Efficient targeting of gene expression in chick embryos by microelectroporation

During vertebrate embryonic development, a key to unraveling specific functions of gene products is the capability to manipulate expression of the gene of interest at the desired time and place. For this, we developed a 'microelectroporation' technique by which DNA can be locally introduced into a targeted site of avian embryos, restricting spatial expression of the protein products during development. This technique involved injection of DNA solution in ovo around the target tissue and pinpoint application of an electric field by tungsten electrodes, allowing efficient and reproducible targeted gene transfer, for example, into an optic vesicle, somites, cranial mesoderm and limb mesenchyme. Because of the locality of gene introduction and its expression, survival rates of the embryos were high: approximately 90% of the embryos injected in optic vesicles were alive for at least 1 day after microelectroporation. The instantaneous gene transfer into embryonic cells allowed rapid expression of protein products such as green fluorescence protein within 2.5 h with fluorescence maintained for 3 days of incubation. This improved technique provides a convenient and efficient way to express transgenes in a spatially and temporally restricted manner in chicken embryos.     

Ribosome-like granules within areas of the perinuclear space in cells of 13–14 somite chick embryos

Summary Studies of the ultrastructure of somite cells from 13–14 somite chick embryos have indicated the presence of ribosome-like granules in polysome-like configurations in the perinuclear space within nuclear blebs. The average size of the nuclear blebs is approximately 0.3 by 0.24 microns. Some of the the blebs are contiguous with elements of the endoplasmic reticulum. The lumen of other blebs is continuous with the lumen of tubules of endoplasmic reticulum. The possible significance of these structures is discussed.I wish to thank Mrs. Marjorie K. Grifftih for her technical assistance. The work was supported by the budget of the Department of Anatomy, Ohio State University.     

Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos

Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including beta-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes beta-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling.     

Osteoclast precursor cells are present in the blood of preossification chick embryos

In an attempt to ascertain the time of appearance of circulating osteoclast precursor cells, we have transplanted quail bone rudiments into or onto the extraembryonic membranes of variously aged chick embryos. We observed that osteoclast precursor cells (1) are present in the embryonic circulation prior to the onset of osteogenesis, (2) differentiate precociously in response to a factor or factors present in developing bone rudiments, and (3) increase in number until about midway in embryonic life.     

Pair-rule gene expression in the somitic stage chick embryo: association with somite segmentation and border formation

Abstract. In vertebrates, metameric organization is highlighted by the formation of somites from mesenchymal cells of the segmental plate which then differentiate into dermamyotomal and sclerotomal tissues. The resegmentation of the sclerotome into rostral and caudal halves follows, coincident with the production of specific extracellular matrix molecules at the abutment of these two cell types. Ultimately, cells from the caudal sclerotome migrate ventrally and contribute to the chondrogenic prevertebrae. The objective of this work is to investigate the molecular steps regulating these events. Our study is focused on the paired-box containing genes, which have been implicated in delineating boundaries early in development. A chick embryo system, which is readily accessible to manipulation and observation during early development, is used in this study. We have identified the existence of the paired-box motif in the chicken genome by polymerase chain reaction and hybridization with the mouse Pax 1 paired-box sequence. Expression of paired-box genes occurs early in development as shown by Northern analysis, and is localized by in situ hybridization to the edge of each somite, a patch at the central core of each somite, and the periphery of the neural tube. This specific spatial pattern of expression is consistent with the hypothesis that the pair-rule genes function as effecters of border formation in the early embryo. Moreover, the patch of positive cells at the center of a resegmenting somite appear to migrate ventrally, and may contribute to structures of the prevertebrae. These findings are relevant to our understanding of the mechanism of somite resegmentation and implicate the involvement of pair-rule genes in the process.     

Pair-rule gene expression in the somitic stage chick embryo: association with somite segmentation and border formation

Abstract. In vertebrates, metameric organization is highlighted by the formation of somites from mesenchymal cells of the segmental plate which then differentiate into dermamyotomal and sclerotomal tissues. The resegmentation of the sclerotome into rostral and caudal halves follows, coincident with the production of specific extracellular matrix molecules at the abutment of these two cell types. Ultimately, cells from the caudal sclerotome migrate ventrally and contribute to the chondrogenic prevertebrae. The objective of this work is to investigate the molecular steps regulating these events. Our study is focused on the paired-box containing genes, which have been implicated in delineating boundaries early in development. A chick embryo system, which is readily accessible to manipulation and observation during early development, is used in this study. We have identified the existence of the paired-box motif in the chicken genome by polymerase chain reaction and hybridization with the mouse Pax 1 paired-box sequence. Expression of paired-box genes occurs early in development as shown by Northern analysis, and is localized by in situ hybridization to the edge of each somite, a patch at the central core of each somite, and the periphery of the neural tube. This specific spatial pattern of expression is consistent with the hypothesis that the pair-rule genes function as effecters of border formation in the early embryo. Moreover, the patch of positive cells at the center of a resegmenting somite appear to migrate ventrally, and may contribute to structures of the prevertebrae. These findings are relevant to our understanding of the mechanism of somite resegmentation and implicate the involvement of pair-rule genes in the process.     

Epithelia are interchangeable between facial primordia of chick embryos and morphogenesis is controlled by the mesenchyme

The embryonic chick face is composed of a series of facial primordia, epithelium-covered buds of mesenchyme, which surround the presumptive mouth. The protruding adult upper beak containing the prenasal cartilage is formed from the frontonasal mass, the paired maxillary primordia form the sides of the face, while the lower beak is derived from the paired mandibular primordia which contain the two Meckel's cartilages. When grafted to a host wing bud, the frontonasal mass and the mandibular primordia both form elongated outgrowths, whereas the maxillary primordium forms a ball of tissue. Facial epithelium is required for growth and morphogenesis of all primordia. Recombinations between epithelium and mesenchyme from different primordia show that the epithelia are interchangeable and appear to be equivalent. Even the epithelium from the maxillary primordium that does not grow out in a polarized fashion can support outgrowth of the frontonasal mass and mandibular mesenchyme. The form of the recombined graft is determined by the mesenchymal component.     

Gain- and loss-of-function in chick embryos by electroporation

It remained very difficult to manipulate gene expression in chick embryos until the advent of in ovo electroporation which enabled the induction of both gain-of-function, and recently loss-of-function, of a gene of interest at a specific developmental stage. Gain-of-function by electroporation is so effective that it has become widely adopted in developmental studies in the chick. Recently, it became possible to induce loss-of-function by introducing an siRNA expression vector by electroporation. In this review, the methods of electroporation for gain-of-function and for loss-of-function by siRNA are discussed.     

Floral development and expression of floral homeotic genes are influenced by cytokinins

Tobacco plants that are somatic mosaics for the expression of a cytokinin-synthesizing gene have viviparous leaves. Epiphyllous buds can be either vegetative or floral. Floral adventitious buds can be either normal or abnormal. Abnormalities of floral development correlate with: (i) a local activation of the cytokinin-synthesizing gene, (ii) a drastic increase in floral cytokinin content, and (iii) a decrease in the steady-state levels of mRNA homologues of the homeotic genes DEFA , GLO and PLENA of Antirrhinum majus . Thus, these data show in planta that cytokinins, a class of phytohormones, are able to alter the development of floral organs and to decrease the expression of three homeotic floral genes.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Tbx18 and boundary formation in chick somite and wing development

The chicken Tbx gene, Tbx18, is expressed in lateral plate mesoderm, limb, and developing somites. Here we show that Tbx18 is expressed transiently in axial mesenchyme during somite segmentation. We present evidence from overexpression and transplantation experiments that Tbx18 controls fissure formation in the late stages of somite maturation. In presumptive wing lateral plate mesoderm, ectopic Tbx18 expression leads to anterior extension of the wing bud. These results suggest that Tbx18 is involved in producing mesodermal boundaries, generating in paraxial mesoderm morphological boundaries between somites and in lateral plate mesoderm a wing- or non-wing-forming boundary.     

Differentiation in vitro of mouse embryos to the stage of early somite

Mouse blastocysts continuously differentiate in vitro to the early somite stage with reconstituted rat tail collagen as the substrate for the attachment. In order for this to occur, it appears that two differentiation barriers must be overcome. The first, the formation of egg cylinders from the inner cell mass, can be overcome by incubating embryos in heat-inactivated fetal calf serum. The second, the formation of the early somite from the presomite stage, can be overcome by replacing fetal calf serum with human cord serum.Mouse blastocysts were initially incubated with calf serum in Eagle's minimum essential medium. After shedding the zona pellucida, the denuded blastocysts lay flat on the surface of the collagen. Soon thereafter, trophoblastic cells invaded the underlying collagen leaving the rounded inner cell mass protruding from the surface of the collagen. By replacing calf serum in the medium with fetal calf serum the inner cell mass differentiated into endoderm and ectoderm to form an egg cylinder.The egg cylinder rapidly became elongated and formed extraembryonic and embryonic regions. However, the embryonic region shrank from this point on in the fetal calf serum, and the resulting yolk sac formation did not contain the embryo proper. When fetal calf serum was replaced with human cord serum at the end of the egg cylinder stage (equivalent to embryos of about 7.5 days gestation) neural tissue, cardiac chambers, and somites were formed.