The m883 mutation causes left-right isomerism of endodermal organs. Formation of endodermal organs in wild-type (A,C,E,G) and m883 mutant embryos (B,D,F,H). A,B: The mutation m883 was initially isolated based on the split appearance of the bilateral preproinsulin expression domain, here in a 3-dpf embryo. C,E: In wild type embryos, as judged from trypsin expression, the right-sided exocrine pancreas extends over several somites along the anterior-posterior axis. D,F: In mutant embryos, the exocrine pancreas is frequently found bilateral and appears shortened. G,H: Expression analysis of prox1 reveals the liver primordium, and demonstrates that liver tissue is also forming on both sides of m883 mutant embryos. I-L: Morphological phenotypes of m883 mutant embryos. Homozygous mutant embryos (J) can be clearly distinguished from wild-type (I) from the 22-somite stage on; the inflation of the brain ventricles fails to occur, while eyes and ears, notochord, and somites appear to develop largely normally. At day 4 (K, L), mutant embryos have severe heart defects, with no blood circulation, causing blood accumulation in the body and retarded growth. Dorsal views in A-D, G, H, with anterior to the left. Transversal sections in E, F, oriented dorsal at top. K,L: Lateral views, anterior to the left and dorsal up. g, gut; li, liver; no, notochord; pa, pancreas.

Abnormal morphogenesis and bilateral symmetry of endoderm in m883 mutant embryos. Expression of foxA1 at 30 hpf (A,B) and ipf1 at 48 hpf (C,D) in wild-type (A,C) and m883 mutant embryos (B,D). foxA1 expression marks the endodermal sheet and forming rod during morphogenesis. While looping of the wild-type gut is visible at 30 hpf, the expression domain is forked more posteriorly and in a bilateral symmetric fashion in m883 mutant embryos. C,D: The expression of ipf1 in the duodenum and pancreas primordium reveals the looped gut at 48 hpf, with the pancreas to the right (here left due to ventral view; see arrow). In m883 mutant embryos, the expression of ipf1 is detected in a straight duodenum, which has not looped, and on both sides of the duodenum a residual pancreas anlage can be detected (arrows in D). A,B: Dorsal views, anterior at top; C,D: ventral views, anterior at top.

The m883 mutation truncates the NaK-ATPase alpha1a.1 protein in the hydrolase domain. A: The chromatogram reveals that in the m883 allele, a C to T transition introduced a stop at codon 569, which truncates the NaK-ATPase alpha1a.1 protein in the hydrolase domain. The schematic representation reveals that the stop codon is in the cytoplasmic hydrolase domain (yellow). The thick cylinders represent the transmembrane domains. Dark blue indicates the N-terminal cation transporting ATPase domain, grey indicates the C-terminal cation transporting ATPase domain, red indicates the E1-E2-ATPase domain, light blue indicates the binding domain for the beta subunit. B-J: Whole mount in situ hybridization with an atp1a1a.1-specific probe reveals transcript at the 2-cell stage (B,C) and 8-16-cell stage (D,E). After MBT, the time of onset of zygotic expression, expression remains ubiquitous (85% epiboly in F,G). For all early stages. sense mRNA probes were used as controls (C,E,G). The expression of atp1a1a.1 continues to be ubiquitous during early somitogenesis stages. However, during late somitogenesis (H, 14-somite stage), the expression becomes enhanced in the intermediate mesoderm and in the adaxial mesoderm as well as the ear placode and eye field. At around the 20-somite stage (I, 24 hpf) and later, the expression is most pronounced in the pronephric duct, intermediate mesoderm, ear, eye, brain, and heart. At later stages, 48 hpf (J), the expression is reduced in the pronephric duct and nephric primordium but still remains strong in the brain, eyes, heart, and mucous cells.

Expression of left-right asymmetry genes in atp1a1a.1^m883 mutant embryos. A,B: charon is a negative regulator of nodal signaling, expressed around the KV. There is no difference in expression of cha in had^m883 mutant and wild-type embryos at the 14-somite stage. C,D: At the 15-somite stage, southpaw is expressed in left lateral plate mesoderm in wild-type, but bilaterally symmetric in m883 mutants. E,F: lefty1/antivin, another suppressor of nodal signaling, is expressed in the left heart primordium and the left diencephalon in the habenula of wildtype embryos, but reveals a clear isomerism of the heart anlage and the habenula expression domain in the mutant embryos at the 20-somite stage. G: pitx2 is expressed in the left heart anlage and lateral plate mesoderm, as well as a left domain in the diencephalon (I) in wildtype at the 22-somite stage, but is expressed bilaterally symmetric in both domains in atp1a1a.1 mutant embryos (H,J). A: Dorsal view onto the tail primordium (ventral side of embryo) anterior at top; B: lateral view anterior to the left; C-H: dorsal views with anterior at top (E slightly oblique); I,J: posterior-dorsal oblique view of brain with dorsal at top and left at left.

Phenocopy of the m883 phenotype with the Na,K-ATPase pump blocker ouabain. A,B: When wild-type embryos were incubated from the 1,000-cell stage onward with 3 mM ouabain, the morphology of embryos resembled m883 mutant embryos. C,D: In the ouabain-treated embryos, the expression of pitx2 reveals isomerism in the heart anlage at the 26-somite stage. E,F: The expression of pitx2 is found bilateral in the lateral plate mesoderm in the ouabain-treated embryos, similar to embryos mutant for the Na, K ATPase alpha1a.1 subunit. A,B,E,F: Anterior to the left; (A, B) lateral and (E, F) dorsal views. C,D: Anterior to the top, dorsal view.

Kupffer's vesicle in atp1a1a.1 mutant embryos resembles that of wild-type embryos in structure and function. A,B: Morphology of the Kupffer's vesicle at 6-8-somite stage reveals no defects in atp1a1a.1 mutant embryos compared to wild-type. C,D: Detection of monocilia in KV by anti-acetylated tubulin immunohistochemistry. The pictures show flat projections of confocal image stacks. Cilia in m883 mutant embryos appear normal. E-H: Life imaging of monocilia induced fluid flow in embryos injected with 100-nm diameter fluorescent beads. White arrows point at the position of fluorescent beads at successive time points. The red arrows indicate the anticlockwise moving beads in a wild-type embryo (E,G), and in a m883 mutant embryo (F,H). These embryos were allowed to develop and the morphological phenotypes documented (I, J). At 30 hpf, the embryo (I) from E,G can be identified as wild-type, while embryo (J) from F,H reveals a mutant phenotype. Wild-type (A,C,E,G,I), and m883 mutant embryos (B,D,F,H,J). Ventral view, anterior to the right (A,B). Lateral view anterior to the left (I,J).