Search

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.

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.

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.

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.

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., (Copyright 1998 Academic Press.)

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.

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.

The slug gene encodes a zinc finger transcription factor expressed by neural crest cells (Nieto et al., Science 264: 835-839, 1994) and by certain non-crest derived mesenchymal cell populations, such as lateral mesoderm and sclerotome (Mayor et al., Development 121: 767-777, 1995; Buxton et al., Dev. Biol. 183: 150-165, 1997). We report here that slug is also expressed in developing chick limbs. The slug expression domain in the limb bud expands from posterior to anterior and marks cells that are predominantly destined to become chondrocytes but have not yet differentiated. Its expression is maintained in connective tissue, but is never observed in the premuscle masses. We show that removal of the apical ectodermal ridge results in loss of slug expression which can be arrested by the addition of an FGF-4 bead. Retinoic acid bead implants lead to down-regulation of slug expression, again accompanied by abolition of limb outgrowth. Dual bead implants demonstrate antagonism between these two factors, suggesting that a localized antagonistic effect between endogenous RA and FGF-4 on slug expression underlies the molecular mechanism controlling the transition between undifferentiated and differentiated state during normal limb development. The fact that slug expression pattern correlates with areas of growth in the limb, and is maintained by FGF-4 and down-regulated by retinoic acid, indicates that slug-expressing cells play a crucial role in growth and patterning of the chick limb. We propose that slug expression provides the best correlation to date between a molecular marker and the physical concept of the progress zone, defined as "a labile region where new positional values are successively engendered in the course of growth" (Summerbell et al., Nature 244: 492-496, 1973).

This study first shows a striking parallel between the expression patterns of the Bmp4, Msx1 and Msx2 genes in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. We have previously shown that the spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. We show here that mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a 'giant' spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, sclerotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed. In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate.

Bone morphogenetic protein-2 (BMP-2) has been implicated in the polarizing region signalling pathway, which specifies pattern across the antero-posterior of the developing vertebrate limb. Retinoic acid and Sonic Hedgehog (SHH) can act as polarizing signals; when applied anteriorly in the limb bud, they induce mirror-image digit duplications and ectopic Bmp-2 expression in anterior mesenchyme. In addition, the two signals can activate Fgf-4 expression in anterior ridge and HoxD expression in anterior mesenchyme. We tested the role of BMP-2 in this signalling cascade by ectopically expressing human BMP-2 (hBMP-2) at the anterior margin of the early wing bud using a replication defective retroviral vector, and found that ectopic expression of Fgf-4 was induced in the anterior part of the apical ectodermal ridge, followed later by ectopic expression of Hoxd-11 and Hoxd-13 in anterior mesenchyme. This suggests that BMP-2 is involved in regulating Fgf-4 and HoxD gene expression in the normal limb bud. Ectopically expressed hBMP-2 also induced duplication of digit 2 and bifurcation of digit 3, but could not produce the mirror-image digit duplications obtained with SHH-expressing cells. These results suggest that BMP-2 may be involved primarily in maintenance of the ridge, and in the link between patterning and outgrowth of the limb bud.

We examined systematically the ability of chick limb bud stumps to regenerate distal structures when fibroblast growth factor (FGF)-4 is applied. When amputations were made within 600 mu m of the tip and FGF-4 applied either posteriorly or both apically and posteriorly, outgrowth of stump tissues occurred and a virtually complete skeleton developed. 'Regeneration' of distal structures was correlated with reactivation of Msx-1 and Shh expression. At proximal amputation levels where FGF-4 did not lead to 'regeneration', neither Msx-1 nor Shh expression was induced. We also grafted cells from progressively more proximal levels of mouse limb buds to chick wing bud tips beneath the apical ridge and the pattern of reactivation of Msx-1 expression along the proximal distal axis of the limb buds was similar to that found in chick limb buds.

The development of the vertebrate skeleton is under complex genetic control, and good progress is being made towards identifying the genes responsible. A recent paper contributes to this progress by describing transgenic mice in which the homeobox-containing MHox gene has been disrupted. MHox(-/-) mice have a range of skeletal defects, involving loss or shortening of structures in the skull, face and limb. Puzzling features of the MHox(-/-) mutation, which has similar effects on bones with very different embryological origins and yet spares other bones completely, may hold clues to the mechanisms that shape the skeleton. MHox(-/-) mice, used in conjunction with other skeletal mutants, will be important tools for exploring these mechanisms further.

We have used in situ hybridisation to whole chick embryos with digoxygenin-labelled probes to investigate the distribution of RXR-gamma transcripts during neural crest cell migration in the developing head and the anterior of the trunk (the vagal region), where neural crest cells make a substantial contribution. We have found that RXR-gamma transcripts are a good marker for migrating neural crest cells in the chick embryo. RXR-gamma transcripts were first detected in cells that had recently emerged from the neural crest, providing an earlier marker for neural crest cells than the HNK-1 epitope. The pattern of RXR-gamma transcript distribution is dynamic in the developing chick head, and changes in a pattern which is coincident with the migration of cells containing RXR-gamma transcripts and the gradual restriction of RXR-gamma transcripts to specific differentiating neural crest derivatives. Transcripts appeared to be present initially in migrating neural crest cells thoughout the developing head, but gradually became restricted to some crest-derived populations and absent from others. By stage 15, RXR-gamma transcripts were not detectable in neural-crest-derived ectomesenchymal cells, although they were still found in cells contributing to the cranial ganglia and their roots.

Retinoids are metabolites of vitamin A that can regulate gene expression in a range of embryonic and adult cell types. They do this by binding to nuclear receptors belonging to the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. Vertebrates possess two classes of nuclear retinoid-receptor genes, each with three members. These are the RAR-alpha, RAR-beta and RAR-gamma genes and the RXR-alpha, RXR-beta and RXR-gamma genes. In this paper we show by cDNA cloning and ribonuclease protection that the chicken RXR-gamma gene gives rise to two mRNA species (RXR-gamma 1 and RXR-gamma 2) that differ at their 5' ends. The two mRNAs have different tissue distributions in the 10-day-old chick embryo. RXR-gamma 2 mRNA was present in the eye and dorsal root ganglia but was undetectable in the liver. In contrast, RXR-gamma 1 mRNA was present in liver, was undetectable in dorsal root ganglia and was just detectable in the eye, where it was much less abundant than RXR-gamma 2 mRNA. The predicted protein products of the RXR-gamma 1 and RXR-gamma 2 mRNAs differ at their N-termini, in a region thought to modulate transcriptional transactivation by the receptor. These results show that at least one of the retinoid-X-receptor (RXR) genes gives rise to more than one protein product, a principle previously established for the retinoic acid-receptor (RAR) genes. The existence of multiple RXR protein isoforms would increase the range of heterodimers formed between RXRs and other nuclear receptors, including RARs and the receptors for thyroid hormone, vitamin D and peroxisome proliferators. This could increase the diversity of transcriptional responses mediated by these molecules.

The chicken cRXR nuclear receptor is a member of the steroid/thyroid hormone receptor superfamily. In this paper we show that cRXR can transactivate gene expression in response to retinoic acid, but that its sensitivity to retinoic acid is lower than that of the hRAR-beta receptor. We have also compared the ability of cRXR and hRAR-beta to respond to a panel of other retinoids. Unlike hRAR-beta, cRXR failed to respond to the naturally occurring retinoid 3,4-didehydro-retinoic acid or to the synthetic retinoid TTNPB, both of which share the ability of retinoic acid to induce digit duplications when locally applied to chick limb buds.