Hyperglycemia impairs left–right axis formation and PNAS PLUS thereby disturbs heart morphogenesis in mouse embryos

Masahiro Hachisugaa,b, Shinya Okia, Keiko Kitajimaa, Satomi Ikutaa, Tomoyuki Sumia, Kiyoko Katob, Norio Wakeb, and Chikara Menoa,1

aDepartment of Developmental Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and bDepartment of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved August 18, 2015 (received for review March 9, 2015) Congenital heart defects with heterotaxia are associated with The left–right (L–R) asymmetry of visceral organs is generated pregestational diabetes mellitus. To provide insight into the mech- on the basis of the L–R axis established during early embryo- anisms underlying such diabetes-related heart defects, we exam- genesis (18). Disturbance of the L–R axis by genetic alterations in ined the effects of high-glucose concentrations on formation of mice results in the development of CHDs, as well as laterality de- the left–right axis in mouse embryos. Expression of Pitx2, which fects (19–27). In mice, formation of the L–Raxisbeginsasaresult plays a key role in left–right asymmetric morphogenesis and car- of the generation of leftward fluid flow in the node ( flow) at diac development, was lost in the left lateral plate mesoderm of embryonic day (E) 7.75. As a consequence of nodal flow, the L–R embryos of diabetic dams. Embryos exposed to high-glucose con- asymmetric expression of several first appears in crown cells centrations in culture also failed to express Nodal and Pitx2 in the at the periphery of the node. Nodal, which encodes a member of the left lateral plate mesoderm. The distribution of phosphorylated TGF-β family of , is one such , with the level of its Smad2 revealed that Nodal activity in the node was attenuated, expression being greater on the left side than on the right (28, 29). accounting for the failure of left–right axis formation. Consistent Nodal produced in the crown cells forms a heterodimer with a with this notion, Notch signal-dependent expression of Nodal-related TGF-β family member, growth differentiation factor 1 (GDF1), genes in the node was also down-regulated in association with a which increases its activity, and it is thought to diffuse to the lateral MEDICAL SCIENCES reduced level of Notch signaling, suggesting that high-glucose con- plate mesoderm (LPM) (30, 31). Nodal activates intracellular sig- centrations impede Notch signaling and thereby hinder establishment naling by binding to Activin receptors and Cryptic cofactor. In the of the left–right axis required for heart morphogenesis. cytoplasm, Smad2 is phosphorylated by activated Activin receptors and then translocates to the nucleus, where it exerts its trans- diabetes | congenital heart defects | heterotaxia | left–right axis activation activity together with the forkhead box (Fox) transcrip- tion factor Foxh1. The activity of Nodal in crown cells is regulated ongenital heart defects (CHDs) are among the most com- in an L–R asymmetric manner by its inhibitor Cerl2 (32), a DAN Cmon birth defects in humans, affecting 0.8–1.16% of live domain family member, as is reflected by the L > R distribution of births (1, 2). Such defects are not necessarily caused by genetic al- phosphorylated Smad2 (pSmad2) (33). This subtle difference in terations, with epidemiological studies having revealed that various Nodal activity is then amplified in LPM (34, 35). Nodal tran- modifiable factors increase the risk of specific types of CHD (1, 3). scription is regulated by a positive loop via the action of Smad2 Pregestational diabetes mellitus (DM), including both type 1 and and Foxh1 (36). Nodal produced in the node likely initially type 2 DM, is one such modifiable factor (1, 3, 4). Mice and rats have been studied as animal models of diabetic Significance embryopathy. In diabetic patients, metabolic disorders, such as ke- β tosis, accompany hyperglycemia. Glucose and -hydroxybutyrate, a Epidemiological studies have revealed that pregestational di- ketone produced in diabetes, both exhibit teratogenic effects on abetes mellitus increases the risk for congenital anomalies, – cultured postimplantation embryos (5 7). Hyperglycemia induces including congenital heart defects (CHDs). Despite the impor- oxidative stress because of the production of reactive oxygen species tance of preventing diabetes-related congenital malforma- (8),andsuchstresshasbeenshowntocontributetoaspectsofdi- tions, however, the underlying pathogenic mechanisms have abetic embryopathy, such as growth retardation and neural tube remained largely unknown. Pregestational diabetes mellitus is defects in rat and mouse embryos (9). Oxidative stress induced by associated specifically with CHDs accompanied by heterotaxia. hyperglycemia was found to reduce the expression of the paired box We have now examined left–right (L–R) axis formation in em- gene Pax3 in the neuroepithelium and cardiac neural crest, resulting bryos of diabetic female mice as well as in mouse embryos in an increase in p53-dependent apoptosis in these tissues (10–14). exposed to high-glucose concentrations in culture. We found Given that Pax3 is required for formation of the neural tube and that high-glucose levels prevent establishment of the L–R axis septation of the truncus arteriosus, the defects in the neural tube required for heart morphogenesis and the L–R asymmetry of and outflow tract of embryos associated with diabetes are likely a visceral organs. Such a mechanism may thus explain, at least in result of the reduction in Pax3 expression (9). In other tissues, part, the CHDs and accompanying heterotaxia in the offspring however, the mechanisms by which maternal diabetes gives rise to of diabetic mothers. birth defects have remained largely unknown. Author contributions: M.H., S.O., K. Kitajima, and C.M. designed research; M.H., S.O., Pregestational DM gives rise to CHDs associated with hetero- K. Kitajima, S.I., T.S., and C.M. performed research; M.H., S.O., K. Kitajima, T.S., K. Kato, taxia in humans (1, 4, 15, 16). Heterotaxia also occurs in offspring of N.W., and C.M. analyzed data; and M.H. and C.M. wrote the paper. diabetic female nonobese diabetic mice, a model of type 1 DM. The authors declare no conflict of interest. Although the laterality of these mouse offspring varies, CHDs ac- This article is a PNAS Direct Submission. companied by right isomerism are the most common phenotype 1To whom correspondence should be addressed. Email: [email protected]. (17). How such defects develop in association with hyperglycemia This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. has remained unclear, however. 1073/pnas.1504529112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1504529112 PNAS Early Edition | 1of8 Downloaded by guest on September 30, 2021 activates its own expression in the left LPM (27, 30, 33, 37). The localized expression of Nodal in the left LPM then expands to encompass the entire LPM as a result of the operation of the positive loop (27, 38). At the anterior end of the left LPM, Nodal signals in the left side of the heart primordium, which determines the direction of heart looping (19, 24). Nodal sig- naling in the left LPM and heart primordium induces the ex- pression of the homeobox gene Pitx2 (39, 40), as a result of which visceral organs acquire their L–R asymmetric morphol- ogy. In the heart, the asymmetric expression of Pitx2 is not involved in looping but is required for proper morphogenesis, with Pitx2 mutant mice developing CHDs (21–23, 25, 26). To understand the molecular etiology of CHDs and heterotaxia associated with maternal diabetes, we examined formation of the L–R axis in embryos derived from female mice with streptozotocin- induced diabetes, as well as in mouse embryos cultured in high- glucose medium. We found that high glucose attenuates Notch signaling in the node, which is followed by down-regulation of Nodal activity at the initial step of L–R axis formation. The reduction in Nodal activity in the node thus prevents the establishment of Nodal expression in the left LPM and consequent Pitx2 expression in the left LPM and heart primordium. Human CHDs accompanied by right isomerism might be explained by this mechanism. Results The L–R Axis Is Affected in Embryos of Diabetic Female Mice. To provide insight into the pathogenesis of heterotaxia in offspring of mothers with pregestational DM, we first examined embryos of female mice with streptozotocin-induced diabetes as an animal model. All E8.7 embryos (n = 126) derived from diabetic females Fig. 1. Impaired formation of the L–R axis in embryos of female mice with (n = 10) with a blood glucose concentration of 300–490 mg/dL streptozotocin-induced diabetes. (A–D) Whole-mount in situ hybridization manifested normal looping of the heart tube to the right side analysis of Pitx2 expression in E8.7 embryos of female mice without diabetes – (d-loop), as did control embryos of nondiabetic dams (Fig. 1 A and (control) (A) or with severe diabetes (B D). Anterior views are shown. All isoforms of Pitx2 mRNA are detected. Black arrowheads indicate Pitx2 ex- H). In contrast, 6 of 10 litters fromdamswithabloodglucoselevel > pression in the left LPM, whereas arrows indicate expression in the endo- of 490 mg/dL included embryos with abnormal heart looping. In derm layer of the yolk sac. The asterisk marks the left horn of the sinus the affected six litters, 11 of 66 evaluable embryos manifested re- venosus. White arrowheads indicate the absence of Pitx2 expression in the versed heart looping (l-loop) (Fig. 1 B, C,andH), whereas 10 em- left outflow tract. White dashed lines show the direction of heart looping. bryos had the heart tube at the midline without apparent L–R (E–G) Transverse sections at the level of the green dashed lines in A–C,re- asymmetry (Fig. 1D). Given that the embryos without asymmetric spectively. Arrowheads show Pitx2 expression in the left outflow tract. (Scale heart looping were at around the seven- to eight-somite stage, when bars, 100 μm.) (H) The percentage of E8.7 embryos manifesting reversed the heart tube begins to elongate, the looping process may have been heart looping or embryonic turning among the offspring of diabetic female mice according to the maternal blood glucose concentration. Litters with delayed in these embryos. Embryonic turning is another indicator of – different percentages of embryos showing reversed heart looping or turning the L R axis, with normal embryos showing clockwise rotation along are indicated by blue vertical lines. Parentheses show the number of em- the long axis as viewed from the cranial to caudal portion. Seven of bryos for evaluation of heart looping. When litters included embryos at 52 evaluable embryos in the six litters showing abnormal heart stages before the turning process, the number for evaluation of turning is looping also manifested reversed embryonic turning (Fig. 1H). separately indicated in brackets. Embryos with the heart tube at the midline Taken together, these observations suggested that L–R asymmetric were excluded from the number of embryos evaluable for heart looping. morphogenesis might be disturbed as a result of aberrant L–Raxis formation in the offspring of female mice with streptozotocin- – induced diabetes. High Glucose Affects L R Axis Formation in Mouse Embryos. The loss – To examine whether L–R axis formation was indeed affected in of Pitx2 expression indicated that the L R axis is affected in such embryos, we performed in situ hybridization analysis of Pitx2 embryos of diabetic female mice. To understand the mechanism – expression. Pitx2 is L–R asymmetrically expressed, with its expres- by which the L R axis is affected by hyperglycemia, we examined sion being localized predominantly to the left LPM and heart pri- embryos exposed to a high-glucose concentration in whole- mordium, as well as the head mesenchyme at E8.7 (Fig. 1 A and E). embryo culture (hereafter referred to as high-glucose embryos). We The expression of Pitx2 was altered in the litters that included first determined the D-glucose concentration at which Pitx2 ex- embryos with abnormal heart looping or turning, being lost either in pression in the LPM is lost. Embryos at the head-fold stage both the left LPM and heart primordium (8 of 66 embryos) (Fig. 1 (E7.75), before the onset of L–R axis formation, were cultured B and F) or in only the left outflow tract (8 of 66 embryos) (Fig. for 16 h in control (140 mg/dL) or high-glucose (390, 530, or 1D), whereas the expression in head mesenchyme was not affected. 720 mg/dL) medium, during which time the embryos developed to Of note, four embryos with Pitx2 expression in the left LPM and the six- to seven-somite stage. Whereas most (21 of 23) control heart primordium showed an l-loop of the heart tube (Fig. 1 C and embryos expressed Pitx2 in the left LPM (Fig. 2A), the expression G). Given that the asymmetric expression of Pitx2 is required for was lost in high-glucose embryos in a concentration-dependent heart morphogenesis and L–R asymmetric organogenesis (20–23, manner [390 mg/dL, 7 of 16; 530 mg/dL, 12 of 16 (P < 0.001); 25, 26, 41), CHDs accompanied by heterotaxia in diabetic embry- 720mg/dL,21of21P < 0.001)] (Fig. 2B). To confirm that the opathy are likely explained by the loss of Pitx2 expression in the left change in osmolarity of the medium caused by glucose addition did LPM and heart primordium (Discussion). not affect L–R axis formation, we added L-glucose at 580 mg/dL

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1504529112 Hachisuga et al. Downloaded by guest on September 30, 2021 which time the L–R axis is established in control embryos, and in PNAS PLUS normal medium for the subsequent 34 h. The embryos still man- ifested reversed heart looping (6 of 12; P < 0.01) with the placenta positioned on the left side (5 of 12; P < 0.01) (Fig. 2L), suggesting that transient elevation of the glucose level at this initial stage can induce heterotaxia. We next explored why Pitx2 expression in the left LPM is lost in high-glucose embryos. Embryos at the head-fold stage were cultured for 12 h to early somite stages. Transient expression of Nodal in the left LPM not only determines the direction of heart looping and embryonic turning, but also induces the expression of Pitx2 (19, 24, 39). In most (16 of 20) control embryos at the four- to five-somite stage, Nodal was normally expressed in the left LPM (Fig. 2D). However, Nodal expression was not detected in the LPM of any (23 of 23; P < 0.001) of the high-glucose embryos examined (Fig. 2E). We simultaneously examined the expression of the homeobox gene Uncx as an indicator of somite number in this experiment. Although the same numbers of bands of Uncx expression were observed in control and high-glucose embryos at the end of the culture period, the successive stripes of Uncx expression became narrower in high-glucose embryos (Fig. 2 D–G). In addition, newly generated somites were positioned more posteriorly in the high-glucose embryos, suggesting that somitogenesis was affected. We next examined the expression of β Fig. 2. Loss of Nodal expression in the LPM of embryos exposed to high genes, which encode TGF- family ligands. Lefty1 is mainly glucose in culture. (A–G) Whole-mount in situ hybridization analysis of Pitx2 expressed in the floor plate, whereas Lefty2 is expressed in the left (A–C), Nodal (D, E), and Lefty1 and Lefty2 (F, G) expression in embryos cultured LPM. The expression of these genes for Nodal inhibitors is in- in control (A, D, F), high–D-glucose (B, E, G), or high–L-glucose (C) medium duced by Nodal signaling, resulting in restriction of the Nodal from the head-fold stage for 16 h (A–C)or12h(D–G). The expression of expression domain to the left side (24, 27, 36, 42, 43). Lefty1 and MEDICAL SCIENCES Uncx wassimultaneouslyexaminedasanindicatorforsomitenumberinD–G). Lefty2 were normally expressed in most control embryos at the Anterior views (A–C)orventralviewswithanteriortothetop(D–F)are four- to five-somite stage (9 of 14 for both Lefty1 and Lefty2)(Fig. – shown. All isoforms of Pitx2 mRNA are detected (A C). Lefty1 and Lefty2 2F). In contrast, Lefty2 expression was lost (14 of 14; P < 0.001) expression is simultaneously detected by corresponding specific probes (F, G). Black arrowheads indicate expression of Pitx2, Nodal,orLefty2 in the and Lefty1 expression was detected in only a few cells of the node left LPM. White arrowheads indicate Lefty1 expression. Asterisks mark head (11 of 14) in high-glucose embryos (Fig. 2G). The loss of Lefty mesenchyme. (Scale bar, 200 μm.) (H–K) Embryos cultured for 48 h from the expression in high-glucose embryos was likely because of the ab- head-fold stage in control (H, J) or high-glucose (I, K) medium. The placenta sence of Nodal expression in the left LPM. Taken together, these and yolk sac of the embryos in (H) and (I) were removed to show the ventral results thus indicated that high glucose inhibits Nodal expression view of the heart in (J) and (K), respectively. White dashed lines show the in the left LPM, which in turn results in the loss of Pitx2 expression direction of heart looping. (Scale bars in H and J, 400 μm.) (L) Summary of the and randomization of heart looping and embryonic turning. numbers of embryos examined for the direction of heart looping and em- bryo turning after culture for 48 h. Numbers in parentheses indicate em- LPM Is Competent to Express Nodal in High-Glucose Embryos. Es- bryos cultured in high-glucose medium for the initial 14 h and in control tablishment of concordant L–R asymmetry of visceral organs medium for the subsequent 34 h. requires expansion of Nodal expression in the entire LPM. Nodal expression in LPM is first induced locally by a signal from the as well as D-glucose at 140 mg/dL Most (five of seven) embryos node and then expands along the anteroposterior axis through – cultured in this L-glucose medium showed normal expression of the action of the Nodal positive loop (27 29, 36). Cryptic, which Pitx2 (Fig. 2C), suggesting that D-glucose itself was responsible encodes a cofactor of Nodal signaling, is expressed in the LPM, for the observed disturbance of L–R axis formation. floor plate, and node, and its expression is required for Nodal – expression in the LPM (24). We found that Cryptic was normally To reproduce the L R morphological defects observed in the = offspring of female mice with streptozotocin-induced diabetes, we expressed in these tissues in both control (n 7) and high-glu- cose (n = 10) embryos (Fig. 3 A and B). To examine whether the subsequently analyzed embryos cultured in high-glucose medium LPM is competent to express Nodal in high-glucose embryos, we containing glucose at 720 mg/dL. Despite the presence of this high subjected embryos at the head-fold stage to local transfection concentration of glucose, embryos at the head-fold stage achieved with Nodal and GFP expression vectors in the right posterior the stage corresponding to E9.25 after culture for 48 h, although mesoderm and then cultured the embryos in control or high- the high-glucose embryos were smaller than control embryos (Fig. – glucose medium. In this assay, endogenous Nodal expression is 2 H and I). With regard to L R asymmetric morphogenesis, heart expected to be induced by ectopic Nodal derived from the expres- looping occurred normally in control embryos (20 of 20), whereas sion vector (27, 34). In control embryos, endogenous expression of + reversed heart looping was observed in 9 of 14 high-glucose em- Nodal spread from the GFP transfection site, resulting in Nodal < – bryos (P 0.001) (Fig. 2 J L). As a consequence of normal em- expression in the right LPM (three of six) or both the left and right bryonic turning, the placenta becomes positioned on the right side LPM (three of six) (Fig. 3 C and E). In contrast, Nodal expression of the embryo. For control embryos (19 of 20), the placenta was spread from the transfection site but did not appear in the left LPM normally positioned on the right side, whereas the placenta was in five of seven high-glucose embryos; the remaining two of seven found on the left side for half (7 of 14; P < 0.01) of high-glucose embryos did not express Nodal in the LPM (Fig. 3 D and E). These embryos (Fig. 2 H, I,andL), suggesting that the turning process results suggested that LPM of high-glucose embryos retains com- was randomized in these latter embryos. To further confirm that petency to express Nodal through the action of the positive loop. high glucose affects the process of L–R axis formation, we cul- To further exclude the possibility that high glucose inhibits the tured embryos in high-glucose medium for the initial 14 h, during , we examined the phosphorylation of

Hachisuga et al. PNAS Early Edition | 3of8 Downloaded by guest on September 30, 2021 Nodal expression domain was found to be narrowed and slightly shortened and to have lost its crescent shape (n = 8; P < 0.001) (Fig. 4B). The product of Gdf1 expressed in crown cells and the LPM enhances the activity of Nodal through formation of a Nodal-GDF1 heterodimer (31). Gdf1 expression in the node appeared normal in control embryos (n = 10), whereas its do- main in high-glucose embryos was also narrowed (7 of 12) and showed various degrees of shortening (n = 12) (Fig. 4 C, D,andD′). These results thus suggested that the amount of Nodal-GDF1 produced in the node is reduced in the high-glucose condition. Expression of Nodal in the left LPM requires the activity of Nodal in crown cells to be regulated in an L–R asymmetric manner, with Cerl2 playing an essential role in such regulation through suppres- sion of Nodal activity (32, 33, 35). Cerl2 is expressed in crown cells, with the level of expression being higher on the right side. In control embryos (13 of 14), the depth of staining for Cerl2 expression be- came symmetric as a result of culture, whereas the expression do- main was larger on the right side than on the left side. In high- glucose embryos, however, Cerl2 expression was down-regulated, although the extent of this change was not as great as that for Nodal or Gdf1 (n = 15; P < 0.001) (Fig. 4 E and F). The right-sided dominance of the Cerl2 expression domain remained apparent in most high-glucose embryos (12 of 15). Collectively, these results indicated that the expression of genes that determine the activity of Nodal in the node is attenuated in high-glucose embryos. The ac- tivity of Nodal in the node might thus be reduced as a result of a change in the balance of the expression of these genes. Fig. 3. Competence of the LPM of high-glucose embryos to express Nodal. We next examined Nodal activity by detecting the distribution (A and B) Whole-mount in situ hybridization analysis of Cryptic expression in of pSmad2 in high-glucose embryos by immunohistofluorescence embryos cultured from the head-fold stage for 12 h in control (A) or high- analysis. The phosphorylation of Smad2 at early somite stages glucose (B) medium. Ventral views with anterior to the top are shown. The depends on Nodal signaling, which is thought to occur first in the node and LPM are indicated by asterisks and arrowheads, respectively. (Scale μ node and then to spread to the left LPM before the onset of Nodal bar, 200 m.) (C and D) Whole-mount in situ hybridization analysis of Nodal and expression in LPM (33). In cultured control embryos, pSmad2 was GFP expression in embryos subjected to local transfection with corresponding detected in crown cells and the left LPM at the four-somite stage expression vectors at the head-fold stage and then cultured for 12 h in control = (C) or high-glucose (D) medium. Transfected cells (dark blue) visualized with the (n 17) (Fig. 4 G and I). In high-glucose embryos, however, GFP probe are present within the region demarcated by the dashed line. Nodal pSmad2 was not detected in either crown cells or the left LPM at expression (brownish red) was detected in the node and LPM (arrows). Right the four- to five-somite stage (n = 23) (Fig. 4 H and J). Instead, ventral views are shown. (Scale bar, 200 μm.) (E) Summary of Nodal expression pSmad2 was detected only in pit cells of the posterior node (16 of patterns in the LPM of transfected embryos. (F–H) Confocal immunofluores- 23 embryos; P < 0.001). These results suggested that the activity of cence images of pSmad2 expression (red) in ES cell colonies cultured for 12 h Nodal produced in the node of high-glucose embryos is markedly either in control medium without (F)orwith(G) SB431542 or in high-glucose reduced, resulting in the failure to express Nodal in the LPM. medium (H). Nuclei were stained with TO-PRO3 (blue). (Scale bar, 100 μm.) Notch Signaling Is Attenuated in the High-Glucose Condition. We Smad2 in mouse embryonic stem (ES) cells exposed to high next explored why the expression of Nodal-related genes is at- glucose. Binding of Nodal to Activin receptors induces the tenuated in crown cells of high-glucose embryos. To exclude the possibility that formation of the node is impaired in these em- phosphorylation of Smad2 and its translocation to the nucleus, bryos, we examined the expression of sonic hedgehog (Shh) and where it exerts its transactivation activity. Smad2 is phosphory- Foxa2, representative marker genes for the node and floor plate. lated in mouse ES cells as a result of the autonomous production Both Shh and Foxa2 were normally expressed in the node of of Nodal and Activin (44). As expected, pSmad2 was detected in high-glucose embryos (n = 9 and 11, respectively) at the three- to the nuclei of ES cells cultured in standard medium (Fig. 3F). four-somite stage at levels similar to those observed in control Whereas SB431542, a potent inhibitor of type I - embryos (n = 10 and 10) (Fig. 5 A–D). Expression of Foxj1 in the like kinase receptors, attenuated Smad2 phosphorylation in ES node is required for ciliogenesis and the function of monocilia in cells, culture of the cells in high-glucose medium (containing D- the node (45). Expression of this gene appeared normal in the glucose at 850 mg/dL) for 12 h had no such effect (Fig. 3 G and H), node of both control (n = 11) and high-glucose (n = 12) embryos indicating that high glucose does not interfere with Nodal-Activin (Fig. 5 E and F). We next examined the morphology of the node signaling in ES cells. Taken together, these results suggested that in cultured embryos at the three- to four-somite stage by scanning high glucose impairs L–R axis formation before the induction of electron microscopy. In some cultured embryos, large cells Nodal expression in LPM rather than at the amplification stage. lacking a cilium were apparent among the pit cells (4 of 11 for control; 5 of 9 for high glucose) (Fig. S1 B and C). Although High Glucose Reduces Nodal Activity in the Node. Nodal produced in most high-glucose embryos (7 of 9) manifested ciliated pit cells the crown cells of the node is required for the subsequent ex- similar to those of control embryos (n = 11) (Fig. S1 A–C), the pression of Nodal in the left LPM (30, 32, 37). We therefore next pit cells lacking a cilium occupied a large area of the node in two examined whether the expression of Nodal-related genes, in- high-glucose embryos (Fig. S1D). Leftward nodal flow was ob- cluding Nodal, Gdf1, and Cerl2, was affected in the node of high- served in high-glucose embryos as well as in control embryos (n = glucose embryos. In cultured control embryos, Nodal expression 11 for control; 7 of 9 for high glucose) (Movies S1 and S2). Even was normally observed in crown cells at the three- to four-somite in the two high-glucose embryos with defective pit cells, a weak stage (n = 8) (Fig. 4A). In high-glucose embryos, however, the nodal flow was observed in the ciliated region (Movie S3). Taken

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Fig. 4. Down-regulation of Nodal activity in the node by high glucose. (A–F) Whole-mount in situ hybridization analysis of Nodal (A, B), Gdf1 (C, D, D′), and Cerl2 (E, F) expression in embryos cultured in control (A, C, E) or high-glucose (B, D, D′, F) medium from the head-fold to early somite stages. Expression of Uncx was detected simultaneously with Nodal or Cerl2 expression as an indicator for somite number. The node region is shown with anterior to the top. (Scale bar, 100 μm.) (G and H) Confocal immunohistofluorescence detection of pSmad2 (red) in embryos cultured in control (G) or high-glucose (H) medium. Nuclei were stained with TO-PRO3 (blue). Ventral views around the node region of embryos at the four-somite stage are shown with anterior to the top. (Scale bar, 200 μm.) (I and J) Enlarged views of optical cross-sections at the level of the node shown by the dashed lines in G and H, respectively. Arrows indicate crown cells. (Scale bar, 100 μm.) MEDICAL SCIENCES

together, these results suggested that node formation was not accounts for CHDs and abnormal L–R asymmetric morphogenesis severely impaired in most embryos exposed to high glucose. apparent in the offspring of diabetic mothers. Expression of Nodal in the node is induced by Notch signaling (46–48). We have also previously shown that Gdf1 and Cerl2 ex- Discussion pression in crown cells is extinguished by a Notch signaling inhibitor Although case-control studies have shown that CHDs and ac- (48). We examined whether Notch signaling is affected in high- companying laterality defects are associated with pregestational glucose embryos at early somite stages. Delta-like 1 (Dll1) induces DM (1, 4, 15, 16), the molecular etiology of these defects has Nodal expression in crown cells through interaction with its re- remained largely unknown. We have now shown that maternal ceptors Notch1 and Notch2 (46, 47). There was no apparent dif- ference in the expression of Dll1 around the node (n = 12 and 11, respectively), in that of Notch1 in crown cells and paraxial meso- derm (n = 12 and 12), and in that of Notch2 in the node and paraxial mesoderm (n = 9 and 9) between control and high-glucose embryos (Fig. 5 G–L). We also examined the distribution of Notch1 in control and high-glucose embryos by immunohistofluorescence analysis. Notch1 was detected predominantly around the node in- cluding the crown cells, in the nascent mesoderm at the anterior primitive streak, and in newly generated somites in control embryos (n = 5) (Fig. 6A). In high-glucose embryos, although the staining in the nascent mesoderm was irregular, we did not detect an apparent change in Notch1 distribution in crown cells (n = 6) (Fig. 6 B and E). We next examined whether Notch signaling might be affected in high-glucose embryos by immunostaining for the cleaved Notch1 intracellular domain (cNICD1) (49). We found that the amount of cNICD1 was significantly reduced in crown cells of high-glucose embryos (n = 13; P < 0.001) at early somite stages compared with that in control embryos (n =14) (Fig. 6 C–E). These results thus suggested that high glucose impairs Notch signaling in the node at the level or upstream of cleavage of the Notch intracellular domain, possibly accounting for the down-regulation of Nodal-related gene expression in crown cells. In summary, high glucose was found to directly affect L–R axis formation in mouse embryos. Attenuation of Notch signaling Fig. 5. Normal Notch-related gene expression around the node of high-glucose in crown cells of the node may prevent the establishment of a embryos. Embryos cultured in control (A, C, E, G, I, K) or high-glucose (B, D, F, H, J, L) medium from the head-fold to early somite stages were subjected to whole- sufficient level of Nodal and Gdf1 expression in the node of high- mount in situ hybridization analysis of Shh (A, B), Foxa2 (C, D), Foxj1 (E, F), Dll1 glucose embryos. Expression of Nodal is therefore not initiated (G, H), Notch1 (I, J), and Notch2 (K, L) expression. Expression of Uncx was de- in the left LPM of these embryos, leading to failure of Pitx2 tected simultaneously with that of Dll1 as an indicator for somite number. The expression. Thus, the loss of Nodal and Pitx2 expression likely node region is shown with anterior to the top. (Scale bar, 100 μm.)

Hachisuga et al. PNAS Early Edition | 5of8 Downloaded by guest on September 30, 2021 high-glucose embryos. Despite its small size, the node plays a fun- damental role in L–R axis formation. First, Nodal produced by crown cells induces Nodal expression in the LPM (37). Second, the L–R asymmetric activity of Nodal in the node determines the side on which Nodal is expressed in the LPM (32, 33, 35). Notch sig- naling is required for Nodal expression in the node (46, 47). In addition, the expression of Gdf1, which encodes a heterodimeriza- tion partner of Nodal, is also likely induced by Notch signaling (48). The attenuated expression of Nodal and Gdf1 inthenodeofhigh- glucose embryos is thus likely attributable to the down-regulation of Notch signaling. The activity of Nodal was virtually undetectable in the node of high-glucose embryos, which probably accounts for the loss of Nodal expression in the LPM. Although the mechanism by which Notch signaling is suppressed by high glucose remains to be determined, our findings indicate that high glucose affects the initial stage of L–R axis formation. In addition, these findings suggest that a transient deviation of glycemic control at this stage, rather than continuous hyperglycemia at later stages of L–R axis formation, may give rise to CHDs in human patients. Reversed heart looping or embryonic turning frequently occurred in litters of dams with a blood glucose concentration of >490 mg/dL. Our finding that the L–R axis is affected only at such high-glucose levels might explain the rarity of heterotaxia associated with pre- gestational diabetes in humans. It does not necessarily suggest, however, that heterotaxia does not occur at less-pronounced glu- cose levels in humans. Somitogenesis progresses faster in mice than in human embryos, suggesting that the period of L–Raxisformation is also longer in humans. If the sum of excess glucose is the key determinant at this stage, then longer exposure to lower glucose levels may affect L–R axis formation in human embryos. In mice, the failure to acquire Nodal expression in the LPM results in right isomerism of viscera and specific types of CHD. − − For example, in Cryptic / mutant mice, which do not establish Nodal expression in the LPM, the position of the stomach is L–R randomized in association with asplenia or hyposplenia in the abdomen, whereas right isomerism of the lung and atrium is apparent in the thorax in association in about half of neonates − − with dextrocardia or mesocardia (24). Of note, Cryptic / neo- Fig. 6. Inhibition of Notch signaling in the node by high glucose. Embryos nates manifest CHDs, including transposition of the great ar- cultured in control (A, C) or high-glucose (B, D) medium from the head-fold teries (TGA) and atrial septal defect (ASD). TGA associated to early somite stages were subjected to immunohistofluorescence analysis with right isomerism has also been described in Nodal mutants of Notch1 (red) (A, B) or cNICD1 (red) (C, D). Nuclei were also stained with (19, 37). Pitx2c, whose expression is induced by Nodal, plays a TO-PRO3 (blue). Ventral views of the node region are shown with anterior to fundamental role in heart morphogenesis. Pitx2 mutant mice the top. Arrowheads indicate staining in crown cells, whereas dashed lines thus develop double-outlet right ventricle (DORV), complete demarcate the node. Insets in C and D show optical cross-sections at the level atrioventricular canal defect (CAVC), ASD, and ventricular including the node shown by the white lines in the merged images. Arrows – indicate crown cells. (Scale bar, 200 μm.) (E) Comparison of fluorescence septal defect (20 23, 25, 26). Unfortunately, given that the cul- intensity for Notch1 or cNICD1 in crown cells between control (C) and high- ture period that can support embryonic development is limited, it glucose (H) embryos. In the panel on the right, average fluorescence in- was not possible to determine which type of CHDs would be- tensity of Notch1 or cNICD1 is normalized by that of TO-PRO3 in each em- come manifest in embryos cultured in high-glucose medium. The < = bryo [log2(red/blue)]. P 0.001; NS, not significant (P 0.082). loss of Nodal expression in the LPM of these embryos, however, would be expected to result in TGA and DORV associated with heterotaxia based on right isomerism. In support of this notion, – hyperglycemia in mice impairs formation of the L R axis. The DORV, TGA, and CAVC associated with right isomerism are often directions of cardiac looping and embryonic turning were thus apparent in fetuses of female nonobese diabetic mice (17). On the found to be reversed in embryos of dams with streptozotocin- other hand, cases of heterotaxia in offspring of human diabetic induced diabetes. In addition, Pitx2 expression in the left LPM and mothers include left isomerism (50). The mechanism underlying the heart primordium was lost in these embryos, indicative of failure to development of such left isomerism remains unknown. establish the L–R axis. Furthermore, culture of embryos from the Nodal expression spreads in the left LPM to provide L–R axis head-fold stage in high-glucose medium also resulted in the loss of information to precursor cells of visceral organs that are con- Pitx2 expression, with this loss being attributable to the loss of Nodal structed along the anteroposterior axis of embryos. The heart expression in the left LPM. Given that the asymmetric morphology primordium at the anterior end of the LPM is the cell population of viscera including the heart is determined by the Nodal–Pitx2 that receives the Nodal signal last. Nodal expression expands in pathway, CHDs and accompanying laterality defects in diabetic the LPM at the four-somite stage, when the heart primordium is embryopathy can be explained, at least in part, by the loss of Nodal crescent-shaped. Its morphology subsequently undergoes a and Pitx2 expression. marked change associated with the formation of the heart tube With the use of whole-embryo culture, we found that the target and loops. The time course of Nodal expression indicates that of high glucose is Notch signaling in crown cells of the node. The the heart primordium receives the Nodal signal at a specific stage amount of cNICD1 in crown cells was thus significantly reduced in of heart development. Given that high glucose reduces Nodal

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1504529112 Hachisuga et al. Downloaded by guest on September 30, 2021 activity in the node, hyperglycemia at a threshold level may delay glucose solution was given as drinking water from E7.5. Control female mice PNAS PLUS Nodal expression in LPM rather than completely abolish it. Al- were fed at libitum until E6.5 and at 5 g/d thereafter. The pregnant female though it is not known whether such a delay in Nodal expansion mice were killed by cervical dislocation to collect embryos at the indicated in the LPM would affect heart morphogenesis, the precise area stages. The blood glucose concentration of streptozotocin-treated mice was measured with a glucometer (Glucose Pilot, Aventir Biotech). The study was Pitx2c of expression might not be established in the heart pri- approved by the Animal Care and Use Committee of Kyushu University. mordium at more advanced stages. This scenario would be expected to give rise to CHDs without laterality defects of vis- Whole-Embryo Culture. Embryos were removed from the decidua in DMEM ceral organs. In support of this notion, four embryos with l-loop supplemented with 10% (vol/vol) FBS. After removal of Reichert’s membrane, derived from dams with streptozotocin-induced diabetes were the developmental stage was determined on the basis of morphological found to have lost Pitx2 expression in the outflow tract but not in criteria. Three to nine embryos at the early to late head-fold stage were the left LPM. In these embryos, the heart primordium might not transferred to a 50-mL conical tube containing 1 mL of DMEM with a have received the Nodal signal as a result of delayed Nodal ex- D-glucose concentration of 100 mg/dL and supplemented with 75% (vol/vol) tension in the left LPM. Given that the direction of heart looping rat serum. The embryos were then subjected to rotation culture at 37 °C in is randomized in the absence of Nodal signaling, d-loop would a humidified incubator containing 5% CO2. The glucose concentration of also be expected to occur in embryos that express Pitx2 only in the culture medium as measured with a glucometer (GF501, TANITA) was 144 ± 16.5 mg/dL, depending on the lot of rat serum (mean ± SD for 32 lots). the left LPM, although we did not detect such embryos in the For high-glucose culture, D-glucose was added to the medium up to a final present study. Of note, we also observed that four embryos with measured concentration of 720 mg/dL. For long-term culture (48 h), the l-loop expressed Pitx2 in the left outflow tract and LPM (Fig. 1 C medium was replaced with fresh every 12 h after the initial 14 h. Where and G). In these embryos, the spread of Nodal expression in the indicated, some embryos were returned to control medium after the initial LPM might not have reached the heart primordium before the 14 h of high-glucose culture. High-glucose culture was always performed looping event, with Nodal signaling not occurring in the left side simultaneously with control culture. of the heart tube until later. Alternatively, glucose might directly inhibit the Nodal-dependent looping process but not the in- ES Cell Culture. Mouse ES cells (G4) were cultured in a gelatin-coated dish with duction of Pitx2 by the Nodal signal. If this is the case, threshold 2i + LIF culture medium (53). The medium was based on DMEM containing glucose concentrations would permit Nodal expression in the left D-glucose at 450 mg/dL. For high-glucose culture, ES cells were incubated for 12 h in medium containing D-glucose at 850 mg/dL SB431542 (Sigma-Aldrich) LPM, but the looping would be randomized, albeit in the pres- was added to the basal medium to a final concentration of 10 μM to sup- ence of Pitx2 expression in the left heart primordium. Various press Nodal-Activin signaling. types of CHDs, including conotruncal malformations, ASD, and MEDICAL SCIENCES ventricular septal defect have been described in human offspring Local Transfection of Embryos. Lipofection solution (Lipofectamine2000, of diabetic mothers. It will be important to address whether such Invitrogen) containing Nodal and GFP expression vectors was injected in the human CHDs without laterality defects are caused by delayed right posterior mesoderm with the use of a glass capillary connected to an Nodal expression in the LPM. injector (Narishige) (27). Injected embryos were then developed by whole- Human pregestational DM is associated with multiple defects (4). embryo culture. Although apoptosis induced by oxidative stress likely accounts for neural tube defects and persistent truncus arteriosus (9), the path- Whole-Mount in Situ Hybridization. Embryos were fixed overnight with ogenic mechanisms underlying many defects have remained un- 4% (wt/vol) paraformaldehyde in PBS and then subjected to whole-mount in clear. We have now shown that Notch signaling is affected by high situ hybridization according to standard protocols. In brief, fixed embryos were treated with PBS containing 0.1% Tween 20 (PBST) and dehydrated with glucose, suggesting that the multiple defects associated with diabetic increasing concentrations of methanol. After exposure to 6% (vol/vol) H2O2 pregnancy may be explained by impairment of separate de- in methanol, the embryos were rehydrated with PBST, treated first with velopmental processes and different signaling cascades. Notch sig- proteinase K (10 μg/mL) in PBST and then with glycine (2 mg/mL) in PBST, naling plays a key role in various processes of development, and its and fixed again with 4% paraformaldehyde and 0.2% glutaraldehyde in disturbance causes human congenital defects, including CHDs and PBST. After prehybridization, the embryos were incubated overnight with skeletal abnormalities (51). It will thus be important to investigate hybridization solution containing the various digoxigenin-labeled probes, whether Notch signaling is affected in developmental processes washed extensively, treated with RNase A (50 μg/mL), and washed again. other than L–R axis formation in diabetic embryopathy. Of note, They were then exposed to 10% (vol/vol) sheep serum, incubated overnight we also found that the somites of high-glucose embryos are smaller with alkaline phosphatase-conjugated antibodies to digoxigenin (Roche), and washed extensively, after which the color was developed with nitroblue along the anteroposterior axis compared with those in control tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. Probes included embryos. The smaller somites observed in high-glucose embryos those for Pitx2, Nodal, Lefty1, Lefty2, Uncx, Gdf1, Cryptic, Cerl2, Shh, Foxa2, might possibly be related to the observation that the risk for cost- Foxj1, Notch1,andNotch2. overtebral defects and VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and Immunostaining of Whole Embryos. Embryos were fixed for 20 min at 4 °C with limb abnormalities) is increased in human diabetic embryopathy 4% paraformaldehyde in PBS, washed with Tris-buffered saline containing (52). Somitogenesis is regulated by oscillation of Notch signaling in 0.1% Tween 20 (TBST), treated with 100% methanol, and rehydrated with presomitic mesoderm (49). Given that the expression pattern for TBST. For the immunodetection of pSmad2, the embryos were bleached with cNICD1 itself appeared normal in presomitic mesoderm of high- 10% H2O2 in methanol for 20 min after the treatment with 100% methanol. glucose embryos at the resolution level of our experiments, the Nonspecific sites were blocked by incubation for 30 min with 0.5% TSA reason why somitogenesis is affected is unclear. A Notch reporter Blocking Reagent (Perkin-Elmer). The embryos were then incubated with rabbit primary antibodies to cNICD1 (#2421, ), to the COOH assay performed in HEK293T cells failed to reveal a suppressive terminus of Notch1 (sc-6014, Santa Cruz Biotechnology), or to pSmad2 effect of high glucose on reporter activity (Fig. S2), suggesting that (#3101, Cell Signaling), all of which were diluted with blocking buffer (1:50). the suppression of Notch signaling by high glucose might depend on After washing with TBST, the embryos were incubated with Alexa Fluor 488- cell type. conjugated goat antibodies to rabbit IgG (Molecular Probes) and with TO- PRO3 (Molecular Probes) to detect nuclei. Immunostaining of control and Materials and Methods high-glucose embryos was always performed simultaneously. Confocal im- Mice. Imprinting control region mice were mated to obtain embryos. Noon of ages were acquired with a Leica TCS SP5 system, and fluorescence of Alexa the day on which a vaginal plug was detected was designated as E0.5. Dams at Fluor 488 and TO-PRO3 was converted to red and blue colors, respectively. E3.5 were injected intraperitoneally with streptozotocin (200 μg per gram of body weight) (Wako) to induce type 1 DM. Food (CR-LPF, Oriental Yeast) Quantitation of Immunofluorescence. Crown cells of the node were identified intake was limited to 5 g/d beginning 3 d after the injection, and 5% (wt/vol) by 3D projection of TO-PRO3 confocal images (Leica LAS AF). Crown cells

Hachisuga et al. PNAS Early Edition | 7of8 Downloaded by guest on September 30, 2021 adjacent to the depression of the node were then marked on the z-stacked there being no differences between control and high-glucose embryos. A P images as the region of interest (depth of 18 μm), for which eight-bit pixel in- value < 0.05 was considered statistically significant. tensities of Alexa Fluor 488 and TO-PRO3 fluorescence were measured with the “Analyze Particles” function of ImageJ. The pixel intensities of crown cells on ACKNOWLEDGMENTS. We thank Y. Honda for technical assistance; Y. Saga both sides of the node were summed and averaged. The averaged value of and D. Watanabe for advice on immunohistofluorescence analysis and Alexa Fluor 488 was then divided by that of TO-PRO3 to correct for the bias preparation of samples for scanning electron microscopy, respectively; because of the distance between the coverslip and the specimen. A. Nagy and M. Gertsenstein for providing G4 embryonic stem cells; and the Research Support Center, Graduate School of Medical Sciences, Kyushu University, for technical support. This work was supported by grants Statistical Analysis. The Wilcoxon rank sum test (R package) for comparison of from the Ministry of Education, Culture, Sports, Science, and Technology immunofluorescence intensity or two-tailed Fisher’s exact probability test for of Japan (26291051, 25670491, 26461637), as well as by the Takeda Science the other experiments was performed according to the null hypothesis of Foundation.

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