Chromosomal localization of the genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma, and VIM in chicken by fluorescence in situ hybridization.

Six structural genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma and VIM were mapped in the chicken by fluorescence in situ hybridization (FISH) using genomic and cDNA clones as probes. The genes were found to be located on four different macrochromosomes: chromosome 1 (IFNG and FABP), chromosome 2 (VIM and ALDH), chromosome 3 (BMP2) and a smaller macrochromosome, most probably chromosome 7 (RXRG). With the exception of IFNG none of the newly mapped sites corresponds to known orthologous regions between chicken and human chromosomes.

Characterization of a chicken retinoid X receptor-gamma gene promoter and identification of sequences that direct expression in retinal cells.

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-gamma (RXR-gamma2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-gamma genomic clone containing the RXR-gamma2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-gamma2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-gamma2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

Wiskott-Aldrich syndrome protein is necessary for efficient IgG-mediated phagocytosis.

Interactions between the Wiskott-Aldrich (WAS) protein (WASp), small GTPases, and the cytoskeletal organizing complex Arp2/3 appear to be critical for the transduction of signals from the cell membrane to the actin cytoskeleton in hematopoietic cells. This study shows that Fcgamma-receptor (FcgammaR)-mediated phagocytosis is impaired in WASp-deficient peripheral blood monocytes, and that in macrophages, formation of the actin cup and local recruitment of tyrosine phosphorylated proteins is markedly attenuated. Results also show that, in normal macrophages, WASp itself is actively recruited to the cup, suggesting that assembly of this specialized cytoskeletal structure is dependent on its expression. (Blood. 2000;95:2943-2946)

Loss of heterozygosity and microsatellite instability at the MLL locus are common in childhood acute leukemia, but not in infant acute leukemia.

Rearrangements involving the MLL gene at chromosome 11q23 are associated with leukemia and are present in up to 70% of infant leukemias. Loss of heterozygosity (LOH) has been shown for anonymous polymorphic markers at 11q23 in adult leukemias. To study LOH at the MLL locus, we have identified two new polymorphic microsatellite markers: a GAA repeat (mllGAAn) in intron 6 of the MLL gene and a GA (mllGAn) repeat in the 5' flanking region of the gene, approximately 2 kb upstream of the translation initiation codon. The heterozygosity index of mllGAAn is 0.54, which renders it useful for analyzing LOH. We screened two groups of leukemia patients to study LOH at the mllGAAn marker. Group A (n = 18) was selected on the basis of presentation before 18 months. Cytogenetic and reverse transcription-polymerase chain reaction analysis showed that 9 of these 18 children had translocations involving MLL. No LOH was observed. Group B (n = 36) were randomly selected children who had presented with leukemia between 1993 and 1994. Cytogenetic analysis of this group showed a variety of different chromosomal abnormalities. LOH was shown in 9 of 20 individuals (45%) who were informative. Microsatellite instability (MSI) was demonstrated in 1 of 18 individuals in group A and 5 of 36 individuals (13.9%) in group B. MSI and LOH were observed simultaneously in three individuals. Loss of an allele was confirmed in one individual by fluorescence in situ hybridization. Individuals with MSI or LOH at mllGAAn were selected for analysis at anonymous polymorphic markers D11S1364 and D11S1356, which flank the MLL gene. No LOH or MSI was observed at these markers in those individuals who were informative. These results show that LOH at the MLL gene locus is a common event during leukemogenesis. Furthermore, the presence of MSI at this locus suggests that the region is a hotspot for genetic instability.

Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer.

The definitive long-term repopulating human hematopoietic stem cell, which seeds the adult blood system, was previously thought to derive from the extra-embryonic yolk sac. However, there is now considerable evidence that in both avian and murine systems, yolk sac hematopoietic cells are largely a transient, embryonic population and the definitive stem cell, in fact, derives from a distinct region within the embryonic mesoderm, the aorta-gonad-mesonephros region. In the human embryo, an analogous region has been found to contain a cluster of cells distinct from, but closely associated with, the ventral endothelium of the dorsal aorta, the appearance of which is restricted both spatially and temporally. We have used antibodies recognising hematopoietic regulatory factors to further characterise this region in the human embryo. These studies indicate that all factors examined, including vascular endothelial growth factor and its receptor FLK-1, Flt-3 ligand and its receptor STK-1, and stem cell leukemia transcription factor, are expressed by both hematopoietic cells in the cluster and endothelial cells. However, there is some discontinuity in cells directly underlying the cluster. Furthermore, we have identified a morphologically distinct region of densely-packed, rounded cells in the mesenchyme directly beneath the ventral wall of the dorsal aorta, and running along its entire length. In the preumbilical AGM region, directly underlying the hematopoietic cluster, but not at more rostral and caudal levels, this region of mesenchyme expresses tenascin-C, an extracellular matrix glycoprotein known to facilitate cell-cell interactions and migration. This region of cells may therefore provide the microenvironmental support for the intraembryonic development of definitive hematopoietic stem cells, a process in which tenascin-C may play a pivotal role.

Intrinsic dendritic cell abnormalities in Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immune deficiency, eczema, and microthrombocytopenia. Biochemical evidence indicates that the Wiskott-Aldrich syndrome protein (WASp) is involved in regulating the actin cytoskeleton. Here we report that WAS dendritic cells (DC) have an immunophenotype very similar to normal DC. However, as a consequence of an intrinsically abnormal cytoarchitecture, they are unable to polarize normally and have severely reduced translocational motility in vitro. These findings indicate that WASp is an essential effector for Cdc-42-mediated polarization of primary hematopoietic cells, and suggest that a significant component of the clinical phenotype of WAS could arise from peripheral DC dysmotility and aberrant immune cell trafficking in vivo. Intrinsic dysfunction of the DC population may also have an important role in the pathogenesis of other primary immunodeficiency syndromes, while induced changes in DC cytoskeletal signaling pathways may contribute to the initiation of acquired immunological and inflammatory disorders.

Chemotaxis of macrophages is abolished in the Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare disease characterized by microthrombocytopenia, eczema and immune deficiency. In this study a direct-viewing chemotaxis chamber was used to analyse chemotactic responses of WAS neutrophils and macrophages in stable linear concentration gradients. In five patients with classic WAS, chemotaxis of macrophages but not of neutrophils was found to be abolished, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells. This supports the existence of an essential functional link, previously suggested by biochemical studies, between Cdc42, WAS protein (WASp) and the actin cytoskeleton in primary human macrophages. Moreover, these data suggest that Cdc42-WASp-mediated filopodial extension is a requirement for chemotaxis but not for chemokinesis in these cells. Abnormal directional cell motility of macrophages and related antigen-presenting cells may play a significant part in the immune deficiency and eczema of WAS.

Retinoid X receptor-gamma gene expression is developmentally regulated in the embryonic rodent peripheral nervous system.

The retinoid X receptor-gamma (RXRgamma) is a transcription factor that belongs to the thyroid hormone/retinoid family of nuclear receptors. Previous studies have shown that RXRgamma is expressed in the developing peripheral nervous system (PNS) of chick embryos, but such expression has not been reported previously in rodent embryos. Indeed, the pattern of RXRgamma expression appears to be different between avian and rodent species. Using in situ hybridisation and northern blot experiments we show that RXRgamma is expressed in elements of the PNS in rat and mouse embryos and in postnatal rats. However, unlike the chick, where RXRgamma is expressed from the onset of neural crest migration, rat RXRgamma expression in the dorsal root ganglia and trigeminal ganglia was detectable at 14.5 days post coitum (dpc), but not at 13, 12, or 11 dpc. These data suggest that RXRgamma may have a role in the dorsal root ganglia and trigeminal ganglia of late embryos, and that this could be evolutionarily conserved between chick and rodent. On the other hand, rat RXRgamma transcripts in the facio-acoustic (VII-VIII) ganglion were detectable at 11 dpc in neural crest cells condensing to form this ganglion. We discuss the possible significance of the timing of RXRgamma expression in the developing PNS and suggest that closer examination of the structure and function of the PNS of RXRgamma null mutant mice would be of interest.

Retinoid X receptor gamma gene transcripts are expressed by a subset of early generated retinal cells and eventually restricted to photoreceptors.

We have examined the distribution of the retinoid X receptor gamma (RXRgamma) in the developing chicken retina by using in situ hybridization and RNase protection assays. We detected RXRgamma transcripts as early as 4 days of embryonic development (d4) in central regions of the retina, spreading to more peripheral regions by d8. The first few RXRgamma-positive cells were scattered within the depth of the retinal neuroepithelium, but as they increased in number they became localized predominantly to the apical (outer, ventricular) layer. The identity of the RXRgamma-positive cells at these stages is unknown, due to the lack of cell type-specific markers. By d10, when photoreceptors and ganglion cells have been generated and begun to establish their definitive layers, RXRgamma-positive cells were virtually restricted to the photoreceptor layer, and maintained this distribution to posthatch stages. RNase protection assays were performed on whole retinae to verify the temporal pattern of in situ hybridization results and showed that between d5 and d16 there was a significant increase in the mRNA levels of the RXRgamma2 isoform. Between d16 and early posthatch stages the level of RXRgamma2 mRNA did not change significantly. Consistent with previous studies, mRNA levels of the RXRgamma1 isoform were substantially lower than mRNA levels of the RXRgamma2 isoform at all time points examined. These results demonstrate that RXRgamma mRNA is expressed in photoreceptors in the developing chicken retina and implicate RXRgamma as the earliest marker of photoreceptor differentiation documented to date.

Regulation of retinoid X receptor-gamma gene transcript levels in rat heart cells.

Retinoid X receptor-gamma (RXRgamma) is a transcription factor that mediates retinoid signalling and is expressed in rat heart during adult life. However, its expression in embryonic and neonatal heart has not been investigated and it is not known whether ventricular cardiomyocytes express RXRgamma or whether all- trans -retinoic acid (tRA) and thyroid hormone (T3) could influence RXRgamma transcript levels in these cells. First, in situ hybridization experiments were used to test for any spatio-temporal correlation between RXRgamma gene expression and the previously shown requirement for retinoid signalling in embryonic ventricular cardiomyocytes. It was shown that RXRgamma transcripts are not detectable in embryonic heart at all developmental stages examined under conditions where they are detectable in other embryonic tissues. Second, Northern blotting was used to examine whether there is a difference in RXRgamma transcript levels between neonatal and adult heart. We show that levels of two RXRgamma transcripts are developmentally regulated during the postnatal period because they differ between neonatal and adult hearts. Third, it was demonstrated that primary cultures of neonatal rat ventricular cardiomyocytes express RXRgamma transcripts, making them a novel in vitro system for the study of RXRgamma gene regulation in heart-derived cells. Finally, this system was used to examine whether tRA and T3can influence levels of RXRgamma transcripts because they have been shown to have antagonistic effects in this system and to influence RXRgamma RNA levels in other systems. It was shown by Northern blot experiments, that in this system, RXRgamma transcript levels are differentially influenced by these two hormones. The significance of these findings in relation to previously published work is discussed.

Differential expression of the rat retinoid X receptor gamma gene during skeletal muscle differentiation suggests a role in myogenesis.

Even though previous studies have shown that transcripts encoding the murine retinoid X receptor gamma (RXRgamma) are present in skeletal muscle of mouse embryos and that cultured myoblasts are induced to differentiate upon retinoid treatment, a function for RXRgamma and retinoids in mammalian myogenesis has not yet been identified. To begin to understand the possible role of RXRgamma during mammalian myogenesis we isolated novel rat RXRgamma cDNA sequences and examined in detail the spatio-temporal expression pattern of RXRgamma transcripts in relation to skeletal muscle differentiation in rat embryos and cultured myoblasts. We show that the onset of RXRgamma expression coincides with the differentiation of limb myoblasts in vivo. In vitro, RXRgamma is expressed in differentiating myoblasts, but not in proliferating myoblasts. In the myotome, however, RXRgamma is first expressed after myoblast differentiation, with RXRgamma transcripts being confined initially to its ventral region. Subsequently, RXRgamma becomes expressed throughout limb and myotome-derived muscle masses, and by the end of the primary myogenic wave, RXRgamma transcripts are mainly confined to their periphery. This dynamic expression pattern of RXRgamma during myogenesis suggests its possible involvement in the differentiation of limb myoblasts but excludes a role in the differentiation of early myotomal myoblasts.

Stable transfection of U937 cells with sense or antisense RXR-alpha cDNA suggests a role for RXR-alpha in the control of monoblastic differentiation induced by retinoic acid and vitamin D.

Although retinoic acid (RA) has been known for many years to be a modulating agent that plays a role in generating both granulocytes and monocytes, the molecular mechanism underlying this role has not been defined in the monoblast lineage. In particular, the part played by the retinoid X receptors (RXRs), which are members of the steroid/thyroid hormone nuclear receptor family, has not been explored. In this study, therefore, the human monoblastic leukemia cell line U937 has been used as a model system to investigate the role of one of the RXRs, RXR-alpha, in monoblast differentiation. RXR-alpha mRNA was present in untreated U937 cells, and levels increased after induction of differentiation with phorbol ester. The same was found for RXR-beta mRNA. Using plasmids containing sense or antisense RXR-alpha sequences under the control of an inducible promoter, we generated stably transfected cell lines which expressed either increased or decreased levels of RXR-alpha, respectively. The sense cell lines (U alpha S and its clonal derivative alpha G2S) showed increased sensitivity to RA, while the antisense cell lines (U alpha A and its clonal derivative alpha B5A) showed decreased sensitivity to RA, as demonstrated by growth inhibition and by regulation of an RA-responsive reporter gene. Both U alpha A and alpha B5A also failed to respond to another modulating agent, 1 alpha,25-dihydroxycholecalciferol (DHCC), but only U alpha S and not alpha G2S showed an enhanced response to DHCC. The combination of RA and DHCC together inhibited growth of both sense and antisense cell lines. In addition, alpha G2S exhibited increased expression of CD11b and CD54, while alpha B5A cells showed increased expression of CD102, suggesting that RXR-alpha has a role in regulating expression of cell adhesion molecules in U937 cells. These results demonstrate that RXR-alpha has a role in mediating growth inhibition and cell adhesion during myelomonocytic differentiation, and suggest that different species of heterodimers involving RXR-alpha may control the acquisition of different features of mature monocyte/macrophage function.

Cell movements, neuronal organisation and gene expression in hindbrains lacking morphological boundaries.

Rhombomeres are segmental units of the hindbrain that are separated from each other by a specialised zone of boundary cells. Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterations in Hox gene expression. The characteristic morphology of boundary cells, their expression of follistatin and the periodic accumulation of axons normally associated with boundaries are all lost. In the absence of boundaries, we detect no change in anteroposterior dispersal of precursor cells and, in most cases, no substantial cell mixing between former rhombomeric units. This is consistent with the idea that lineage restriction can be maintained by processes other than a mechanical barrier composed of boundary cells. Much of the early organisation of the motor nuclei appears normal despite the loss of boundaries and altered Hox expression.

Replication-competent retroviral vectors for expressing genes in avian cells in vitro and in vivo.

Replication-competent retroviral vectors based on Rous sarcoma virus (RSV) are becoming increasingly popular for expressing genes in both primary cell cultures and embryonic chick tissues in ovo. In this article, we review the features of RSV and its life cycle that make it suitable for use as a vector. We describe the design and use of the RCAS and RCAS (BP) series of vectors, which are currently the most widely used RSV-based vectors, illustrating both their strengths and weakness. Finally, we outline laboratory protocols suitable for the banding of these retroviral vectors.

The role of the Fgr tyrosine kinase in the control of the adhesive properties of U937 monoblastoid cells and their derivatives.

In humans, expression of the cellular proto-oncogene c-fgr is normally restricted to mature cells of the myeloid lineage, mantle zone B cells and various myeloid and B-cell lines. Previous studies of the monoblastoid cell line, U937, showed that c-fgr expression increased following differentiation, but its role in monocytes and related cells has not been defined in functional terms. We therefore investigated the role of c-fgr in U937 cells transfected with the c-fgr gene such that its expression could be manipulated independent of differentiation. Induction of the transfected c-fgr gene by cadmium ions did not affect cell proliferation, responses to phorbol 12-myristate 13-acetate (PMA), dihydroxycholecalciferol (DHCC), tumour necrosis factor-alpha (TNF-alpha) or retinoic acid, or phagocytosis of antibody-coated sheep red blood cells. However, there was increased surface expression of CD54 (intracellular adhesion molecule-1; ICAM-1) and CD102 (ICAM-2) and decreased surface expression of CD50 (ICAM-3) compared with cells that had been transfected with plasmid only and treated in the same way. These findings suggest that the product of the c-fgr gene may be important in control of relative adhesive properties of mature monocytic cells.

Local origin of cells in FGF-4 - induced outgrowth of amputated chick wing bud stumps.

Urodele amphibians are the only vertebrates that can regenerate amputated limbs, even as adults. However, we have previously shown that amputated chick wing bud stumps can be induced to ((regenerate)) and to form a complete set of correctly-patterned skeletal elements, following implantation of beads soaked in fibroblast growth factor-4 (FGF-4). We have now performed Dil injection experiments to determine which cells contribute to FGF-4-induced chick wing bud ((regenerates)). We show that the FGF-4-induced outgrowth of the regenerating wing bud stump is comprised of mesenchyme cells that originate from a region within 200 microm of the FGF-4 bead, and that cells proximal to the bead move distally.