Expression and function of Ccbe1 in the chick early cardiogenic regions are required for correct heart development.

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.

Ccbe1 expression marks the cardiac and lymphatic progenitor lineages during early stages of mouse development.

The mammalian heart is a complex organ composed of diverse components and various cell types. Heart organogenesis requires the contribution of distinct pools of heart progenitors positioned in separate embryonic regions and subject to particular developmental signals. Moreover, these embryonic heart lineages have different transcriptional profiles expressing specific genes which activate pathways involved in heart lineage specification. Understanding the molecular control of heart organogenesis has major implications for treating congenital and adult heart diseases since specific heart lineages have been associated with particular human cardiovascular malformations. Collagen and calcium-binding EGF-like domain 1 (Ccbe1) was identified in our laboratory using an Affymetrix GeneChip system approach to identify the transcriptome of chick heart/hemangioblast precursor cells. Here, we present a detailed and systematic analysis of the expression of Ccbe1 during early mouse development using whole-mount in situ hybridization (WISH), immunohistochemistry and histological techniques. Ccbe1 mRNA was initially detected in the early cardiac progenitors of the two bilateral cardiogenic fields (E7.0) and in the cardiogenic mesoderm (E7.5 to E8.0). Ccbe1 mRNA was then persistently detected in the pericardium and transiently expressed in the myocardial tissue of the primitive heart tube (E8.25), being later expressed in the proepicardium. By E9.5, the Ccbe1 and Prox1 proteins were found to be expressed in common regions, including the septum transversum and in the proximity of the anterior cardinal vein. Here, it is shown that Ccbe1 is expressed in the FHF, SHF and proepicardium during heart organogenesis (E7.0 to E8.75). Later in development, Ccbe1 expression is localized in the septum transversum and in the vicinity of the anterior cardinal vein, embryonic structures related to hepatic and lymphatic development, respectively.

Identification of differentially expressed genes in the heart precursor cells of the chick embryo.

Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their targets and pathways they are involved with, remain largely unknown. Previous studies in the avian embryo showed that at HH4 Cerberus (cCer) transcripts are detected in the anterior endomesoderm including the heart precursor cells and later in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Whole-mount in situ hybridization and sectioning of selected genes showed heart and vascular expression patterns for these transcripts during early chick development. We have developed an effective strategy to specifically identify genes that are differentially expressed in the HPC lineages. Within this set we have identified several genes that are expressed in the heart, blood and vascular lineages, which are likely to play a role in their development. These genes are potential candidates for future functional studies on early embryonic patterning.

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.

Comparative expression of mouse and chicken Shisa homologues during early development.

During vertebrate embryogenesis, fibroblast growth factor (FGF) and Wnt signaling have been implicated in diverse cellular processes, including cell growth, differentiation, and tissue patterning. The recently identified Xenopus Shisa protein promotes head formation by inhibiting Wnt and FGF signaling through its interaction with the immature forms of Frizzled and FGF receptors in the endoplasmic reticulum, which prevents their posttranslational maturation. Here, we describe the mouse and chicken homologues of Xenopus Shisa. The mouse and chicken Shisa proteins share, respectively, 33.6% and 33.8% identity with the Xenopus homolog. In situ hybridization analysis shows that mouse shisa is expressed throughout embryonic development, predominantly in the anterior visceral endoderm, headfolds, somites, forebrain, optic vesicle, and limb buds. Cross-species comparison shows that the expression pattern of cshisa closely mirrors that of mshisa. Our observations indicate that the Shisa family genes are typically expressed in tissues known to require the modulation of Wnt and FGF signaling.