Quail Cells Research Articles

Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo.

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10-20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4-14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK-1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro- and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4-14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart.

Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10–20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4–14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK-1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro- and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4–14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart. Anat Rec 255:407–419, 1999. © 1999 Wiley-Liss, Inc.

Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium

Morris (J. Anat., 1976;121:47–64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morris's hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail-chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail-specific antibodies (QCPN, QH-1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail-derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av-endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. Thistechnique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics. Anat Rec 255:212–226, 1999. © 1999 Wiley-Liss, Inc.

Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium.

Morris (J. Anat., 1976;121:47-64) proposed that the subepicardial mesenchyme might represent a continuing source of myocardioblasts during embryonic and fetal development. Recent studies have shown that the epicardium and subepicardial mesenchyme, and the coronary vasculature are all derived from a region of the pericardial wall, called the proepicardial serosa. In avian embryos, the cells from the proepicardial serosa colonize the heart via a secondary tissue bridge formed by attachment of proepicardial villi to the heart. In the present study, Morris's hypothesis was tested by tracing the fate of the proepicardial serosa. This was achieved by constructing quail-chick chimeras. The proepicardial serosa was transplanted from HH16/17 quail embryos to HH16/17 chick embryos (ED3). A new transplantation technique facilitated an orthotopic attachment of the quail proepicardial villi to the chicken heart, and prevented the attachment of the chicken proepicardial villi to the heart. The fate of the grafted quail cells was traced in chimeras from ED4 to ED18 with immunohistochemistry, using quail-specific antibodies (QCPN, QH-1). From ED4 onward, the transplant was connected to the dorsal heart wall via its proepicardial villi. Starting from the point of attachment of the quail proepicardial villi to the heart, the originally naked myocardium became almost completely covered by quail-derived epicardium, and quail mesenchymal cells populated the subepicardial, myocardial, and subendocardial layers including the av-endocardial cushions. Quail cells formed the endothelial and smooth muscles cells of the coronary vessels, and the perivascular and intramyocardial fibroblasts. Quail myocardial cells were never found in the subepicardial, myocardial, and subendocardial layers. This suggests that the subepicardial mesenchyme normally does not contribute a substantial number of myocardioblasts to the developing avian heart. The new transplantation technique presented facilitates the production of chimeric hearts in which the derivatives of the proepicardial serosa are almost completely of donor origin. This technique might be useful for future studies analyzing the role of certain genes in cardiac development by the creation of somatic transgenics.

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the alpha subunit of the human hypoxia-inducible factor 1 (HIF-1alpha). Anti-HIF-1alpha IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF-1alpha protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF-1alpha signal was weak or absent, respectively. In electrophoretic mobility shift assays, affinity-purified IgY antibody was shown to recognize the native HIF-1 (but not the related HIF-2) complex that specifically binds an oligonucleotide containing the HIF-1 DNA binding site. Furthermore, IgY antibody immunoprecipitated HIF-1alpha from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF-1alpha in the nucleus of hypoxic COS-7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF-1alpha fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies.

Induction of primitive streak and Hensen's node by the posterior marginal zone in the early chick embryo.

In the preprimitive streak chick embryo, the search for a region capable of inducing the organizer, equivalent to the Nieuwkoop Center of the amphibian embryo, has focused on Koller's sickle, the hypoblast and the posterior marginal zone. However, no clear evidence for induction of an organizer without contribution from the inducing tissue has been provided for any of these structures. We have used DiI/DiO labeling to establish the fate of midline cells in and around Koller's sickle in the normal embryo. In the epiblast, the boundary between cells that contribute to the streak and those that do not lies at the posterior edge of Koller's sickle, except at stage X when it lies slightly more posteriorly in the epiblast. Hypoblast and endoblast (a second lower layer formed under the streak) have distinct origins in the lower layer, and goosecoid expression distinguishes between them. We then used anterior halves of chick prestreak embryos as recipients for grafts of quail posterior marginal zone; quail cells can be identified subsequently with a quail-specific antibody. Anterior halves alone usually formed a streak, most often from the posterior edge. Quail posterior marginal zones without Koller's sickle were grafted to the anterior side of anterior halves. These grafts were able to increase significantly the frequency of streaks arising from the anterior pole of stage X-XI anterior halves without contributing to the streak or node. Stage XII anterior halves no longer responded. A goosecoid-expressing hypoblast did not form under the induced streak, indicating that it is not required for streak formation. We conclude that the marginal zone posterior to Koller's sickle can induce a streak and node, without contributing cells to the induced streak.

Vascularization of embryonic adrenal gland grafted onto chorioallantoic membrane.

Vascularization and endothelial phenotype expression were analysed in embryonic adrenal tissue grafted onto chorioallantoic membrane (CAM), by means of routine light microscopy and immunocytochemical staining, and of electron microscopy. Adrenal gland tissue from chick or quail embryos (donors) was grafted onto CAMs of chick or quail embryos (host). Vessels of chick origin were discriminated from those of quail origin by monoclonal antibodies, anti-MB1, specific for quail endothelial and haemopoietic cells, and QCPN, which labels quail cell nuclei. Vessels of adrenal type were distinguished from those of CAM-type by their ultrastructural endothelial phenotype - porous in the former and continuous in the latter. The observations carried out 6 days after implantation indicate that the adrenal gland develops and differentiates according to a virtually normal histological pattern. As regards the adrenal and CAM vascularization, the grafting procedure elicits angiogenic events consisting in the formation of peripheral anastomoses between the graft and the CAM original microvasculature and in new-growth of vessels from the CAM into the grafted tissue and vice versa. As to the endothelial phenotype, the ultrastructural results demonstrate that besides its own native vasculature, the adrenal tissue contains vessels with continuous endothelium and the CAM mesenchyme is supplied by adrenal-type, fenestrated vessels.

Embryonic CNS macrophages and microglia do not stem from circulating, but from extravascular precursors

Invasion of mesoderm-derived cells into the developing spinal cord and brain has been shown to produce early central nervous system (CNS) macrophage and microglia populations in avian embryos. A triplicate mode of entry has been proposed: through the endothelial wall of CNS blood vessels; from the ventricular cavities; and through the pial surface. Invasion of circulating blood cells (monocytes) has not yet been proved in embryonic CNS. This report demonstrates: 1) the use of chick-quail blood chimeras by way of parabiosis (two embryos in one egg); 2) the use of QH1 monoclonal antibody for detection of quail cells circulating in chick blood vessels; 3) the presence of extravascular QH1-positive cells (macrophages) in E7-10 CNS in parabiosis quail, and their absence in parabiosis chick. We conclude that avian macrophages/microglia precursors do not penetrate through the wall of embryonic CNS vessels. In combination with published results, this finding strongly supports the view that invasion of migratory macrophages from the pial surface and proliferation inside the CNS generate all microglia in avian embryos.

TAL1/SCL Is Expressed in Endothelial Progenitor Cells/Angioblasts and Defines a Dorsal-to-Ventral Gradient of Vasculogenesis

In this study we establish that TAL1/SCL, a member of the helix–loop–helix family of transcription factors, and an important regulator of the hematopoietic lineage in mice, is expressed in the endothelial lineage of avians. The earliest events of vascular development were examined using antibodies to TAL1/SCL, and the QH1 antibody, an established marker of quail endothelial cells. Analyses using double immunofluorescence confocal microscopy show that: (i) TAL1/SCL is expressed by both quail and chicken endothelial cells; (ii) TAL1/SCL expression precedes that of the QH1 epitope; and (iii) TAL1/SCL, but not QH1, expression defines a subpopulation of primordial cells within the splanchnic mesoderm. Collectively these data suggest that TAL1/SCL-positive/QH1-negative cells are angioblasts. Further, using TAL1/SCL expression as a marker of the endothelial lineage, we demonstrate that in addition to the previously described cranial-to-caudal gradient, there is a dorsal-to-ventral progression of vasculogenesis.

Host Range and Cytopathogenicity of the Highly Attenuated MVA Strain of Vaccinia Virus: Propagation and Generation of Recombinant Viruses in a Nonhuman Mammalian Cell Line

Modified vaccinia virus Ankara (MVA), attenuated by over 500 passages in primary chick embyro fibroblasts (CEF), is presently being used as a safe expression vector. We compared the host ranges of MVA and the parental Ankara strain in CEF and 15 permanent cell lines. The cells could be grouped into three categories: permissive, semipermissive, and nonpermissive. For MVA, the permissive category consisted of primary CEF, a quail cell line derived from QT6, and the Syrian hamster cell line BHK-21. Only in BHK-21 cells did the virus yield approach that occurring in primary CEF. The semipermissive category included two African green monkey cell lines: BS-C-1 and CV-1. The nonpermissive category for MVA consisted of three human cell lines HeLa, 293, and SW 839; one rhesus monkey cell line FRhK-4; two Chinese hamster cell lines CHO and CHL; one pig cell line PK(15); and three rabbit cell lines RK13, RAB-9, and SIRC. The grouping for MVA with a restored K1L host range gene was similar except for the inclusion of RK13 cells among permissive lines. The grouping for the Ankara strain, however, was quite different with more permissive and semipermissive cell lines. Nevertheless, in cells that were permissive for MVA, the virus replicated to higher levels than Ankara, consistent with both positive and negative growth elements associated with the adaptation of MVA. The cell lines were also characterized according to their susceptibilty to MVA-induced cytopathic effects, expression of a late promoter regulated reporter gene by an MVA recombinant, and stage at which virion morphogenesis was blocked. Finally, the permissive BHK-21 cell line was shown to be competent for constructing and propagating recombinant MVA, providing an alternative to primary CEF.

Selection of an avian retrovirus mutant with extended receptor usage.

Receptor recognition by avian retroviruses is thought to involve the interaction of two regions of the SU protein, hr1 and hr2, with the host cell surface receptor. These regions exhibit considerable variation, concordant with differences in receptor usage among the many avian leukosis virus subgroups. We hypothesize that some retroviruses have altered receptor usage in response to selective pressures imposed by receptor polymorphisms in their hosts. To test this hypothesis, we passaged td-Pr-RSV-B on cocultured permissive chicken (C/E) and nonpermissive quail (QT6/BD) cells. A variant virus with an expanded host range was identified at passage 29 and ultimately shown to be identical in sequence to td-Pr-RSV-B, except for changes at codons 155 and 156 of SU amino acid corresponding to two amino acid changes within hr1. Superinfection resistance studies suggest that the variant virus recognizes the subgroup B receptor on chicken cells and the subgroup E receptor on quail cells. These findings indicate that altered receptor usage can be conferred by small changes in env and may point to a key region for receptor interaction. Further, they demonstrate the evolutionary potential of retroviral env genes to alter receptor usage in response to appropriate selective pressure.

Suppression in transformed avian fibroblasts of a gene (CO6) encoding a membrane protein related to mammalian potassium channel regulatory subunits.

Gene expression patterns in normal and v-myc-transformed quail embryo fibroblasts were compared by mRNA differential display. Displaying approximately 2500 mRNA species by reverse transcription/PCR, reamplification of 73 differential cDNA fragments and rescreening by Northern analysis led to the isolation of a clone, termed CO6, that hybridized to an mRNA species present only in the normal but not in the transformed fibroblasts. Further analyses revealed that the 0.95-kb CO6 mRNA was present in all normal quail and chicken embryo fibroblasts tested, but that it was undetectable in a variety of established quail cell lines transformed by the v-myc, v-myc/v-mil, v-jun/junD or v-src oncogenes or by a chemical carcinogen. Furthermore, CO6 mRNA was not detectable in fibroblasts newly transformed by retroviral constructs carrying v-myc or v-jun alleles or by the avian sarcoma virus ASV17. In fibroblasts transformed by a temperature-sensitive v-src mutant, expression of CO6 was strongly induced at the non-permissive temperature and reduced at the permissive temperature. Nucleotide sequence analysis of quail CO6 cDNA indicated that the corresponding gene encodes a 200-amino acid protein with 46 to 48% amino acid sequence identity to the regulatory beta subunits (K(VCa)beta) of the bovine, human and canine high conductance Ca2+-activated K+ channels. No sequence homology to other ion channel subunits or to any other proteins in the databases was found. Like the K(VCa)beta subunits, the CO6 protein contains two putative transmembrane segments. Based on the relationship to mammalian K(VCa)beta both in primary structure and domain topology, the CO6 protein may represent the regulatory subunit of a yet unidentified avian Ca2+-activated potassium channel or a related membrane protein possibly involved in the regulation of cell proliferation.

Analysis of cleavage site mutations between the NC and PR Gag domains of Rous sarcoma virus.

In retroviruses, the viral protease (PR) is released as a mature protein by cleavage of Gag, Gag-Pro, or Gag-Pro-Pol precursor polypeptides. In avian sarcoma and leukemia viruses (ASLV), PR forms the C-terminal domain of Gag. Based on the properties of a mutation (cs22) in the cleavage site between the upstream NC domain and the PR domain, the proteolytic liberation of PR previously was inferred to be essential for processing of Gag and Pol proteins. To study this process in more detail, we have analyzed the effects that several mutations at the NC-PR cleavage site have on proteolytic processing in virus-like particles expressed in COS and quail cells. Mutant Gag proteins carrying the same mutations also were synthesized in vitro and tested for processing with purified PR. In both types of studies, N-terminal sequencing of the liberated PR domain was carried out to exactly identify the site of cleavage. Finally, synthetic peptides corresponding to the mutant proteins were assessed for the ability to act as substrates for PR. The results were all consistent and led to the following conclusions. (i) In vivo, if normal processing between NC and PR is prevented by mutations, limited cleavage occurs at a previously unrecognized alternative site three amino acids downstream, i.e., in PR. This N-terminally truncated PR is inactive as an enzyme, as inferred from the global processing defect in cs22 and a similar mutant. (ii) In Gag proteins translated in vitro, purified PR cleaves this alternative site as rapidly as it does the wild-type site. (iii) Contrary to previously accepted rules describing retroviral cleavage sites, an isoleucine residue placed at the P1 position of the NC-PR cleavage site does not hinder normal processing. (iv) A proline residue placed at the P2 position in this cleavage site blocks normal processing.

1 The Avian Embryo as a Model in Developmental Studies: Chimeras and in Vitro Clonal Analysis

The avian embryo is a model in which techniques of experimental embryology and cellular and molecular biology can converge to address fundamental questions of development biology. The first part of the chapter describes two examples of transplantation and cell labeling experiments performed in ovo. Thanks to the distinctive histologic and immunocytochemical characteristics of quail and chick cells, the migration and development of definite cells are followed in suitably constructed chimeric quail-chick embryos. Isotopic transplantations of neural tube portions between quail and chick, combined with in situ hybridization with a nucleic probe specific for a quail oligodendrocyte marker, allowed study of the origin and migration of oligodendroblasts in the spinal cord. Heterotopic transplantations of rhombomeres were performed to establish the degree of plasticity of these segments of the hindbrain regarding Hox gene expression, which was revealed by labeling with chick-specific nucleic probes. The second part describes in vitro cell cloning experiments devised to investigate cell lineage segregation and diversification during development of the NC. An original cloning procedure and optimal culture conditions permitted analysis of the developmental potentials of individual NC cells taken at definite migration stages. The results revealed a striking heterogeneity of the crest cell population, which appeared to be composed of precursors at different states of determination. Clonal cultures also provide a means to identify subsets of cells that are the target of environmental factors and to understand how extrinsic signals influence the development of responsive cells.

Embryonic Neural Chimeras in the Study of Vertebrate Brain and Head Development

Construction of neural chimeras between quail and chick embryos has been employed since 1969 when the unique nucleolar structure of the quail nucleus and its use to devise a cell marking technique by associating quail and chick cells in ovo were described in the "Bulletin Biologique de la France et de la Belgique." This method was first applied to the ontogeny of the neural crest, a structure whose development involves extensive cell migration, and, since 1984, to that of the central nervous system (CNS). This chapter highlights some of the most significant findings provided by this approach concerning the CNS, such as (i) demonstration of the common origin of the floor plate and notochord from a group of cells localized in the "organizer", i.e., Hensen's node, and the way in which these two structures become positioned respectively within and under the neural tube during gastrulation and neurulation in Amniotes; (ii) the neural crest origin of the skull vault and the facial and hypobranchial skeleton. This means that the mesodermal contribution to the skull is limited to the occipital and otic regions and extends only to the rostral limit of the notochord. A correlation can be drawn between the development of the telencephalon and the mesectodermally derived skull in the vertebrate phylum; (iii) demonstration that the midbrain-hindbrain junction, at the stage of the encephalic vesicles, acts as an organizing center for tectal and cerebellar structures. This function was correlated with the activity of several developmental genes, thus providing insight into their function during neurogenesis; (iv) the pattern of morphogenetic movements and cell migration taking place in defined brain-to-be areas, as well as the origin of various cell types of nervous tissues; and (v) a new avenue for studying brain localization of either behavioral traits or genetically encoded brain disorders.

Avian models in developmental biology

The avian embryo is uniquely amenable to experimental manipulation. The most widely used models are chimeras resulting from heterotopic or orthotopic exchanges of rudiments between chick and quail embryos, according to Le Douarin's technique (1969). Cell migrations and fates are traced in these chimeras either through the identification of quail cell nuclei stained for DNA or by means of monoclonal antibodies that recognize a particular lineage in only one of the two species. The ontogeny of the hemopoietic and endothelial lineages, as enlightened through appropriately designed chimeras, is reviewed in the present article. Homologies recently disclosed in mouse and human embryo are emphasized. Finally, the possibilities afforded by retroviral somatic transgenesis in the avian embryo will be envisaged.

The U3 region of the long terminal repeat of a subgroup A transformation-defective rous sarcoma virus (tdPH2010) converts a noncytopathic virus to a cytopathic virus.

The molecular basis of the cytopathic effects (CPE) of the transformation-defective avian retrovirus mutant, tdPH2010, was studied. tdPH2010 is a subgroup A virus isolated from the Schmidt-Ruppin (NY) subgroup A strain (SRA(NY)). Subgroup A avian retroviruses are generally considered noncytopathic. Integrated tdPH2010 was molecularly cloned from infected quail cells. A noncytopathic, transformation-defective control strain, BSU, was created by deleting the src gene from the molecularly cloned wild type SRA(NY) virus. Chimeras between tdPH2010 and BSU were constructed and viruses were recovered from transfected chick embryo fibroblasts. Growth curves of cells infected with chimeric viruses indicated that the long terminal repeat (LTR) of tdPH2010 converts BSU to a cytopathic virus. Nucleotide sequencing revealed two point mutations unique to tdPH2010 in the U3 region of LTR at positions -126 and -23 from the transcription start site. Both mutations were located inside or near the promoter/enhancer elements of U3. The mutation at -126 (G to T) converted one of the very well-conserved pentanucleotide repeat (PRE) motifs from GGTGG to GGTGT. The other at -23 (G to A) is located next to the TATA box. The G at this position is conserved in all other known avian retrovirus promoters.

Diastematomyelia and spina bifida can be caused by the intraspinal grafting of somites in early avian embryos.

In this experimental study, an embryological model was created to reproduce diastematomyelia and spina bifida and to investigate new aspects of the origin of spinal cord malformations. A somite was implanted from a donor quail embryo into the neural tube of a 2-day-old chick embryo. The somite was chosen because the septum that characteristically separates the two hemicords consists exclusively of mesodermal derivatives. After 2 days of reincubation, diastematomyelia, spina bifida, or a normal embryo without a graft was observed. If the graft persisted in the neural tube, it formed a septum between the floor and roof plates but never made contact with the lateral walls of the tube. Otherwise, the graft was extruded from the neural tube. In this case, the quail cells often were found in dorsal or dorsolateral positions in the surrounding tissue. Sometimes, the wall of the neural tube formed an extrusion in the direction of the eliminated graft. On many occasions, however, spina bifida aperta was produced and no quail cells could be found in the host. The results suggest that diastematomyelia may be the result of abnormal mesodermal invasion of the neural tube. The development of a septum in the neural tube after implantation of a somite may mimic the process during spontaneous diastematomyelia formation, which could be the consequence of abnormal gastrulation, the process by which the two early germ layers of the blastodisc are converted into the three definitive germ layers.

Long-term culture of avian embryonic cells in vitro.

The pH of the embryonic blood, one of the most important environmental factors for embryonic cells, was found to range from 8.1 to 8.5 in chick embryos until 108 h after incubation. Based on these results, the culture medium adjusted to pH 8.0 was used to culture embryonic chick and quail cells. They were easily subcultured for a long period of time at pH 8.0. This pH culture condition may have wide application for manipulating embryonic cells or tissues and establishing cell lines from avian embryos.

A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains.

Entry of human immunodeficiency virus type 1 (HIV-1) into cells requires binding to CD4 and fusion with a cellular membrane. Fusion does not occur in most nonhuman cells even when they express human CD4, indicating that one or more human accessory factors are required for virus infection. Recently, a seven-transmembrane domain protein has been shown to serve as an accessory factor for T-cell-tropic (T-tropic) HIV-1 isolates (Y. Feng, C. C. Broder, P. E. Kennedy, and E. A. Berger, Science 272:872-877, 1996). Here we show that expression of this glycoprotein, termed fusin, in murine, feline, simian, and quail cell lines, in conjunction with human CD4, rendered these cells fully permissive for HIV-1 envelope glycoprotein (Env)-mediated membrane fusion. Expression of CD4 or fusin alone did not permit fusion. In addition, introduction of fusin and CD4 into a human cell line, U87MG, that is resistant to HIV-1 induced syncytium formation and to infection by HIV-1 when expressing CD4 alone made this cell line permissive for Env-mediated cell-cell fusion. Fusion was observed only with T-tropic Env proteins. Macrophage-tropic (M-tropic) Env proteins from the SF162, ADA, and Ba-L HIV-1 strains did not fuse with cells expressing fusin and CD4, suggesting that M-tropic viruses utilize an accessory molecule other than fusin. Finally, coexpression of fusin and CD4 made both a murine and feline cell line susceptible to virus infection by T-tropic, but not M-tropic, HIV-1 strains.