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- The dynamic right-to-left translocation of Cerl2 is involved in the regulation and termination of nodal activity in the mouse nodePublication . Inacio, Jose Manuel; Marques, Sara; Nakamura, Tetsuya; Shinohara, Kyosuke; Meno, Chikara; Hamada, Hiroshi; Belo, Jose AntonioThe 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 his LPM expression.
- Expression and Function of Ccbe1 in the Chick Early Cardiogenic Regions Are Required for Correct Heart DevelopmentPublication . Furtado, João; Bento, Margaret; Correia, Elizabeth; Inacio, Jose Manuel; Belo, José A.During the course of a differential screen to identify transcripts specific for chick heart/hemangioblast precursor cells, we have identified Ccbe1 (Collagen and calcium-binding EGF-like domain 1). While the importance of Ccbe1 for the development of the lymphatic system is now well demonstrated, its role in cardiac formation remained unknown. Here we show by whole-mount in situ hybridization analysis that cCcbe1 mRNA is initially detected in early cardiac progenitors of the two bilateral cardiogenic fields (HH4), and at later stages on the second heart field (HH9-18). Furthermore, cCcbe1 is expressed in multipotent and highly proliferative cardiac progenitors. We characterized the role of cCcbe1 during early cardiogenesis by performing functional studies. Upon morpholino-induced cCcbe1 knockdown, the chick embryos displayed heart malformations, which include aberrant fusion of the heart fields, leading to incomplete terminal differentiation of the cardiomyocytes. cCcbe1 overexpression also resulted in severe heart defects, including cardia bifida. Altogether, our data demonstrate that although cardiac progenitors cells are specified in cCcbe1 morphants, the migration and proliferation of cardiac precursors cells are impaired, suggesting that cCcbe1 is a key gene during early heart development.
- Xenopus Pkdcc1 and Pkdcc2 Are Two New Tyrosine Kinases Involved in the Regulation of JNK Dependent Wnt/PCP Signaling PathwayPublication . Vitorino, Marta; Silva, Ana Cristina; Inacio, Jose Manuel; Ramalho, Jose Silva; Gur, Michal; Fainsod, Abraham; Steinbeisser, Herbert; Belo, José A.Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly, our knockdown experiments revealed that these two proteins are both involved on blastopore and neural tube closure during gastrula and neurula stages, respectively. In vertebrates, tissue polarity and cell movement observed during gastrulation and neural tube closure are controlled by Wnt/Planar Cell Polarity (PCP) molecular pathway. Our results showed that Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. But surprisingly, they revealed different roles in the induction of a luciferase reporter under the control of Atf2 promoter. While Pkdcc1 induces Atf2 expression, Pkdcc2 does not, and furthermore inhibits its normal induction by Wnt11 and Wnt5a. Altogether our data show, for the first time, that members of the PKDCC family are involved in the regulation of JNK dependent Wnt/PCP signaling pathway.
- Functional analysis of the mouse Nodal antagonist, Cerl2, during left-right axis formationPublication . Inacio, Jose M; Marques, Sara; Belo, José A.Although recently our understanding of how the LR asymmetry is generated in vertebrate embryos has seen rapid progress, many important questions remain to be explained. In mouse embryos, the leftward flow of the extra-embryonic fluid in the node cavity, called nodal flow, seems to be the symmetry-breaking event. However, it is not yet know how this flow functions or how the asymmetric signal(s) generated in the node is/are transferred to the lateral plate mesoderm. The mouse gene cerberus-like2(cerl2) encodes a 20-kDa protein with a predicted signal peptide sequence and a cysteine-rich domain (CRD) containing nine cysteines characteristic of the Cerberus/DAN family. Whole-mount in situ hybridization studies showed that cerl2 transcripts could be first detected in a horseshoe-shaped expression pattern in the perinodal region of the mouse embryo (E7.0), resembling Nodal expression at this stage. At stage E7.5, expression of cerl2 begins to decrease in intensity on the left side, and by early somitogenesis (E8.0), it can be strongly detected in the right side of the node, assuming a complementary expression pattern to that observed in Nodal. Furthermore, it was shown that Cerl2 activity is upstream of the Nodal receptor inhibiting Nodal and its downstream targets. A physical interaction between these two proteins exists, which suggests that Cerl2 is a secreted Nodal antagonist. Here, to elucidate the role of Cerl2 protein in the early events of symmetry breaking the functional activity of this Nodal antagonist will be discussed.