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1.
Figure 1

Figure 1. Pbx1 inactivation in Nkx2-5-positive mesenchyme causes spleen hypoplasia. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A–E) Whole mount (A), transverse (A inset, red box corresponds to plane of section indicated by red line; B–C), and sagittal (D–E) sections of Nkx2-5Cre/+;R26R-LacZ embryos from E9.5 to E12.5, stained by β galactosidase. (F) IHC for Pbx1b and (G) Nkx2-5 (black arrowheads) in E10.5 WT transverse sections. (H–J) IF on E11.5 Nkx2-5Cre/+sagittal sections. Co-localization (orange, J inset) of Pbx1 (red), which is widespread in the spleen, and Cre (green) in spleen mesenchyme (outlined by white dashes). (K–N) IHC with α-Pbx1b on E11.5 and 12.5 sagittal sections from control (K,M) and Pbx1 conditional mutant (L,N) embryos, showing Cre-mediated Pbx1 loss in all but a few positive cells (black arrowheads; insets). (O–R) P1 and P7 control (O,Q) and homozygous mutant spleens (P,R). Ao, aorta; C, coelomic cavity; DP, dorsal pancreas; G, gut; Go, gonad; L, liver; Smp, splanchnic mesodermal plate; Sp, spleen; St, stomach; WT, Wildtype. See also Figures S1 and S2.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.
2.
Figure 2

Figure 2. Pbx1 ablation in spleen mesenchyme causes down-regulation of Nkx2-5, essential for spleen growth. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A,B) IHC (E14.5 sagittal sections) shows few Nkx2-5-positive cells in Pbx1 conditional mutants (B) versus widespread Nkx2-5 in controls (A). (C) qRT-PCR reveals a significant reduction of Nkx2-5 mRNA in Pbx1 mutant versus control spleen mesenchyme. (D–G) Reduced Nkx2-5 levels in Nkx2-5+/− (E), and Nkx2-5Cre/GFP (G) mice result in smaller spleens versus controls (D,F: P0 and E17.5). (H) Diagram of the murine Nkx2-5 locus. Enhancers directing expression in heart (green boxes) and spleen-stomach (red box) indicated (Figure S3). (I) Pbx proteins bind to the Nkx2-5 spleen-stomach enhancer, as assessed by ChIP. Primers located within the enhancer (H, red arrows), but not non-specific primers (lane 5), amplify a specific band from α-Pbx immunoprecipitated chromatin from SPCL2 cells (lane 1; Experimental Procedures). (J) A Luc reporter containing WT Nkx2-5 spleen-stomach enhancer (pGL3 Nkx2-5 WT) is transactivated approximately two-fold when transiently transfected into HEK293T cells versus a control Luc reporter (pGL3-empty). This transactivation is abolished when three predicted Pbx binding sites within the enhancer have been mutated (pGL3 Nkx2-5 Mut). Data are mean ± SEM of three independent experiments performed in triplicate. P, pancreas; Sp, spleen; St, stomach. See also Figure S3.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.
3.
Figure 4

Figure 4. Loss of Pbx1/2 in cultured spleen mesenchymal cells reduces proliferation and increases p15Ink4b levels. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A) SP6 cells after infection with a lentivirus containing short hairpin RNAs specific for Pbx1 (shRNA Pbx1, gray line), exhibit significantly reduced cell number over four passages, versus cells infected with a control lentivirus carrying a scrambled shRNA sequence (shRNA scrambled, black line). (B) Asynchronous SP6 cells infected with shRNA Pbx1 show reduced BrdU incorporation versus scrambled shRNA, and accumulate in G0/G1 and G2/M. Loss of Pbx1 reduces percentage of cells in S phase of the cell cycle (p=0.003), and increases percentage of cells in G0/ G1 (p=0.004) and G2/ M (p=0.032). (C) Asynchronous (I) and serum-released (II) growing SP6 cells infected by shRNA Pbx1 exhibit increased p15 mRNA versus shRNA scrambled. (D) Western blot shows reduced Pbx1b and increased p15 in shRNA Pbx1-infected SP6 cells versus control. α-Vinculin, loading control. (E) Pbx1 inactivation in SpM cells by infection with a Cre-expressing adenovirus (Ad-Cre, gray line) significantly reduces cell number over three passages (P), versus SpM cells infected by control adenovirus (Ad-Null, black line). Data are mean ± SEM of one representative experiment out of a total of three performed in triplicate. (F) Reintroduction of Pbx1b by an adenovirus expressing Pbx1b (Ad-Pbx1b), performed four days after Ad-Cre-mediated Pbx1 inactivation, produces a significant increase in cell number (black line) after two passages (P), versus SpM cells infected by Ad-Null (gray line). Data are mean ± SEM of one representative experiment out of a total of two performed in triplicate. See also Figure S5.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.
4.
Figure 7

Figure 7. Pbx1 is present in the early spleen anlage where it directs a regulatory module that converges on Nkx2-5, controlling organ growth and morphogenesis. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A–C) Present at high levels in E13.5 spleen mesenchyme (A), Pbx1 (green) is markedly reduced by P0 (B), and almost completely absent from 6 week-old mouse spleens (C). (D) During normal development, Pbx1/2 promote spleen expansion via multiple pathways (black lettering and lines; left panel): Pbx genes transactivate Nkx2-5 (and Tlx1, as reported; Brendolan et al., 2005) in the spleen mesenchyme, and repress the CDK-inhibitor p15. Moreover, like Pbx, Nkx2-5 can also bind to the regulatory elements of p15 and repress its transcription. Additionally, Nkx2-5 and Tlx1 likely act cooperatively (solid line) to transactivate target genes in spleen mesenchyme. The pivotal role of Nkx2-5 within the Pbx-directed regulatory module is highlighted by its central position within the depicted network. Disruption in any component of this module (illustrated by hollow fonts and dashed lines; right panel) can result in hyposplenia or asplenia in mice and humans. In particular, complete asplenia results from global loss of Pbx1 or Tlx1 in the mouse (Brendolan et al., 2005; Roberts et al., 1994), and is associated with mutation of NKX2-5 in ICA patients (scintigraphy image of patient II.4: white arrow indicates absence of uptake of 99mTc-labeled autologous red cells in the left abdomen below the liver, where the spleen should be located). Also, both spleen-specific Pbx1/2 inactivation and reduced Nkx2-5 dosage in mouse models yield hyposplenia and perturbed spleen morphogenesis. Green lettering and lines indicate findings described in this study; blue lettering and lines indicate previously reported hierarchical genetic control (Brendolan et al., 2005; Roberts et al., 1994). B, bladder; d, dextrum (right side); KL, left kidney; KR, right kidney; LL, left lobe of liver; LR, right lobe of liver; Sp, spleen; St, stomach.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.
5.
Figure 3

Figure 3. Binding of Pbx1 to the p15Ink4b promoter is associated with p15Ink4b repression. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A) p15 locus on mouse chromosome 4. Region upstream of p15 (red box) with evolutionarily conserved sequences (blue peaks). Oligonucleotides for EMSA assays: colored lines. Primers for ChIP: green arrows. Sequence of the p15 promoter (orange box) shown in Figure S4A. (B) qRT-PCR of total RNA reveals significant upregulation of p15 in mutant embryonic spleens versus controls. GAPDH mRNA, internal control. (C) EMSA with radiolabeled oligo-1 (blue line; panel A) containing a Pbx-Prep/Meis binding site (black box). Spleen mesenchymal cell nuclear extracts probed with oligo-1, and treated with α-Pbx Ab (lane 4), resulted in a supershifted band, indicating Pbx binding. Binding specificity confirmed by competition from excess unlabeled WT (lanes 6–7), but not mutated oligo (lanes 8–9). (D) ChIP shows Pbx binding to the p15 promoter. Primers within the promoter (p15Ink4b IN1; panel A; green arrows), but not non-specific primers (OUT2; lane 5) amplify a distinct band from α-Pbx immunoprecipitated chromatin (lane 1). (E) Transient transfection of NIH 3T3 cells with Pbx1 causes a dose-dependent reduction in p15 promoter activity. Data are mean ± SEM of one out of four independent experiments performed in triplicate. (F) IF on E13.5 WT transverse sections. p15 (red) co-localizes in cells (white arrows, insets) exhibiting high levels of the mesenchymal marker vimentin (green). (G–H) IF on sections from E16.5 WT (G) and Pbx1Δex3/Δex3;Nkx2-5Cre/+ (H) spleens. Insets correspond to areas within red boxes. p15 (red) is not present in Pbx1b-positive cells (green). See also Figure S4 and Table S1.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.
6.
Figure 5

Figure 5. Genetic ablation of p15Ink4b, which is bound by Nkx2-5 in its cis-regulatory elements, partially rescues the spleen phenotypes. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A–F) Partial rescue of spleen size and morphogenesis in E15.5 (A–C) and E16.5 (D–F) Pbx1Δex3/Δex3;Nkx2-5Cre/+ mutants on a p15-deficient background. Embryonic spleens (outlined in white) in which Pbx1 has been deleted in the mesenchyme either alone (E,F) or with Pbx2 (B,C), show a significant increase in spleen size and rescued morphology on a p15-deficient background (medium white arrows; C,F), versus littermates which retain p15 (thin white arrows; B,E). Control spleens (thick white arrows; A,D) for comparison. (G–I) Summary of genetic rescue data at E16.5. (G) Spleen surface area measurements (Experimental Procedures) normalized to controls reveal that p15-null Pbx1 mutant spleens are significantly larger than p15+/+ mutant spleens (p=0.014). (H) All rescued embryos develop one single spleen anlage. (I) Proliferation rate in p15−/− mutant spleen mesenchyme is restored to near WT levels, significantly higher than in p15+/+ Pbx1 mutants (p=0.038). Number of samples (n) indicated on corresponding bar of each graph. P, pancreas; Sp, spleen; St, stomach. (J) ChIP shows Nkx2-5 binding to the p15 promoter. Primers located within the p15 promoter (p15Ink4b IN2), but not non-specific primers (OUT1, OUT2, and OUT3), amplify a specific product from αNkx2-5-immunoprecipitated chromatin from SPCL2 cells, as assessed by qPCR and normalized to amplification of input chromatin. (K) Transient transfection of NIH 3T3 cells with 800ng of Pbx1b, NKX2-5 WT or NKX2-5 P236H, or co-transfection of Pbx1b and NKX2-5 in combination (500ng each), causes a reduction in p15 promoter activity. Transactivation normalized to empty expression vector (pcDNA3). Data are mean ± SEM of one out of three independent experiments performed in triplicate. (L) Semi-qRT-PCR on E16.5 mouse splenic RNA does not reveal perturbations of Nkx2-5 in p15−/− mutants versus controls. E16.5 WT pancreas RNA used as negative control for Nkx2-5 expression. All data are mean ± SEM. TBP, TATA-binding protein. See also Figure S6.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.
7.
Figure 6

Figure 6. Characterization of an inherited NKX2-5 mutation in ICA patients. From: Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module.

(A) Pedigree of the family. Black symbols indicate confirmed ICA cases. Gray symbols indicate siblings who died early in childhood with clinical symptoms similar to those of II.4. Haplotypes of NKX2-5 are indicated: M stands for c.707C>A, and WT for the wild-type allele. E? indicates unknown haplotype. (B) Illumina sequencing reads displayed for patient I.1. Reads overlapping the mutation in exon 2 of NKX2-5 (bp position g.172,659,827 – g.172,659,852, hg19) reveal the heterozygous substitution of C to A (reads are reversed). (C) The P236H mutation is plotted on a schematic diagram of the NKX2-5 protein (drawn to scale) showing conserved and/or functional domains (homeodomain: HD; tinman/Nkx2-5 domain: TN; NK2 specific domain: NK; tyrosine rich domain: YRD). Two other conserved domains, the NKX2-5 box and the GIRAW motif, are indicated by yellow and green boxes, respectively. Evolutionary conservation of the NKX2-5 region containing amino acid P236 is represented below. (D) Depicted are spleens from Nkx2-5Y-A:IRESlacZ/+↔wildtype chimaeric P0 mouse pups (Prall et al., 2007). Chimaeric spleens (right panel), with reduced Nkx2-5 function, are smaller and dysmorphic, compared to WT (left panel). (E) An expression vector encoding a construct consisting of the Gal4 DNA-binding domain fused to the C-terminus of WT human NKX2-5 (NKX2-5 WT) causes a greater than 30-fold increase in transactivation when transiently co-transfected into HEK293T cells with a UAS-Luc reporter. This transactivation is reduced by more than 50% when a fusion construct bearing the P236H mutation (NKX2-5 P236H) is used. Constructs expressing the Gal4-DBD alone, or fusion constructs in which all Tyrosines in the YRD have been substituted with Alanines or Phenylalanines (NKX2-5 Y->A and NKX2-5 Y->F, respectively; Elliott et al., 2006), used as negative controls. Data are mean ± SEM of two independent experiments performed in triplicate. (F) Human NKX2-5 WT expression vector transiently co-transfected into SPCL2 cells with Tlx1 expression vector and a Luc reporter containing the Nkx2-5 spleen-stomach enhancer (pGL3-Nkx2-5-Luc) increased transactivation nearly three-fold relative to reporter alone. Co-transfection of Tlx1 with mutated NKX2-5 expression construct bearing the P236H mutation (NKX2-5 P236H) did not transactivate the Luc reporter, nor did NKX2-5 WT, NKX2-5 P236H, or Tlx1 transfected singly. Data are mean ± SEM of four independent experiments performed in triplicate. See also Figure S7 and Tables S2–S5.

Matthew Koss, et al. Dev Cell. ;22(5):913-926.

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