deletion to facilitate the ablation of <i>Snai3</i> in T cells or the entire animal, we found little to no effect of the loss of <i>Snai3</i> in the entire animal or in T cell lineages. This finding provided the hypothesis that absence of Snai3 was mitigated, in part, by the presence of Snai2. To test this hypothesis we created <i>Snai2/Snai3</i> double deficient mice. The developmental consequences of lacking both of these proteins was manifested in stunted growth, a paucity of offspring including a dramatic deficiency of female mice, and impaired immune cell development within the lymphoid lineages.</p> </div

Conditional <i>Snai3</i> deletion strategy.

<p>(A) The <i>Snai3</i> WT genomic DNA region contains the <i>Rnf166</i> gene 5 kb upstream and the <i>MVD</i> gene 10 kb downstream. The <i>Snai3</i> gene consists of 5’ and 3’ untranslated regions (white boxes), three exons (black boxes), and two introns. Arrow marks the transcriptional start site (TSS). Primers used for genotyping mice and for QT-PCR of <i>Snai3</i> transcript are labeled as P1-P4 and QT, respectfully, and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069216#pone.0069216.s001" target="_blank">Supplemental Table 1</a>. The Targeting Construct had a LoxP site inserted into the PacI site of intron one and the PL451 cassette inserted into the SalI site 2kb upstream of the TSS. (B) Homologous recombination created the <i>Snai3</i> targeted genome containing unique 5’ PL451 and Knockin LoxP PCR products. The Neomycin (Neo) cassette was deleted via FLP-mediated recombination of Frt sites. (C) Cre recombinase activity deleted the <i>Snai3</i> genomic region and created a new PCR product by bringing together primers P1 and P4, which are normally 6kb apart and unable to make a PCR product. Figure is not to scale.</p

Snai1-3 protein expression suggests post-translational regulation.

<p>Whole cell lysates were generated from total thymus, spleen, bone marrow, and testes. Samples were subjected to immunoblot analysis as described in the <i>Materials</i>. (A–C) Samples were probed with primary antibodies specific for Snai1 (A), Snai2 (B), and Snai3 (C). Blots were probed for β-actin to verify equal protein loading. Representative blots are shown but similar results were generated for two independent mice.</p

<i>Snai2</i> and <i>Snai3</i> DKO mice are developmentally stunted with ocular deformities.

<p>(A) Representative photo of age and sex matched (males) WT and DKO mice. Animals are approximately 6 weeks of age and presented alongside a ruler for reference. (B) Representative photo of WT and DKO male facial ocular area. (C) Graphical representation comparing ages (weeks, wks) and weights (grams, g) of WT and DKO animals over 3 week intervals. One-way ANOVA with Bonferroni post hoc test: ** p < 0.01, *** p < 0.001.</p

Circulating hematopoietic profile of six month old mice.

<p>Retro-orbital bleeds were performed three times for each animal assayed. Three mice were analyzed for WT, Snai2^+/- Snai3^-/-, and Snai2^-/- Snai3^+/- genotypes. Only one DKO animal survived to six months of age. FACS was used to assess overall percentages (A) and absolute numbers of each lineage (B) within the peripheral blood. Significance was tested using one-way ANOVA followed by the Bonferroni post hoc test. ** p < 0.01, *** p < 0.001 (C) Snai2^-/- Snai3^+/- and DKO animals display increased lymphocytes and neutrophils in circulating blood. Cytospins were performed with 30 µl of peripheral blood. Slides were stained with Wright-Giemsa to differentiate between blood cell types. 20x images were photographed and representatives for each genotype are shown.</p

Myeloid populations are enhanced in the <i>Snai2</i> and <i>Snai3</i> DKO.

<p>FACS analysis was performed to assess myeloid cell populations in the bone marrow (A), peripheral blood (B), and spleen (C). Cells were stained for CD11b and Gr1. Macrophages are identified by CD11b⁺ Gr1^Int staining. Neutrophils (representative of granulocytes) are distinguished by CD11b⁺ Gr1^Hi staining. Results are presented as a percentage of total cells analyzed. One-way ANOVA with Bonferroni post hoc test: * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Histological analysis of 4 week old DKO spleen and thymus.

<p>Spleen and thymus were dissected from 4 week old Snai2^+/- Snai3^+/- and DKO animals. Organs were processed for histological analysis as described in the <i>Materials</i>. Tissue sections were cut to an approximate thickness of 10 µm. Representative images are shown for all genotypes and tissues assayed. (A) Splenic sections were left unstained and viewed by brightfield microscopy. 4x magnification of spleen sections from the Snai2^+/- Snai3^+/- and DKO. F = lymphoid follicle; R = red pulp. The DKO spleen is reduced in size compared to the Snai2^+/- Snai3^+/- spleen. (B) Thymus sections were stained with hematoxylin and eosin to differentiate between thymic cortex (darker stain in Snai2^+/- Snai3^+/-) and thymic medulla (lighter stain in Snai2^+/- Snai3^+/-): Snai2^+/- Snai3^+/- (left panel) and DKO (right panel). C = cortex; M = medulla. The DKO thymus is reduced in size compared to the Snai2^+/- Snai3^+/- thymus.</p

<i>Snai2</i> and <i>Snai3</i> DKO lymphoid organs are reduced in size but present a healthy appearance.

<p>(A) Representative photo of thymus, spleen, and bone marrow dissected from age and sex matched WT and DKO animals. Organs are presented with a ruler for reference. (B–C) Quantification of thymic (B) and splenic (C) mass among different iterations of <i>Snai2</i> and <i>Snai3</i> knockouts. % of Body Mass = (Organ Weight (mg) / Body Weight (mg)) X 100, One-way ANOVA with Bonferroni post hoc test: * p < 0.05.</p

Double positive thymocytes are reduced in favor of an increased CD4⁺ single positive population in the <i>Snai2</i> and <i>Snai3</i> DKO.

<p>FACS analysis was performed to assess T cell populations in the thymus (A), peripheral blood (B), and spleen (C). Cells were assayed for CD4 and CD8 cell surface staining. DP = CD4⁺CD8⁺ double positive cells, CD4 = CD4⁺ single positive, CD8 = CD8⁺ single positive, Results are presented as a percentage of total cells analyzed. One-way ANOVA with Bonferroni post hoc test: * p < 0.05, *** p < 0.001.</p

<i>Snai2</i> and <i>Snai3</i> DKO mice demonstrate a severe impairment in B cell development.

<p>FACS analysis was performed to assess B cell populations in the bone marrow (A), peripheral blood (B), and spleen (C). B cell populations were assessed using surface staining for B220 and CD19. In the bone marrow (A), B220⁺CD19^- (pre-pro-) and B220⁺CD19⁺ (pro-, pre-, immature, and mature re-circulating) cells were assayed. In the peripheral blood (B) and spleen (C), B cells were defined as B220⁺CD19⁺. Results are presented as a percentage of total cells analyzed. One-way ANOVA with Bonferroni post hoc test: ** p < 0.01, *** p < 0.001.</p