Differential expression of Eya1 and Eya2 during chick early embryonic development

Eyes absent is essential for compound eye formation in Drosophila. Its mammalian homologues of Eya are involved in the development of sensory organs, skeletal muscles and kidneys. Mutations of EYA1 in human cause branchio-oto-renal syndrome, with abnormalities in branchial derivatives, ear and kidney. For an insight into the function of Eya1 and Eya2 in early development, we performed whole-mount in situ hybridization and compared the expression patterns of these two genes in the developing chick embryos. Eya1 was first expressed in the primitive streak at Hamburger and Hamilton stage 4 (HH4) and appeared in the ectoderm and head mesenchyme distinct from migrating neural crest cells at HH6-HH11. At HH15 and HH17, the olfactory, otic and vagal/nodose placodes and cranial ganglia were positive for Eya1. In contrast, Eya2 was already expressed in the endoderm at HH4, and appeared in the endoderm and prospective placodal region at HH6-HH11. Eya2 expression was observed in pharyngeal clefts and pouches as well as cranial placodes at HH15 and HH17. These results indicate differential expression of Eya1 and Eya2, both spatially and temporally, in chick during early development. The expression patterns are somewhat different from those of other species such as Xenopus, zebrafish and mouse. The results suggest distinct and unique functions for Eya1 and Eya2 in early chick development.     

Melanoma Cell Adhesion Molecule (MCAM) expression in the myogenic lineage during early chick embryonic development

We describe the expression pattern of cMCAM, a cell adhesion molecule of the immunoglobulin superfamily, in early chick embryonic development by in situ hybridisation. An initial ectodermal domain of expression is subsequently expanded, and cMCAM is expressed in the neural crest cells, otic vesicle, heart primordium, notochord and endoderm. In addition, cMCAM expression localises in the myotome once the somite cells have been specified. An in vitro murine cellular system allowed us to confirm that MCAM expression coincides with the onset of myogenic cell determination.     

Expression of ASK1 during chick and early mouse development

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.     

Temporal and spatial protease nexin 1 expression during chick development

Protease nexin 1 (Pn-1) or glia derived nexin is a secreted protease inhibitor. By screening a chick embryonic cDNA library, we isolated Pn-1 cDNA and analyzed its expression pattern during development by in situ hybridization. Pn-1 was first observed at HH-stage 3 in the primitive pit. At HH-stage 7, expression was observed in the medial part of the neural folds and asymmetrically in the right lateral plate mesoderm and at the left side of Hensen's node. At HH-stage 10-11, Pn-1 was expressed in the closing neural tube, lateral plate mesoderm and paraxial head mesoderm. From HH-stage 12 onwards, expression was observed caudally in the lateral plate mesoderm and cranially in the Wolffian duct. At the level of the compartmentalized somite, expression was seen in the sclerotome. Pn-1 was also expressed in the anterior wall of the pharynx and still in the paraxial head mesoderm. At HH-stage 15, the expression in the Wolffian duct remained caudally while the expression in the sclerotome extended along the whole body axis. A stronger expression was observed in the cranial four somites. From HH-stage 17-18 onwards, expression became visible in the mesenchyme of the developing limb buds. At these stages, expression was no longer observed in the Wolffian duct. At HH-stage 36, Pn-1 was expressed in the vertebral bodies, in the neural tube, and in the metanephros.     

CXCL14 expression during chick embryonic development

Chemokines are small secreted signalling molecules best known for their roles as chemoattractants for cells of the immune system. CXCL12 and its receptor CXCR4 comprise one chemokine signalling pathway with essential functions in non-immune cell types during embryonic development. CXCL14, a chemokine-encoding gene related to CXCL12, is developmentally regulated in zebrafish and Xenopus embryos, but its role during embryogenesis remains unknown. Here we describe the embryonic expression pattern of CXCL14 in an amniote, the chick. Although expression in some regions is conserved with that of fish and frog, chick CXCL14 displays a complex pattern of expression in several novel sites. We analyse the expression pattern in the branchial arches, trigeminal placode and ganglion, inner ear, dorsal midline of the brain, somites, trunk neural tube and limb bud. Expression in several domains raises the possibility that CXCL14 may be involved in some of the same developmental events during which CXCL12-CXCR4 signalling is known to play a role.     

Frizzled-4 expression during chick kidney development

Wnts have been implicated in metanephric kidney development. To determine whether Frizzleds, the genes that encode Wnt receptors, are present at early stages of nephrogenesis, we examined the expression of several recently identified Frizzled genes in the chick by in situ hybridization. Here we report the cloning and characterization of chick Frizzled-4 (cFz-4), which we found to be expressed in the developing chick kidney. cFz-4 was first expressed in the pronephros caudal to the third somite at Hamburger and Hamilton stage 10. Its expression increased with maturation, becoming restricted to the newly induced glomeruli and tubules in the mesonephros and metanephros. Within the metanephros, cFz-4 and Wnt-4 expression patterns were similar, whereas Wnt-11 was expressed solely in the tips of the branching ureteric bud. cFz-4 expression was compared with that of known kidney markers. It preceded that of Lmx-1, but was similarly restricted to developing glomeruli and tubules. In contrast, Pax-2 expression and Lim 1/2 antibody labeling occurred in intermediate mesoderm caudal to the fifth somite in the early pronephros, and each persisted in both the tubules and nephric ducts throughout further development.     

Stromal-derived factor 1 expression in the human thymus

Stromal-derived factor-1 (SDF-1), the only known ligand for the chemokine receptor CXCR4, is broadly expressed in cells of both the immune and central nervous systems, and it can induce the migration of resting leukocytes and hemopoietic progenitors. SDF-1 mRNA was previously detected in human thymus-derived stromal cells, but its role in thymopoiesis was unknown. Here we show that SDF-1 is expressed in medullar epithelial cells forming Hassall's corpuscles (HC). In search of the cell type that may be attracted by SDF-1(+) cells in the medulla, we determined that dendritic cells (DC) could be found in situ in close proximity to SDF-1(+) epithelial cells in HC. In HIV-1-infected SCID-hu thymuses, DC contained apoptotic cells and were located within enlarged HC. It was further demonstrated that uptake of apoptotic thymocytes by immature DC induced an increase in CXCR4 expression and SDF-1-mediated chemotaxis. Our data suggest a role for SDF-1 in the elimination of apoptotic thymocytes.     

Retrovirus-mediated gene expression during chick visual system development

The chick embryo is an excellent model for studying eye morphogenesis, retinal cell fate determination, and retinotectal projections due to its accessibility and the available molecular tools. Avian replication-competent retroviruses allow efficient infection of proliferating cells and stable integration of the viral genome, including up to 2.3kb of foreign cDNA, into the host chromosome. High-titer retroviruses are produced by transient transfection of avian DF-1 cells followed by centrifugation of the culture medium. Targeted infection of the optic vesicle, the lens vesicle, the retina and pigmented epithelium, the periocular mesenchyme, and the tectum can be performed at different developmental stages in ovo. In addition, retroviruses can be used to transduce genes of interest into various ocular tissue explants or cells in vitro. Virus-mediated gene expression can be detected within 12h of infection. Therefore, avian replication-competent retroviruses serve as powerful tools to misexpress wild-type and mutant gene products and to study molecular mechanisms underlying vertebrate visual system development.     

Endogenous patterns of BMP signaling during early chick development

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily signaling molecules that play important roles in a wide variety of developmental processes. In this study, we have used an antibody specific for the phosphorylated and activated form of Smad1 to examine endogenous patterns of BMP signaling in chick embryos during early development. We find complex spatial and temporal distributions of BMP signaling that elucidate how BMPs may function in multiple patterning events in the early chick embryo. In the pregastrula embryo, we find that BMP signaling is initially ubiquitous and is extinguished in the epiblast at the onset of primitive streak formation. At the head process stage, BMP signaling is inactivated in prospective neural plate, while it is strongly activated at the neural plate border, a region which is populated by cells that will give rise to neural crest. During later development, we find a dynamic spatiotemporal activation of BMP signaling along the rostrocaudal axis, in the dorsal neural tube, in the notochord, and in the somites during their maturation process. We discuss the implication of our results for endogenous functions of BMP signaling during chick development.     

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.     

Cloning and expression analysis of Fgf5, 6 and 7 during early chick development

FGFs with similar sequences can play different roles depending on the model organisms examined. Determining these roles requires knowledge of spatio-temporal Fgf gene expression patterns. In this study, we report the cloning of chick Fgf5, 6 and 7, and examine their gene expression patterns by whole mount in situ hybridization. We show that Fgf5's spatio-temporally restricted expression pattern indicates a potentially novel role during inner ear development. Fgf6 and Fgf7, although belonging to different subfamilies with diverged sequences, are expressed in similar patterns within the mesoderm. Alignment of protein sequences and phylogenetic analysis demonstrate that FGF5 and FGF6 are highly conserved between chick, human, mouse and zebrafish. FGF7 is similarly conserved except for the zebrafish, which has considerably diverged.