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P M Brickell

Publications and source records attributed to P M Brickell.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways.

Wiskott-Aldrich syndrome is an X-linked combined immunodeficiency affecting cells of several different hemopoietic lineages. The Wiskott-Aldrich syndrome protein (WASP), which has no homology with any other known protein families, is rich in proline motifs known to contribute to Src homology 3 binding sites. However, its function has not been determined. The Tec family of cytoplasmic tyrosine kinases, which include Btk (the X-linked agammaglobulinemia gene), Itk, and Tec, is thought to be involved in lymphoid cell signaling pathways. In this work, we show binding of WASP to the Src homology 3 domains of Btk, Itk, Tec, Grb2, and phospholipase C-gamma, which suggests a function for WASP in lymphoid cell signaling.

Amino Acid Sequence↗
PubMed1996

Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.

BACKGROUND: Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS: We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS: Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells.

Blotting, Western↗
PubMed1996

Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells.

The active vitamin D3 metabolite 1,25-dihydroxycholecalciferol (1,25(OH)2D3) acts as an antiproliferative and differentiating agent for the monoblastoid cell line U937 and as an important immunologic mediator implicated particularly in the function of cells belonging to the monocyte/macrophage lineage. These effects are controlled by the vitamin D receptor (VDR), a member of the steroid hormone receptor family. The objective of this study was to develop U937 transfectants expressing antisense VDR mRNA, and to use these to examine the role of 1,25(OH)2D3-VDR interaction in this lineage. A 2-kb VDR cDNA insert (including the complete VDR coding region) was cloned in an antisense orientation into the EBV episomal vector pMEP4 under the control of an inducible promoter and transfected into U937. The resultant cell line, DH42, was hygromycin resistant, contained VDR cDNA, expressed fewer VDRs than controls, and showed a substantial decrease in antiproliferative response to 1,25(OH)2D3. However, 1,25(OH)2D3 increased the number of cells expressing macrophage cell surface Ags, including CD14 and CD11b. A subpopulation of smaller cells did not express the differentiation markers after cadmium stimulation. Cell cycle analysis showed shifts in the distribution of cells from G1 to S phase, which were more pronounced after cadmium treatment. A considerable proportion of cells were outside the cycle and DNA fragmentation confirmed apoptosis. Thus, the functional outcome of the VDR antisense transfection suggests that in the myelomonocytic lineage, VDR expression may act as a protective mechanism against programmed cell death.

Actins↗
PubMed1996

Bone morphogenetic protein-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures.

Bone morphogenetic proteins (BMPs) induce ectopic cartilage and bone when implanted intramuscularly in adult rats. Expression data suggest that BMPs signal skeletal development in embryos. An important question is which cells are targets of BMP signaling in adult and embryonic tissues. Here, we examined the effect of BMP-2 on micromass cultures of chick limb bud mesenchyme. We report that BMP-2 promotes formation of cartilage and simultaneously inhibits development of muscle cells. To follow the fate of presumptive muscle cells, we replaced chick somites with quail somites at the wing level and made micromass cultures from the chimeric wings. In untreated cultures, quail cells formed muscle but not cartilage. In BMP-2-treated cultures, quail cells disappeared altogether. This suggests that BMP-2 may simultaneously promote cartilage differentiation and reduce the presumptive myogenic cell populations in regions of skeletal development.

Animals↗
PubMed1996

Nucleotide sequence and genomic structure of the chicken insulin-like growth factor-II (IGF-II) coding region.

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide with structural similarity to insulin that appears to be involved in regulating embryonic growth. While mammalian IGF-II proteins and IGF-II genes have been well characterized, little is known about avian IGF-II proteins and the genomic sequences that encode them. In this paper we report the isolation and characterization of chicken IGF-II cDNA and genomic clones. The predicted chicken prepro-IGF-II protein contains 187 residues and comprises a signal peptide (24 residues), IGF-II peptide (67 residues), and C-terminal peptide (96 residues), which share 33, 82, and 25/28% amino acid sequence identity, respectively, with their rat/human counterparts. Like the human and rat IGF-II genes, the chicken IGF-II gene has three coding exons. These are interrupted by introns at similar positions to those found in the human and rat IGF-II genes.

Amino Acid Sequence↗
PubMed1996

Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb.

Bone morphogenetic proteins are members of the transforming growth factor beta (TGF beta) superfamily which are involved in a range of developmental processes including modelling of the skeleton. We show here that Bmp-2 is expressed in mesenchyme surrounding early cartilage condensations in the developing chick limb, and that Bmp-4 is expressed in the perichondrium of developing cartilage elements. To investigate their roles during cartilage development, BMP-2 and BMP-4 were expressed ectopically in developing chick limbs using retroviral vectors. Over-expression of BMP-2 or BMP-4 led to a dramatic increase in the volume of cartilage elements, altered their shapes and led to joint fusions. This increase in volume appeared to result from an increase in the amount of matrix and in the number of chondrocytes. The latter did not appear to be due to increased proliferation of chondrocytes, suggesting that it may result from increased recruitment of precursors. BMP-2 and BMP-4 also delayed hypertrophy of chondrocytes and formation of the osteogenic periosteum. These data provide insights into how BMP-2 and BMP-4 may model and control the growth of skeletal elements during normal embryonic development, suggesting roles for both molecules in recruiting non-chondrogenic precursors to chondrogenic fate.

Animals↗
PubMed1996

Synthesis of TNF alpha and TGF beta mRNA in the different micro-environments within atheromatous plaques.

OBJECTIVE: To examine localisation of tumour necrosis factor (TNF alpha) and transforming growth factor beta (TGF beta) mRNA synthesis in human coronary artery atheromatous plaques, to explore how synthesis of these cytokines relates to distribution of macrophages and smooth muscle cells, and to correlate this with plaque micro-environments. METHOD: In situ hybridisation with digoxigenin-labelled sense and anti-sense riboprobes was used, combined with immunohistochemistry to detect TNF alpha protein, macrophage, lymphocyte and smooth muscle cell markers. RESULTS: In the intimal plaque TNF alpha mRNA is synthesised by monocytes/macrophages as well as by smooth muscle cells. Both TNF alpha and TGF beta mRNAs were present at the margins of the lesions and in reactive areas, where there was little lipid and fibrosis. Focal aggregates of macrophages in the adventitia expressed both TNF alpha mRNA and protein and TGF beta mRNA. CONCLUSION: Synthesis of these two cytokines by macrophages as well as smooth muscle cells contributes to the pathobiology of the plaque and that this is part of the 'reaction to injury', rather than a feature of a specific cell, or a specific layer, within the vessel wall.

Coronary Disease↗
PubMed1996

Bone morphogenic protein-4.

Bone morphogenic protein-4 (BMP-4) is one of nine structurally related BMPs belonging to the transforming growth factor-beta (TGF-beta) superfamily of secreted proteins. Mature BMP-4 is a dimer that binds to a multimeric transmembrane receptor with serine/threonine kinase activity. Although discovered because it stimulates bone formation in adult mammals, BMP-4 has important roles as a signalling molecule in embryonic tissues, including the developing central and peripheral nervous system, musculature and skeleton. It participates in an ancient signalling pathway also found in insects and worms. Nevertheless, the main practical application of BMPs is for stimulating repair of bone, and their use in humans is currently being assessed.

Animals↗
PubMed1996

Coexpression of H2-Mb and H2-Ab genes during fetal and postnatal development.

The major histocompatibility complex (MHC) class II-like molecules, H2-M, have an essential role in processing and presentation of antigens by the MHC class II molecules, because functional inactivation of these genes lead to surface expression of MHC class II molecules devoid of associated foreign peptides. We have used in situ hybridization to examine the expression of MHC class II and H2-Mb genes in embryonic and neonatal mice and show here that expression of H2-Ab and H2-Mb mRNA is absent in 13.5-day-old mice. However, mRNA for both genes could be detected in the thymus of 14.5- and 15.5-day-old embryos, and the patterns of hybridization suggested that the two genes were expressed in the same cells. In spleen and thymus of neonatal mice the H2-Ab and H2-Mb genes were also coexpressed, with expression being localized to the white pulp of the spleen and to the thymic medulla, which are rich in antigen-presenting cells. The steady-state levels of H2-Ab mRNA appeared to be approximately 10 to 14 times greater that the level of H2-Mb mRNA molecules, irrespectively of the tissue and age, reflecting the different functions of the two molecules.

Animals↗
PubMed1996

Activation of Fgf-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb.

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.

Animals↗
PubMed1996

Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons.

The neurotransmitter phenotype of sympathetic neurons is specified by interactions with the surrounding embryonic tissues. Adrenergic differentiation is elicited early during development in the vicinity of notochord and dorsal aorta and the importance of axial midline tissues for adrenergic differentiation has been well documented. We now provide evidence that bone morphogenetic proteins, BMP-4 and BMP-7 are signals produced by the dorsal aorta that direct sympathetic neuron differentiation. BMP-4 and BMP-7 are expressed in the dorsal aorta at critical times during sympathetic neuron differentiation and have the ability to enhance the formation of adrenergic sympathetic neurons both in cultures of neural crest cells and when ectopically expressed in the developing embryo.

Adrenergic Fibers↗
PubMed1995

Identification of a phorbol ester responsive region in the myeloid-specific promoter of the c-fgr proto-oncogene.

Expression of the c-fgr proto-oncogene is activated during differentiation of myeloid cells. We used a luciferase reporter assay to identify sequences that regulate c-fgr gene transcription during differentiation of human U937 promonocytic cells, induced by phorbol 12-myristate 13-acetate (PMA) or by tumour necrosis factor-alpha (TNF-alpha) and 1,25-dihydroxycholecalciferol (1,25-DHCC). We found that the region from nucleotides -344 to -116, with respect to the transcriptional start site, is required for basal activity of the c-fgr promoter in U937 cells, and that the region from nucleotides -1211 to -752 is responsive to PMA. No sequence elements responsive to TNF-alpha and 1,25-DHCC were found, suggesting that these agents induce c-fgr mRNA accumulation by a mechanism differing from that mediating the effects of PMA.

Bone Marrow↗
PubMed1995

Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: their relationships in the signalling cascade involved in limb patterning.

The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.

Animals↗
PubMed1995

MHox and vertebrate skeletogenesis: the long and the short of it.

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.

Abnormalities, Multiple↗
PubMed1995

Expression of the chicken retinoid X receptor-gamma gene in migrating cranial neural crest cells.

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.

Animals↗