Sara Marques

Reversal of left-right asymmetry induced by aberrant Nodal signaling in the node of mouse embryos

The node at the anterior tip of the primitive streak serves as an initial generator of the left-right (L-R) axis in mammalian embryos. We now show that a small disturbance in molecular signaling at the node is responsible for the L-R reversal of visceral organs in the inv mutant mouse. In the node of wild-type embryos, the expression of Nodal and Cerl2 (Dand5), which encodes an inhibitor of Nodal, is asymmetric, with the level of Nodal expression being higher on the left side and that of Cerl2 expression higher on the right. In inv/inv embryos, however, a localized reduction in the level of Cerl2 expression results in upregulation of the Nodal signal and a consequent induction of Lefty expression in the node. The ectopic expression of Lefty1 delays the onset of Nodal expression in the lateral plate mesoderm. L-R asymmetry of Cerl2 expression in the node also becomes reversed in a manner dependent on the Nodal signal. Nodal expression in the lateral plate mesoderm then appears on the right side, probably reflecting the balance between Nodal and Cerl2 in the node. The inhibition of Cerl2 expression by the Nodal signal suggests a mechanism for amplification of the cue for L-R asymmetry provided by nodal flow and for stabilization of asymmetric gene expression around the node. In inv/inv embryos, this system may function in reverse as a result of ectopic production of Lefty, which inhibits the Nodal signal on the left side in a manner dependent on leftward nodal flow.

The Dynamic Right-to-Left Translocation of Cerl2 Is Involved in the Regulation and Termination of Nodal Activity in the Mouse Node

The determination of left-right body asymmetry in mouse embryos depends on the interplay of molecules in a highly sensitive structure, the node. Here, we show that the localization of Cerl2 protein does not correlate to its mRNA expression pattern, from 3-somite stage onwards. Instead, Cerl2 protein displays a nodal flow-dependent dynamic behavior that controls the activity of Nodal in the node, and the transmission of the laterality information to the left lateral plate mesoderm (LPM). Our results indicate that Cerl2 initially localizes and prevents the activation of Nodal genetic circuitry on the right side of the embryo, and later its right-to-left translocation shutdowns Nodal activity in the node. The consequent prolonged Nodal activity in the node by the absence of Cerl2 affects local Nodal expression and prolongs its expression in the LPM. Simultaneous genetic removal of both Nodal node inhibitors, Cerl2 and Lefty1, sustains even longer and bilateral this LPM expression.

Identification and functional analysis of novel genes expressed in the Anterior Visceral Endoderm

During early vertebrate development, the correct establishment of the body axes is critical. The anterior pole of the mouse embryo is established when Distal Visceral Endoderm (DVE) cells migrate to form the Anterior Visceral Endoderm (AVE). Symmetrical expression of Lefty1, Cer1 and Dkk1 determines the direction of DVE migration and the future anterior side. In addition to the establishment of the Anterior-Posterior axis, the AVE has also been implicated in anterior neural specification. To better understand the role of the AVE in these processes, we have performed a differential screening using Affymetrix GeneChip technology with AVE cells isolated from cer1P-EGFP transgenic mouse embryos. We found 175 genes which were upregulated in the AVE and 36 genes in the Proximal-posterior sample. Using DAVID software, we characterized the AVE cell population regarding cellular component, molecular function and biological processes. Among the genes that were found to be upregulated in the AVE, several novel genes were identified. Four of these transcripts displaying high-fold change in the AVE were further characterized by in situ hybridization in early stages of development in order to validate the screening. From those four selected genes, one, denominated Adtk1, was chosen to be functionally characterized by targeted inactivation in ES cells. Adtk1 encodes for a serine/threonine kinase. Adtk1 null mutants are smaller and present short limbs due to decreased mineralization, suggesting a potential role in chondrogenesis during limb development. Taken together, these data point to the importance of reporting novel genes present in the AVE.

Generating asymmetries in the early vertebrate embryo: the role of the Cerberus-like family.

One fundamental aspect of vertebrate embryonic development is the formation of the body plan. For this process, asymmetries have to be generated during early stages of development along the three main body axes: Anterior-Posterior, Dorso-Ventral and Left-Right. We have been studying the role of a novel class of molecules, the Cerberus/Dan gene family. These are dedicated secreted antagonists of three major signaling pathways: Nodal, BMP and Wnt. Our studies contribute to the current view that the fine tuning of signaling is controlled by a set of inhibitory molecules rather than by activators. In this context, the Cerberus-like molecules emerge as key players in the regulation and generation of asymmetries in the early vertebrate embryo.

Targeted Inactivation of Cerberus Like-2 Leads to Left Ventricular Cardiac Hyperplasia and Systolic Dysfunction in the Mouse

Previous analysis of the Cerberus like 2 knockout (Cerl2−/−) mouse revealed a significant mortality during the first day after birth, mostly due to cardiac defects apparently associated with randomization of the left-right axis. We have however, identified Cerl2-associated cardiac defects, particularly a large increase in the left ventricular myocardial wall in neonates that cannot be explained by laterality abnormalities. Therefore, in order to access the endogenous role of Cerl2 in cardiogenesis, we analyzed the embryonic and neonatal hearts of Cerl2 null mutants that did not display a laterality phenotype. Neonatal mutants obtained from the compound mouse line Cer2−/−::Mlc1v-nLacZ24+, in which the pulmonary ventricle is genetically marked, revealed a massive enlargement of the ventricular myocardium in animals without laterality defects. Echocardiography analysis in Cerl2−/− neonates showed a left ventricular systolic dysfunction that is incompatible with a long lifespan. We uncovered that the increased ventricular muscle observed in Cerl2−/− mice is caused by a high cardiomyocyte mitotic index in the compact myocardium which is mainly associated with increased Ccnd1 expression levels in the left ventricle at embryonic day (E) 13. Interestingly, at this stage we found augmented left ventricular expression of Cerl2 levels when compared with the right ventricle, which may elucidate the regionalized contribution of Cerl2 to the left ventricular muscle formation. Importantly, we observed an increase of phosphorylated Smad2 (pSmad2) levels in embryonic (E13) and neonatal hearts indicating a prolonged TGFβs/Nodal-signaling activation. Concomitantly, we detected an increase of Baf60c levels, but only in Cerl2−/− embryonic hearts. These results indicate that independently of its well-known role in left-right axis establishment Cerl2 plays an important role during heart development in the mouse, mediating Baf60c levels by exerting an important control of the TGFβs/Nodal-signaling pathway.

The Role of Cerl2 in the Establishment of Left-Right Asymmetries during Axis Formation and Heart Development

The formation of the asymmetric left-right (LR) body axis is one of the fundamental aspects of vertebrate embryonic development, and one still raising passionate discussions among scientists. Although the conserved role of nodal is unquestionable in this process, several of the details around this signaling cascade are still unanswered. To further understand this mechanism, we have been studying Cerberus-like 2 (Cerl2), an inhibitor of Nodal, and its role in the generation of asymmetries in the early vertebrate embryo. The absence of Cerl2 results in a wide spectrum of malformations commonly known as heterotaxia, which comprises defects in either global organ position (e.g., situs inversus totalis), reversed orientation of at least one organ (e.g., situs ambiguus), and mirror images of usually asymmetric paired organs (e.g., left or right isomerisms of the lungs). Moreover, these laterality defects are frequently associated with congenital heart diseases (e.g., transposition of the great arteries, or atrioventricular septal defects). Here, reviewing the knowledge on the establishment of LR asymmetry in mouse embryos, the emerging conclusion is that as necessary as is the activation of the Nodal signaling cascade, the tight control that Cerl2-mediates on Nodal signaling is equally important, and that generates a further regionalized LR genetic program in the proper time and space.

CCBE1 is required for coronary vessel development and proper coronary artery stem formation in the mouse heart: CCBE1 and Coronary Vasculature Development

Background: Proper coronary vasculature development is essential for late‐embryonic and adult heart function. The developmental regulation of coronary embryogenesis is complex and includes the coordinated activity of multiple signaling pathways. CCBE1 plays an important role during lymphangiogenesis, enhancing VEGF‐C signaling, which is also required for coronary vasculature formation. However, whether CCBE1 plays a similar role during coronary vasculature development is still unknown. Here, we investigate the coronary vasculature development in Ccbe1 mutant embryos. Results: We show that Ccbe1 is expressed in the epicardium, like Vegf‐c, and also in the sinus venosus (SV) at the stages of its contribution to coronary vasculature formation. We also report that absence of CCBE1 in cardiac tissue inhibited coronary growth that sprouts from the SV endocardium at the dorsal cardiac wall. This disruption of coronary formation correlates with abnormal processing of VEGF‐C propeptides, suggesting VEGF‐C–dependent signaling alteration. Moreover, Ccbe1 loss‐of‐function leads to the development of defective dorsal and ventral intramyocardial vessels. We also demonstrate that Ccbe1 mutants display delayed and mispatterned coronary artery (CA) stem formation. Conclusions: CCBE1 is essential for coronary vessel formation, independent of their embryonic origin, and is also necessary for peritruncal vessel growth and proper CA stem patterning. Developmental Dynamics 247:1135‐1145, 2018.