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NDR Kinases Are Essential for Somitogenesis and Cardiac Looping during Mouse Embryonic Development.

Schmitz-Rohmer D, Probst S, Yang ZZ, Laurent F, Stadler MB, Zuniga A, Zeller R, Hynx D, Hemmings BA, Hergovich A - PLoS ONE (2015)

Bottom Line: In addition, Ndr1/2-double embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping.The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double embryos around E10.Ndr1/2-double embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT
Studies of mammalian tissue culture cells indicate that the conserved and distinct NDR isoforms, NDR1 and NDR2, play essential cell biological roles. However, mice lacking either Ndr1 or Ndr2 alone develop normally. Here, we studied the physiological consequences of inactivating both NDR1 and NDR2 in mice, showing that the lack of both Ndr1/Ndr2 (called Ndr1/2-double mutants) causes embryonic lethality. In support of compensatory roles for NDR1 and NDR2, total protein and activating phosphorylation levels of the remaining NDR isoform were elevated in mice lacking either Ndr1 or Ndr2. Mice retaining one single wild-type Ndr allele were viable and fertile. Ndr1/2-double embryos displayed multiple phenotypes causing a developmental delay from embryonic day E8.5 onwards. While NDR kinases are not required for notochord formation, the somites of Ndr1/2-double embryos were smaller, irregularly shaped and unevenly spaced along the anterior-posterior axis. Genes implicated in somitogenesis were down-regulated and the normally symmetric expression of Lunatic fringe, a component of the Notch pathway, showed a left-right bias in the last forming somite in 50% of all Ndr1/2-double embryos. In addition, Ndr1/2-double embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping. The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double embryos around E10. Taken together, we show that NDR kinases compensate for each other in vivo in mouse embryos, explaining why mice deficient for either Ndr1 or Ndr2 are viable. Ndr1/2-double embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.

No MeSH data available.


Related in: MedlinePlus

Murine NDR kinases are essential for cardiac looping.(A) OPT 3D reconstruction of wild-type (Ai) and Ndr1/2-double  (Aii) embryos at E8.5. Blue: anatomy. Red: Nkx2.5 whole mount in situ hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two Ndr1/2-double  and two control embryos were examined. Using bright field microscopy ten Ndr1/2-double  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 Ndr1/2-double  (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 Ndr1/2-double  and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and Ndr1/2-double  (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 Ndr1/2-double  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 Ndr1/2-double  (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 Ndr1/2-double  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.
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pone.0136566.g006: Murine NDR kinases are essential for cardiac looping.(A) OPT 3D reconstruction of wild-type (Ai) and Ndr1/2-double (Aii) embryos at E8.5. Blue: anatomy. Red: Nkx2.5 whole mount in situ hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two Ndr1/2-double and two control embryos were examined. Using bright field microscopy ten Ndr1/2-double 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 Ndr1/2-double (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 Ndr1/2-double and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and Ndr1/2-double (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 Ndr1/2-double 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 Ndr1/2-double (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 Ndr1/2-double 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.

Mentions: Comparative analysis of developing hearts at E8.5, showed that while wild-type hearts had progressed to LS-III with prominent rightward looping (Fig 6Ai), the looping of Ndr1/2-double hearts remained in stage LS-II (Fig 6Aii). In addition, Ndr1/2-double hearts displayed a bulbous morphology and appeared less transparent than wild-type controls (data not shown), which pointed to either a thickened myocardium or a reduced heart lumen. To determine whether this striking heart phenotype was due to a developmental delay or arrest, E9.5 embryos were analyzed (Fig 6B and 6C). While the looping process had progressed in wild-type hearts (Fig 6Bi and 6Ci), the heart looping of Ndr1/2-double embryos remained arrested at LS-II at E9.5 (Fig 6Bii and 6Cii). In agreement with this observation, no rightward looping of Ndr1/2-double embryonic hearts was observed. In addition, Ndr1/2-double embryos appeared smaller than their wild-type litter mates at E9.5, but organogenesis was not arrested at this stage (Fig 3). OPT (Optical Projection Tomography) analysis established that the lumen of the developmentally arrested Ndr1/2-double embryonic hearts had not formed properly (Fig 6D and 6E). Indeed, analysis of histological sections revealed that the myocardium of Ndr1/2-double embryonic hearts was thickened (Fig 6Fii, 6Gii and 6Hii) and the cardiac jelly and/or lumen contained additional cells (indicated by arrows in Fig 6Fii, 6Gii and 6Hii). Taken together, the pericardial edema (Fig 3H), arrest of cardiac looping (Fig 6A, 6B and 6C) and defects in the heart lumen (Fig 6D to 6H) indicated that these cardiac defects are the likely cause of the embryonic lethality of Ndr1/2-double embryos during early mouse organogenesis.


NDR Kinases Are Essential for Somitogenesis and Cardiac Looping during Mouse Embryonic Development.

Schmitz-Rohmer D, Probst S, Yang ZZ, Laurent F, Stadler MB, Zuniga A, Zeller R, Hynx D, Hemmings BA, Hergovich A - PLoS ONE (2015)

Murine NDR kinases are essential for cardiac looping.(A) OPT 3D reconstruction of wild-type (Ai) and Ndr1/2-double  (Aii) embryos at E8.5. Blue: anatomy. Red: Nkx2.5 whole mount in situ hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two Ndr1/2-double  and two control embryos were examined. Using bright field microscopy ten Ndr1/2-double  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 Ndr1/2-double  (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 Ndr1/2-double  and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and Ndr1/2-double  (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 Ndr1/2-double  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 Ndr1/2-double  (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 Ndr1/2-double  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.
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pone.0136566.g006: Murine NDR kinases are essential for cardiac looping.(A) OPT 3D reconstruction of wild-type (Ai) and Ndr1/2-double (Aii) embryos at E8.5. Blue: anatomy. Red: Nkx2.5 whole mount in situ hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two Ndr1/2-double and two control embryos were examined. Using bright field microscopy ten Ndr1/2-double 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 Ndr1/2-double (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 Ndr1/2-double and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and Ndr1/2-double (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 Ndr1/2-double 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 Ndr1/2-double (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 Ndr1/2-double 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.
Mentions: Comparative analysis of developing hearts at E8.5, showed that while wild-type hearts had progressed to LS-III with prominent rightward looping (Fig 6Ai), the looping of Ndr1/2-double hearts remained in stage LS-II (Fig 6Aii). In addition, Ndr1/2-double hearts displayed a bulbous morphology and appeared less transparent than wild-type controls (data not shown), which pointed to either a thickened myocardium or a reduced heart lumen. To determine whether this striking heart phenotype was due to a developmental delay or arrest, E9.5 embryos were analyzed (Fig 6B and 6C). While the looping process had progressed in wild-type hearts (Fig 6Bi and 6Ci), the heart looping of Ndr1/2-double embryos remained arrested at LS-II at E9.5 (Fig 6Bii and 6Cii). In agreement with this observation, no rightward looping of Ndr1/2-double embryonic hearts was observed. In addition, Ndr1/2-double embryos appeared smaller than their wild-type litter mates at E9.5, but organogenesis was not arrested at this stage (Fig 3). OPT (Optical Projection Tomography) analysis established that the lumen of the developmentally arrested Ndr1/2-double embryonic hearts had not formed properly (Fig 6D and 6E). Indeed, analysis of histological sections revealed that the myocardium of Ndr1/2-double embryonic hearts was thickened (Fig 6Fii, 6Gii and 6Hii) and the cardiac jelly and/or lumen contained additional cells (indicated by arrows in Fig 6Fii, 6Gii and 6Hii). Taken together, the pericardial edema (Fig 3H), arrest of cardiac looping (Fig 6A, 6B and 6C) and defects in the heart lumen (Fig 6D to 6H) indicated that these cardiac defects are the likely cause of the embryonic lethality of Ndr1/2-double embryos during early mouse organogenesis.

Bottom Line: In addition, Ndr1/2-double embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping.The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double embryos around E10.Ndr1/2-double embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.

View Article: PubMed Central - PubMed

Affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

ABSTRACT
Studies of mammalian tissue culture cells indicate that the conserved and distinct NDR isoforms, NDR1 and NDR2, play essential cell biological roles. However, mice lacking either Ndr1 or Ndr2 alone develop normally. Here, we studied the physiological consequences of inactivating both NDR1 and NDR2 in mice, showing that the lack of both Ndr1/Ndr2 (called Ndr1/2-double mutants) causes embryonic lethality. In support of compensatory roles for NDR1 and NDR2, total protein and activating phosphorylation levels of the remaining NDR isoform were elevated in mice lacking either Ndr1 or Ndr2. Mice retaining one single wild-type Ndr allele were viable and fertile. Ndr1/2-double embryos displayed multiple phenotypes causing a developmental delay from embryonic day E8.5 onwards. While NDR kinases are not required for notochord formation, the somites of Ndr1/2-double embryos were smaller, irregularly shaped and unevenly spaced along the anterior-posterior axis. Genes implicated in somitogenesis were down-regulated and the normally symmetric expression of Lunatic fringe, a component of the Notch pathway, showed a left-right bias in the last forming somite in 50% of all Ndr1/2-double embryos. In addition, Ndr1/2-double embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping. The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double embryos around E10. Taken together, we show that NDR kinases compensate for each other in vivo in mouse embryos, explaining why mice deficient for either Ndr1 or Ndr2 are viable. Ndr1/2-double embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.

No MeSH data available.


Related in: MedlinePlus