Enu Mutagenesis Identifies a Novel Platelet Phenotype in a Loss-Of-Function Jak2 Allele

<div><p>Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (<i>Jak2</i>^<i>K915X</i>), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (<i>Jak2</i>^<i>+/-</i>). Timed pregnancy experiments revealed that <i>Jak2</i>^{<i>K915X/K915X</i>} and <i>Jak2</i>^<i>-/-</i> displayed embryonic lethality; however, <i>Jak2</i>^{<i>K915X/K915X</i>} embryos were viable an additional two days compared to <i>Jak2</i>^<i>-/-</i> embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.</p> </div

Strain 7254 is defined by a K915X mutation and generates a non-functional truncated Jak2 protein.

<p>(A) The Jak2 domain structure is indicated. The DNA sequence from <i>7254</i> splenocytes and corresponding protein sequence are also shown. (B) Splenocytes were harvested from phenylhydrazine primed <i>Jak2</i>^<i>K915X</i> and <i>Jak2</i>^<i>+/-</i> mice and their wild type littermates. HA-tagged Jak2 and Jak2^K915X were also expressed in 293T cells. A Western blot was performed with a peptide-specific JAK2 antibody. (C) 293T cells were transfected with cDNAs encoding HA-Jak2 or HA-Jak2 K915X. Western blotting was performed with phosphorylation-specific antibodies that recognize pSer-523, pTyr-570 and pTyr-1007/1008 in Jak2. The membranes were stripped and reprobed with an anti-HA antibody. (D) HA-tagged versions of Jak2 and Jak2^K915X with or without the JAK2 3’ UTR were expressed in 293T cells. Western blots were performed with 4G10 anti-phosphotyrosine and HA antibodies. Immunoblotting with anti β-tubulin was performed to demonstrate equal loading.</p

The K915X mutation in Jak2 enhances viability of mouse embryos.

<p>Embryos from timed matings were sacrificed at E12.5-E14.5. Representative embryos from E12.5 are shown. A summary of the results is provided in tabular format.</p

<i>Jak2</i>^{<i>K915X/+</i>} mice have elevated megakaryocytes and platelets.

<p>(A) Bone marrow sections were prepared from 12 week <i>Jak2</i>^<i>+/+</i> and <i>Jak2</i>^{<i>K915X/+</i>} mice and stained with H and E. Representative sections are illustrated at 20x magnification. Megakaryocytes are indicated by an asterisk. (B) Platelets from male and female wild type, <i>Jak2</i>^<i>+/-</i> and <i>Jak2</i>^<i>K915X</i> mice at 8 wk of age were monitored. (C) Red blood cells were evaluated from male and female mice at 8 wk of age from WT, <i>Jak2</i>^<i>+/-</i> and <i>Jak2</i>^{<i>K915X/+</i>} mice. <i>Jak2</i>^<i>+/+</i> or <i>JAK2</i>^{<i>Control</i>} mice were littermate controls of <i>Jak2</i>^<i>+/-</i> or <i>Jak2</i>^{<i>K915X/+</i>} breedings, respectively. Statistically significant differences between groups are denoted as *, p< 0.05 and **, p<0.0001. Each group has n=20-30.</p

Cardiac edema in zebrafish embryos injected with <i>klf3</i> morpholinos.

<p>(A,C) Enlarged GFP images of embryonic hearts at 72 hpf. GFP expression was driven by the <i>myl7</i> regulatory element (cardiac myosin light chain 2) and marks the myocardium of the ventricle (rostral) and atrium (caudal). (B,D) Light microscopy images of the corresponding zebrafish embryos with the GFP heart image shown in (A,C) superimposed. The GFP heart is in the same orientation as in (A) but has not yet been enlarged. Most embryos injected with <i>klf3</i> morpholino exhibited cardiac abnormalities as shown in (C) and cardiac edema shown by arrows in (D) in comparison to wild type embryos shown in (A,B). Injection of the mismatch control morpholino had no effect on the developing zebrafish heart (not shown).</p

<i>Klf3^H275R</i> mRNA, protein, and impairment in DNA binding.

<p>(A) Similar levels of <i>Klf3</i> mRNA by qRT-PCR (P>0.05) and (B) KLF3 protein by Western blot (WB) in whole H275R homozygotes (Homo) and heterozygotes (Het) versus wild-type (WT) embryos at E12.5. (C) Bacterial expression of GST-zinc finger 1–3 of normal and mutant protein showing that H275R impaired DNA binding to CCACACCCT (canonical β-globin promoter) in electrophoretic mobility shift assay (coomassie blue-stained SDS PAGE gel (below)). (D) Dose-response showing H275R also impaired binding to CCACACCT in electrophoretic mobility shift assays when full length <i>Klf3</i> was expressed in COS cells (western blot with αKlf3 antibody below). Protein identity was validated by eliminating binding with a KLF3 antibody (αKlf3).</p

Morphological evidence for aortic valvular stenosis in <i>Klf3</i>^H275R/+ mice.

<p>(A) Histological sections showing thickened aortic valve leaflets in adult <i>Klf3</i>^H275R/+ (arrow in center and right panels) vs. WT (arrow in left panel). Blebs or hematomas were sometimes observed on aortic valve leaflets of <i>Klf3</i>^H275R/+ mice (e.g. arrow in right panel). (B) Scanning electron microscopy images of adult aortic valves (arrows) from WT (left) and <i>Klf3</i>^H275R/+ (center), with magnified images of valve leaflets (right), that show thickened leaflets in <i>Klf3</i>^H275R/+ mice. (C) Micro-ultrasound images of aortic arch (Ao) in adult <i>Klf3</i>^H275R/+ (below) vs. WT (above) showing aortic dilatation in the left ventricular outflow tract (LVOT) typical of <i>Klf3</i>^H275R/+ mice. (D) Elevated peak blood velocity in the ascending aorta of <i>Klf3</i>^H275R/+ (solid circles) relative to WT controls (open circles). Mean ± SE are shown for each group. The lower dashed line shows the WT mean + 2SD and the upper dashed line the WT mean + 3SD. 20 of 31 <i>Klf3</i>^H275R/+ had peak velocities >3SD above WT.</p

Abnormal cardiac histology in <i>Klf3^H275R</i> embryos.

<p>In homozygotes, histological images showed thinned and disorganized ventricular myocardium and septum (A) at E12.5 and (B) in a rare homozygous survivor at E14.5 (two section levels from same specimen are shown). At E14.5, ventricular (VSD) and atrial septation defects (ASD) were observed in some homozygote and heterozygote hearts. (B) The interventricular septum of heterozygotes at E14.5 appeared thickened relative to WT and homozygotes (black arrows). (C) Heterozygous <i>Klf3</i>^H275R hearts at E14.5 exhibited thickened myocardial and septal wall thicknesses (arrows), diminished left and right ventricular lumens (*), and (D) abnormal hyperplasia of atrioventricular cushion tissue (arrows) (enlargement from (C)). All homozygotes died prenatally. About half of the heterozygotes died before weaning.</p

Abnormal hearts and aortic valves in <i>Klf3</i>^H275R/+ neonates.

<p>(<b>A</b>) Thoracic images of neonates delivered by cesarean-section at E18.5 (term). The heterozygous <i>Klf3</i>^H275R neonate breathed occasionally for 30 min then stopped breathing and died (right). The wild type (WT) neonate (left) breathed and appeared normal before being euthanized for MRI. The ventricular myocardium (V) and septum (S) were markedly thickened in the heterozygous neonate that died. (<b>B</b>) Aortic valve leaflets (arrows) of a heterozygous <i>Klf3</i>^H275R neonate found dead on postnatal day 0 (P0) were thickened (right) relative to a same age control (left). (C) Optical Projection Tomographic images of aortic valves from apparently healthy neonates on day 1 showing thickened aortic valve leaflets in <i>Klf3</i>^H275R/+ (right) compared to WT (left). (<b>D</b>) Serial measurements of aortic peak blood velocity in <i>Klf3^H275R</i>/+ as neonates on day 1 and at 8 wk as adults (n = 7; solid lines join points). Box plots show WT values for day 1 neonates (n = 14) and 8–14 wk adults (n = 21). At day 1, all 7 <i>Klf3^H275R</i>/+ mice had aortic peak velocities within the normal range whereas 6 of 7 were elevated by 8 wk.</p

Gene trap insertion sites and effects on <i>Klf3</i> mRNA and protein.

<p>(A) Schematic of <i>Klf3</i> gene showing the DNA insertion site of the gene trap marker in the XS line (XS0187) and in the CH line (CH0516). SD = splice donor site; β-geo = β-galactosidase and neomycin fusion protein; pA = polyadenylation. (B) <i>Klf3</i> mRNA in E12.5 whole embryos from the XS (grey squares) and CH (black circles) gene trap lines measured by qRT-PCR was significantly decreased in homozygotes (homo) but not heterozygotes (het). (C) KLF3 protein in spleens of Klf3^H275R/+ adults was similar to WT (i.e. Klf3+/+, XS WT, CH WT) whereas it was undetectable in XS and CH homozygote spleens by western blot.</p