NDR kinases are essential for growth, cardiac development and blood vessel remodeling from about embryonic day 8 onward in mouse embryos.

<p>(A, B) Bright field images of wild-type (A) and <i>Ndr1/2</i>-double null (B) littermates at E8.5. Both embryos are of the 6-somite stage. Note that the <i>Ndr1/2</i>-double null somites are small and irregularly shaped. Scale bars correspond to 500μm. (C) Average somite numbers of wild-type and <i>Ndr1/2</i>-double null littermates at E8.5. Data correspond to the analysis of a total of 15 litters and are blotted as box and whisker chart illustrating the distribution of somite numbers per litter and genotype. Average somite numbers are indicated. (D, E) Distribution of <i>Shh</i> (D) and <i>T/brachyury</i> (E) transcripts in wild-type (left) and <i>Ndr1/2</i>-double null littermate embryos (right) at E8.5. Four animals per genotype were analyzed, and all embryos displayed the staining shown in Fig 3D and 3E. (F, G, H) Bright field images of wild-type (F) and <i>Ndr1/2</i>-double null (G, H) littermate embryos at E9.5. 56 <i>Ndr1/2</i>-double null and 163 control embryos at E9.5 were analyzed. White arrow in H points to the pericardial edema. Scale bars correspond to 500μm. (I, J) Bright field images of the yolk sacs of wild-type (I) and <i>Ndr1/2</i>-double null (J) littermate embryos at E9.5. All <i>Ndr1/2</i>-double null yolk sacs (n = 56) displayed defective vascular development as illustrated in Fig 3J.</p

The CDK inhibitor p21/Cip1 is up-regulated in <i>Ndr1/2</i>-double null embryos and required for survival.

<p>(A) Changes in transcript levels between wild-type and <i>Ndr1/2</i>-double null embryos at E8.5 as revealed by microarray analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s007" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s008" target="_blank">S2</a> Tables). (B) Validation of the alterations in <i>p21</i> and <i>p27</i> expression in wild-type (control) and <i>Ndr1/2</i>-double null embryos by qRT-PCR (at E8.5). Data shown represent the average gene expression levels obtained by analyzing three independent embryos per genotype. Each embryo was analyzed in triplicate. Statistical analysis was performed using a two-tailed Student t-test assuming unequal variance. (C) Genotype distribution of offspring from <i>Ndr1</i>^+/-;<i>Ndr2</i>^-/-;<i>p21</i>^+/- intercrosses. Embryos were isolated at E8.5 and genotyped. Top panel: <i>p21</i> genotype of the <i>Ndr1/2</i>-double null embryos; bottom panel: <i>Ndr1</i> genotype of the <i>p21</i>-null embryos. The p-values are indicative of the probabilities to obtain the observed genotype distribution by chance.</p

Somitogenesis is altered in <i>Ndr1/2</i>-double null embryos.

<p>(A) Changes in gene expression between wild-type and <i>Ndr1/2</i>-double null embryos at E8.5 determined by microarray analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s007" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s008" target="_blank">S2</a> Tables). (B, C, D) Distribution of <i>Meox1</i> (B), <i>Tbx6</i> (C) <i>and Mesogenin1</i> (D) transcripts in wild-type (wt) and <i>Ndr1/2</i>-double null embryos at E8.5. Four animals per genotype were analyzed for <i>Meox1</i> staining, and two embryos per genotype were analyzed for <i>Tbx6</i> and <i>Mesogenin</i> staining. All embryos displayed the staining patterns as shown in Fig 5B, 5C and 5D. (E) Hematoxylin/Eosin staining of transversal (left) and parasagittal (right) sections of wild-type (top panels) and <i>Ndr1/2</i>-double null (bottom panels) embryos at the 6-somite stage. Six embryos of each genotype were analyzed. PSM: presomitic mesoderm; SI, SII, SIII: last, second to last and third to last formed somite. Scale bars = 50μm. (F, G, H) Distribution of <i>Snail1</i> (F), <i>Axin2</i> (G) <i>and Lnfg</i> (H) transcripts in wild-type (wt) and <i>Ndr1/2</i>-double null embryos. Asterisk (*) indicates aberrant asymmetrical expression (right) of <i>Lnfg</i> in the last formed somite pair. Seven embryos were analyzed per genotype for <i>Snail1</i> staining, and five embryos were analyzed per genotype for <i>Axin2</i> staining. The expression patterns of <i>Snail1</i> and <i>Axin2</i> appeared indistinguishable between all mutant and control embryos. Nine mutant and four control embryos were analyzed for <i>Lnfg</i> staining. All four control embryos showed the expected symmetric expression pattern as illustrated in Fig 5H. In contrast, four of nine mutant embryos displayed strongly asymmetric <i>Lnfg</i> expression and one mutant embryo showed mild asymmetric expression.</p

<i>Ndr1/2</i>-double null mice are embryonic lethal, but a single <i>Ndr</i> allele is sufficient to sustain normal development.

<p>Genotype (GT) distribution of offspring from <i>Ndr-</i>single allele intercrosses (<i>Ndr1</i>^+/-;<i>Ndr2</i>^-/- x <i>Ndr1</i>^-/-;<i>Ndr2</i>^+/-) at weaning. Actual offspring numbers are indicated, together with the expected and obtained Mendelian ratios. No <i>Ndr1/2</i>-double null mice were recovered. All other genotypes were obtained at approximately the expected Mendelian ratios. A total of 415 offspring were analyzed (n = 415).</p><p><i>Ndr1/2</i>-double null mice are embryonic lethal, but a single <i>Ndr</i> allele is sufficient to sustain normal development.</p

Murine NDR kinases are essential for cardiac looping.

<p>(A) OPT 3D reconstruction of wild-type (Ai) and <i>Ndr1/2</i>-double null (Aii) embryos at E8.5. Blue: anatomy. Red: <i>Nkx2</i>.<i>5</i> whole mount <i>in situ</i> hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two <i>Ndr1/2</i>-double null and two control embryos were examined. Using bright field microscopy ten <i>Ndr1/2</i>-double null and ten control embryos were analysed at E8.5 to confirm the observed phenotype (data not shown). (B, C) Bright field images of wild-type (Bi, Ci) and <i>Ndr1/2</i>-double null (Bii, Cii) developing hearts at E9.5. B: frontal view; C: lateral view. Scale bars = 100μm. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right, d: dorsal, v: ventral. Five <i>Ndr1/2</i>-double null and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and <i>Ndr1/2</i>-double null (Dii, Eii) embryos shown in 6A. Panel D: coronal plane; panel E: transversal plane. Labels: a: anterior, p: posterior, d: dorsal, v: ventral. Arrows point to the heart. Two <i>Ndr1/2</i>-double null and two control embryos were analyzed for OPT as shown in 6D and 6E. (F, G, H) Hematoxylin and Eosin stained transversal sections of a wild-type (Fi, Gi, Hi) and <i>Ndr1/2</i>-double null (Fi, Gi, Hi) hearts at the 6-somite stage. The myocardium (MC) and headfolds (HF) are indicated in (Fi) and (Fii). Arrows in (Fii), (Gii) and (Hii) point to remaining cells in the cardiac jelly and lumen. Note the similar section plan between the embryos shown in (E) and (G). Three <i>Ndr1/2</i>-double null and three control embryos were analyzed. (J) Scheme showing the approximate level of the sections within the embryo. The distance between sections is about 30μm.</p

<i>Ndr1/2</i>-double null embryos die around mid-gestation.

<p>Genotype distribution in embryos derived from intercrosses of <i>Ndr1</i>^+/-;<i>Ndr2</i>^-/- with <i>Ndr1</i>^-/-; <i>Ndr2</i>^+/- mice at indicated time points. Note (a): all <i>Ndr1/2</i>-double null embryos recovered at E10.5 were dead and were in progress of resorption.</p><p><i>Ndr1/2</i>-double null embryos die around mid-gestation.</p