Sapropterin Treatment Prevents Congenital Heart Defects Induced by Pregestational Diabetes Mellitus in Mice.

Background Tetrahydrobiopterin is a cofactor of endothelial NO synthase ( eNOS ), which is critical to embryonic heart development. We aimed to study the effects of sapropterin (Kuvan), an orally active synthetic form of tetrahydrobiopterin on eNOS uncoupling and congenital heart defects ( CHD s) induced by pregestational diabetes mellitus in mice. Methods and Results Adult female mice were induced to pregestational diabetes mellitus by streptozotocin and bred with normal male mice to produce offspring. Pregnant mice were treated with sapropterin or vehicle during gestation. CHD s were identified by histological analysis. Cell proliferation, eNOS dimerization, and reactive oxygen species production were assessed in the fetal heart. Pregestational diabetes mellitus results in a spectrum of CHD s in their offspring. Oral treatment with sapropterin in the diabetic dams significantly decreased the incidence of CHD s from 59% to 27%, and major abnormalities, such as atrioventricular septal defect and double-outlet right ventricle, were absent in the sapropterin-treated group. Lineage tracing reveals that pregestational diabetes mellitus results in decreased commitment of second heart field progenitors to the outflow tract, endocardial cushions, and ventricular myocardium of the fetal heart. Notably, decreased cell proliferation and cardiac transcription factor expression induced by maternal diabetes mellitus were normalized with sapropterin treatment. Furthermore, sapropterin administration in the diabetic dams increased eNOS dimerization and lowered reactive oxygen species levels in the fetal heart. Conclusions Sapropterin treatment in the diabetic mothers improves eNOS coupling, increases cell proliferation, and prevents the development of CHD s in the offspring. Thus, sapropterin may have therapeutic potential in preventing CHD s in pregestational diabetes mellitus.

Simulated diabetic ketoacidosis therapy in vitro elicits brain cell swelling via sodium-hydrogen exchange and anion transport.

A common complication of type 1 diabetes mellitus is diabetic ketoacidosis (DKA), a state of severe insulin deficiency. A potentially harmful consequence of DKA therapy in children is cerebral edema (DKA-CE); however, the mechanisms of therapy-induced DKA-CE are unknown. Our aims were to identify the DKA treatment factors and membrane mechanisms that might contribute specifically to brain cell swelling. To this end, DKA was induced in juvenile mice with the administration of the pancreatic toxins streptozocin and alloxan. Brain slices were prepared and exposed to DKA-like conditions in vitro. Cell volume changes were imaged in response to simulated DKA therapy. Our experiments showed that cell swelling was elicited with isolated DKA treatment components, including alkalinization, insulin/alkalinization, and rapid reductions in osmolality. Methyl-isobutyl-amiloride, a nonselective inhibitor of sodium-hydrogen exchangers (NHEs), reduced cell swelling in brain slices elicited with simulated DKA therapy (in vitro) and decreased brain water content in juvenile DKA mice administered insulin and rehydration therapy (in vivo). Specific pharmacological inhibition of the NHE1 isoform with cariporide also inhibited cell swelling, but only in the presence of the anion transport (AT) inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid. DKA did not alter brain NHE1 isoform expression, suggesting that the cell swelling attributed to the NHE1 was activity dependent. In conclusion, our data raise the possibility that brain cell swelling can be elicited by DKA treatment factors and that it is mediated by NHEs and/or coactivation of NHE1 and AT.

Understanding early organogenesis using a simplified in situ hybridization protocol in Xenopus.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Rac1 Signaling Is Required for Anterior Second Heart Field Cellular Organization and Cardiac Outflow Tract Development.

BACKGROUND: The small GTPase Rac1 regulates diverse cellular functions, including both apicobasal and planar cell polarity pathways; however, its role in cardiac outflow tract (OFT) development remains unknown. In the present study, we aimed to examine the role of Rac1 in the anterior second heart field (SHF) splanchnic mesoderm and subsequent OFT development during heart morphogenesis. METHODS AND RESULTS: Using the Cre/loxP system, mice with an anterior SHF-specific deletion of Rac1 (Rac1(SHF)) were generated. Embryos were collected at various developmental time points for immunostaining and histological analysis. Intrauterine echocardiography was also performed to assess aortic valve blood flow in embryos at embryonic day 18.5. The Rac1(SHF) splanchnic mesoderm exhibited disruptions in SHF progenitor cellular organization and proliferation. Consequently, this led to a spectrum of OFT defects along with aortic valve defects in Rac1(SHF) embryos. Mechanistically, it was found that the ability of the Rac1(SHF) OFT myocardial cells to migrate into the proximal OFT cushion was severely reduced. In addition, expression of the neural crest chemoattractant semaphorin 3c was decreased. Lineage tracing showed that anterior SHF contribution to the OFT myocardium and aortic valves was deficient in Rac1(SHF) hearts. Furthermore, functional analysis with intrauterine echocardiography at embryonic day 18.5 showed aortic valve regurgitation in Rac1(SHF) hearts, which was not seen in control hearts. CONCLUSIONS: Disruptions of Rac1 signaling in the anterior SHF results in aberrant progenitor cellular organization and defects in OFT development. Our data show Rac1 signaling to be a critical regulator of cardiac OFT formation during embryonic heart development.

Expression of Ski can act as a negative feedback mechanism on retinoic acid signaling.

BACKGROUND: Retinoic acid signaling is essential for many aspects of early development in vertebrates. To control the levels of signaling, several retinoic acid target genes have been identified that act to suppress retinoic acid signaling in a negative feedback loop. The nuclear protein Ski has been extensively studied for its ability to suppress transforming growth factor-beta (TGF-ß) signaling but has also been implicated in the repression of retinoic acid signaling. RESULTS: We demonstrate that ski expression is up-regulated in response to retinoic acid in both early Xenopus embryos and in human cell lines. Blocking retinoic acid signaling using a retinoic acid antagonist results in a corresponding decrease in the levels of ski mRNA. Finally, overexpression of SKI in human cells results in reduced levels of CYP26A1 mRNA, a known target of retinoic acid signaling. CONCLUSIONS: Our results, coupled with the known ability of Ski to repress retinoic acid signaling, demonstrate that Ski expression is a novel negative feedback mechanism acting on retinoic acid signaling.

Retinoic acid is a key regulatory switch determining the difference between lung and thyroid fates in Xenopus laevis.

BACKGROUND: The lung and thyroid are derived from the anterior endoderm. Retinoic acid and Fgf signalling are known to be essential for development of the lung in mouse but little is known on how the lung and thyroid are specified in Xenopus. RESULTS: If either retinoic acid or Fgf signalling is inhibited, there is no differentiation of the lung as assayed by expression of sftpb. There is no change in expression of thyroid gland markers when retinoic acid signalling is blocked after gastrulation and when Fgf signalling is inhibited there is a short window of time where pax2 expression is inhibited but expression of other markers is unaffected. If exogenous retinoic acid is given to the embryo between embryonic stages 20 and 26, the presumptive thyroid expresses sftpb and sftpc, specific markers of lung differentiation and expression of key thyroid transcription factors is lost. When the presumptive thyroid is transplanted into the posterior embryo, it also expresses sftpb, although pax2 expression is not blocked. CONCLUSIONS: After gastrulation, retinoic acid is required for lung but not thyroid differentiation in Xenopus while Fgf signalling is needed for lung but only for early expression of pax2 in the thyroid. Exposure to retinoic acid can cause the presumptive thyroid to switch to a lung developmental program.

Fgf is required to regulate anterior-posterior patterning in the Xenopus lateral plate mesoderm.

Given that the lateral plate mesoderm (LPM) gives rise to the cardiovascular system, identifying the cascade of signalling events that subdivides the LPM into distinct regions during development is an important question. Retinoic acid (RA) is known to be necessary for establishing the expression boundaries of important transcription factors that demarcate distinct regions along the anterior posterior axis of the LPM. Here, we demonstrate that fibroblast growth factor (Fgf) signalling is also necessary for regulating the expression domains of the same transcription factors (nkx2.5, foxf1, hand1 and sall3) by restricting the RA responsive LPM domains. When Fgf signalling is inhibited in neurula stage embryos, the more posterior LPM expression domains are lost, while the more anterior domains are extended further posterior. The domain changes are maintained throughout development as Fgf inhibition results in similar domain changes in late stage embryos. We also demonstrate that Fgf signalling is necessary for both the initiation of heart specification, and for maintaining heart specification until overt differentiation occurs. Fgf signalling is also necessary to restrict vascular patterning and create a vascular free domain in the posterior end of the LPM that correlates with the expression of hand1. Finally, we show cross talk between the RA and Fgf signalling pathways in the patterning of the LPM. We suggest that this tissue wide patterning event, active during the neurula stage, is an initial step in regional specification of the LPM, and this process is an essential early event in LPM patterning.

Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut.

Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates.

Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin.

BACKGROUND: Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation. RESULTS: Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a beta-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative beta-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4. CONCLUSION: RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating beta-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.

Retinoic acid regulates anterior-posterior patterning within the lateral plate mesoderm of Xenopus.

The lateral plate mesoderm (LPM) lines the body cavities, gives rise to the heart and circulatory system and is responsible for patterning the underlying endoderm. We describe gene expression domains within the lateral plate mesoderm of the neurula stage Xenopus embryo that demonstrate a marked anterior posterior pattern in that tissue. FoxF1 and Nkx-2.5 are expressed in the anterior LPM, Hand1 in the middle and Xsal-1 in the posterior LPM. Since retinoic acid is known to pattern many tissues during development, and RALDH2, the enzyme primarily responsible for retinoic acid synthesis, is expressed in the anterior and dorsal LPM, we hypothesized that retinoic acid is necessary for correct patterning of the LPM. Exposure to exogenous retinoic acid during neurulation led to an expansion of the anterior and middle expression domains and a reduction of the posterior domain whereas exposure to a retinoic acid antagonist resulted in smaller anterior and middle expression domains. Furthermore, inhibition of RALDH2, which should decrease endogenous RA levels, caused a reduction of anterior domains indicating that endogenous RA is necessary for regulating their size. After altering retinoic acid signaling in a temporally restricted window, the displaced anterior-posterior pattern is maintained until gut looping, as demonstrated by permanently altered Hand1, FoxF1, xHoxC-10, and Pitx2 expression domains. We conclude that the broad expression domains of key transcription factors demonstrate a novel anterior-posterior pattern within the LPM and that retinoic acid can regulate the size of these domains in a coordinated manner.

Retinoic acid signaling is essential for formation of the heart tube in Xenopus.

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.

Early expression of thyroid hormone receptor beta and retinoid X receptor gamma in the Xenopus embryo.

The role of thyroid hormone in Xenopus metamorphosis is particularly well understood as it plays an essential role in that process. However, recent evidence suggests that thyroid hormone may play an earlier role in amphibian embryogenesis. We demonstrate that Xenopus thyroid hormone receptor beta (XTR beta) is expressed shortly after neural fold closure, and that its expression is localized to the developing retina. Retinoid X receptor gamma (RXR gamma), a potential dimerization partner for XTR beta, was also found to be expressed in the retina at early stages, and at later stages RXR gamma was also expressed in the liver diverticulum. Addition of either thyroid hormone or 9-cis retinoic acid, the ligands for XTR beta and RXR gamma, respectively, did not alter the expression of their receptors. However, the addition of thyroid hormone and 9-cis retinoic acid did alter rhodopsin mRNA expression. Addition of thyroid hormone generates a small expansion of the rhodopsin expression domain. When 9-cis retinoic acid or a combination of thyroid hormone and 9-cis retinoic acid was administered, there was a decrease in the expression domain of rhodopsin in the developing retina. These results provide evidence for an early role for XTR beta and RXR gamma in the developing Xenopus retina.

Regulation of heart size in Xenopus laevis.

The region with the potential to form the heart has traditionally been called the heart field. This region can be approximated by, but is not identical to, the expression domain of the early cardiac gene Nkx2.5. The region expressing Nkx2.5 does not change in size, although there are major shape changes and a subdivision of the region into non-myogenic and myogenic lineages. Using a variety of embryo manipulations, we have sought to determine whether cellular interactions could change the size of the initial Nkx2.5-expressing region and thus change the size of the heart. We have shown that if the heart is isolated from the dorsal half of the embryo, the volume of tissue expressing myocardial differentiation markers increases, indicating that signals restricting the size of the heart come from the dorsal side. Despite the change in myocardial volume, the non-myogenic heart lineages are still present. The ability of dorsal tissues to restrict the size of the heart is further demonstrated by fusing two Xenopus embryos shortly after gastrulation, generating twinned embryos where the heart of one embryo would develop adjacent to different tissues of the second embryo. The final size of the differentiated heart was markedly reduced if it developed in close proximity to the dorso-anterior surface of the head but not if it developed adjacent to the flank or belly. In all cases, the manipulations that restricted the size of the myocardium also restricted the expression of Nkx2.5 and GATA-4, both key regulatory genes in the cardiogenic pathway. These results provide evidence for a model in which signals from dorso-anterior tissues restrict the size of the heart after gastrulation but before neural fold closure.

Expression of muscle LIM protein during early development in Xenopus laevis.

We have isolated the Xenopus homologue of Muscle LIM protein (MLP, CRP3) and examined its expression during early embryonic development. MLP is only expressed in the differentiated heart during early development and is expressed in a subset of other striated muscles during later stages. There is no MLP expression during primary myogenesis in the somites, although it is found in adult skeletal muscle.

Developmental expression of cardiac myosin-binding protein C in Xenopus.

We have isolated the Xenopus homologue of cardiac myosin-binding protein C ( cMyBP-C) and describe its expression during early embryogenesis. cMyBP-C is expressed in both somites and heart at the time these tissues differentiate. Expression in the somites declines in older tadpoles. There is a high degree of conservation in residues where mutations causing hypertrophic cardiomyopathy have been identified in humans.

Cell Migration and Induction in the Development of the Surface Ectodermal Pattern of the Xenopus laevis Tadpole: (Xenopus/ciliated cell/hatching gland/cement gland/ectodermal differentiation).

The surface of the Xenopus tadpole contains three specialized, transient cell types; the ciliated, hatching gland, and cement gland cells. To distinguish whether the appearance of these cell types on the surface is due to induction of surface cells or due to migration of deep ectodermal cells into the surface, we transplanted labelled surface or deep cells to unlabelled hosts at early to mid-gastrulae. After raising the host to a tadpole (Stage 28), we examined the embryo's surface for ciliated, hatching gland, and cement gland cells, and observed which cells were labelled. We find that all ciliated cells move into the surface from the deep ectodermal layer along with other cells of unknown function. Hatching gland cells arise by induction of surface cells as do the majority of cement gland cells. A few deep cells give rise to cement gland cells. Therefore, migration of deep cells to the surface and localized induction of surface cells contribute to the final surface patterning of the Xenopus tadpole.