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Biomedical subjects

L F Reichardt

Publications and source records attributed to L F Reichardt.

At least 55 records · Page 3Linked to original sources
PubMed1997

Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis.

We present genetic evidence that integrins regulate epithelial-mesenchymal interactions during organogenesis. Mice with a mutation in the alpha8 gene do not express the integrin alpha8 beta1 and exhibit profound deficits in kidney morphogenesis. In wild-type animals, inductive interactions between the ureteric epithelium and metanephric mesenchyme are essential for kidney morphogenesis. In alpha8 mutant homozygotes, growth and branching of the ureteric bud and recruitment of mesenchymal cells into epithelial structures are defective. Consistent with these phenotypes, alpha8 expression is induced in mesenchymal cells upon contact with the ureter. Since none of its previously identified ligands appears likely to mediate the essential functions of alpha8 beta1 in kidney morphogenesis, we have used an alpha8 beta1-alkaline phosphatase chimera to localize novel ligand(s) in the growing ureter. The distribution of these ligand(s) makes them strong candidates for regulators of kidney morphogenesis.

Alleles↗
PubMed1997

A sensory axon repellent secreted from ventral spinal cord explants is neutralized by antibodies raised against collapsin-1.

During embryogenesis, different subclasses of sensory neurons extend central projections to specific locations in the spinal cord. Muscle and cutaneous afferents initially project to the same location in the dorsal cord. Later, specific muscle afferents leave other afferents behind and project into the ventral cord. Previous studies have shown that ventral spinal cord explants secrete a repellent for sensory neurites. We now find that antibodies to collapsin-1 neutralize this repellent activity. Additional data suggest that all afferents respond to collapsin-1 when they are first confined to the dorsal cord, but that ventrally projecting muscle afferents become collapsin-1 insensitive as they project into the ventral cord. Our results suggest that the transient dorsal expression of collapsin-1 prevents all efferents from entering the cord early and sustained ventral expression prevents dorsally terminating afferents from entering the ventral cord later.

Age Factors↗
PubMed1996

Neurotrophin-3 is a survival factor in vivo for early mouse trigeminal neurons.

Mice lacking neurotrophin-3 (NT-3) have been shown previously to be born with severe sensory deficits. This study characterizes the developmental course of this deficit in the trigeminal sensory ganglion, which in NT-3 homozygous mutants contains only 35% of the normal number of neurons at birth. At embryonic day 10.5 (E10.5), normal numbers of neurons, as assessed by expression of neurofilament protein and of total cells, are present in the ganglia of mutant homozygotes. During the next 3 d (E10.5-E13.5), virtually all of the deficit develops, after which mutant animals retain only approximately 30% the normal number of neurons. Quantification of neuronal and neuronal precursor numbers in normal and mutant animals reveals that neurons are specifically depleted in the absence of NT-3. A deficiency in precursor proliferation is only seen after most of the neuronal deficit has developed. Numbers of apoptotic cells in the ganglia of mutant animals are elevated during this same interval, indicating that the neuronal deficit is caused, in large part, by increased cell death of embryonic neurons. To determine sources of NT-3 in the trigeminal system, we examined the expression pattern of beta-galactosidase in mice, in which lacZ has replaced the NT-3 coding exon. E10.5-E11.5 embryos exhibit intense reporter expression throughout the mesenchyme and epithelia of the first branchial arch. Beta-galactosidase expression in E13.5 embryos is largely confined to the oral epithelium and the mesenchyme underlying the skin. Throughout the E10.5-E13.5 interval, the trigeminal ganglion and its targets in the CNS do not express reporter activity. We conclude that NT-3 acts principally as a peripherally derived survival factor for early trigeminal neurons.

Animals↗
PubMed1996

Renal and neuronal abnormalities in mice lacking GDNF.

Glial cell-line derived neurotrophic factor (GDNF) is a potent survival factor for embryonic midbrain dopaminergic, spinal motor, cranial sensory, sympathetic, and hindbrain noradrenergic neurons, and is available to these cells in vivo. It is therefore considered a physiological trophic factor and a potential therapeutic agent for Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Here we show that at postnatal day 0 (P0), GDNF-deficient mice have deficits in dorsal root ganglion, sympathetic and nodose neurons, but not in hindbrain noradrenergic or midbrain dopaminergic neurons. These mice completely lack the enteric nervous system (ENS), ureters and kidneys. Thus GDNF is important for the development and/or survival of enteric, sympathetic and sensory neurons and the renal system, but is not essential for catecholaminergic neurons in the central nervous system (CNS).

Animals↗
PubMed1996

Inhibition of the NT-3 receptor TrkC, early in chick embryogenesis, results in severe reductions in multiple neuronal subpopulations in the dorsal root ganglia.

To assess functions of neurotrophins at defined times in development, we have prepared antibodies of the extracellular domains of each of the trk receptors. Here, antibodies to trkC, the major receptor for NT-3, are used to examine trkC expression and function during the formation and maturation of the chick dorsal root ganglion (DRG). Our results show that in the immature DRG, the majority of cells express trkC, and inhibition of trkC activation results in reductions in neuronal numbers before the period of target-mediated cell death, the time when neurotrophins previously have been shown to regulate survival. Furthermore, blockade of trkC in ovo induced reductions in subpopulations of DRG neurons known to be dependent on NGF, in addition to those dependent on NT-3 during the target-regulated cell death period. An early function for NT-3 on immature DRG neurons is supported further by data presented here that demonstrate that whereas BDNF and NGF can support a subset of immature DRG neurons in vitro, activation of the trkC receptor either by NT-3 binding or via antibody-mediated cross-linking induces the most robust survival response. When all three neurotrophins are combined, the number of surviving neurons does not exceed that supported by NT-3 alone. Together, these data are consistent with coexpression of more than one trk receptor family member on immature sensory neurons, and they demonstrate that inhibition of trkC activation has surprisingly early and pleiotrophic effects on the development of spinal sensory ganglia.

Animals↗
PubMed1996

Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling.

trkB encodes a receptor tyrosine kinase activated by three neurotrophins--brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4/5. In vivo, three isoforms of the receptor are generated by differential splicing--gp145trkB or the full-length trkB receptor, and trkB.T1 and trkB.T2, two cytoplasmically truncated receptors that lack kinases, but contain unique C termini. Although the truncated receptors appear to be precisely regulated during nervous system development and regeneration, their role in neurotrophin signaling has not been directly tested. In this paper, we studied the signaling properties and interactions of gp145trkB, trkB.T1, and trkB.T2 by expressing the receptors in a Xenopus oocyte microinjection assay. We found that oocytes expressing gp145trkB, but not trkB.T1 or trkB.T2, were capable of eliciting 45Ca efflux responses (a phospholipase C-gamma-mediated mechanism) after stimulation by BDNF. When trkB.T1 and trkB.T2 were coexpressed with gp145trkB, they acted as dominant negative receptors, inhibiting the BDNF signal by forming nonfunctional heterodimers with the full-length receptors. An ATP-binding mutant of gp145trkB had similar dominant inhibitory effects. Our data suggest that naturally occurring truncated trkB receptors function as inhibitory modulators of neurotrophin responsiveness. Furthermore, the homodimerization of gp145trkB appears to be an essential step in activation of the BDNF signaling cascade.

Animals↗
PubMed1996

Retrograde transport of neurotrophins from the eye to the brain in chick embryos: roles of the p75NTR and trkB receptors.

The receptors involved in retrograde transport of neurotrophins from the retina to the isthmo-optic nucleus (ION) of chick embryos were characterized using antibodies to the p75 neurotrophin receptor and trkB receptors. Survival of neurons in the ION has been shown previously to be regulated by target-derived trophic factors with survival promoted or inhibited by ocular injection of brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF), respectively. In the present paper, we show that during the period of target dependence, these neurons express trkB and p75 neurotrophin receptor but not trkA or trkC mRNAs. We also show that BDNF and NT-3 were transported efficiently at low doses, whereas NGF was transported significantly only at higher doses. The transport of BDNF and NT-3 was reduced by high concentrations of NGF or by antibodies to either trkB or the p75 neurotrophin receptor. Thus both receptors help mediate retrograde transport of these neurotrophins. Ocular injection of the comparatively specific trk inhibitor K252a did not reduce transport of exogenous BDNF, but did induce significant neuronal death in the ION, which could not be prevented by co-injection of BDNF. Thus, transport of BDNF alone does not generate a trophic signal at the cell body when axonal trkB is inactivated. In summary, our results indicate that both p75 neurotrophin and trkB receptors can mediate internalization and retrograde transport of BDNF, but activation of trkB seems to be essential for the survival-promoting actions of this neurotrophin.

Animals↗
PubMed1996

A reciprocal cell-cell interaction mediated by NT-3 and neuregulins controls the early survival and development of sympathetic neuroblasts.

Neurotrophin 3 (NT-3) can support the survival of some embryonic sympathetic neuroblasts before they become nerve growth factor dependent. We show that NT-3 is produced in vivo by nonneuronal cells neighboring embryonic sympathetic ganglia. NT-3 mRNA is produced by these nonneuronal cells in vitro and is up-regulated by platelet-derived growth factor, ciliary neurotrophic factor, and glial growth factor 2 (a neuregulin). Nonneuronal cell-conditioned medium promotes survival and induces TrkA expression in isolated sympathetic neuroblasts, and this activity is blocked by anti-NT-3 antibody. Neuroblasts also enhance NT-3 production by nonneural cells. Neuroblasts synthesize several forms of neuregulin, and antibodies to neuregulin attenuate the effect of the neuroblasts on the nonneuronal cells. These data suggest a reciprocal cell-cell interaction, in which neuroblast-derived neuregulins promote NT-3 production by neighboring nonneuronal cells, which in turn promotes neuroblast survival and further differentiation.

Animals↗
PubMed1996

TrkA activation is sufficient to rescue axotomized cholinergic neurons.

To test the molecular nature of the NGF receptor responsible for the ability of NGF to rescue septal cholinergic neurons following axotomy, we infused polyclonal antibodies that act as specific agonists of trkA (RTA) into the lateral ventricle of fimbria-fornix lesioned animals. Rats receiving chronic intraventricular infusions of RTA showed significantly more low affinity NGF receptor immunoreactive (p75NGFR-IR) neurons on the lesioned side than did control animals 2 weeks following unilateral fimbria-fornix lesion. RTA also initiated cholinergic sprouting. Infusions of RTA in combination with an antibody that blocks p75NGFR (REX) did not reduce the cell savings effect observed with RTA alone. However, animals infused with RTA plus REX demonstrated significantly less sprouting. These findings suggest that antibody-induced trkA activation is sufficient to mediate NGF-promoted survival of axotomized cholinergic neurons in vivo.

Animals↗
PubMed1996

Lack of neurotrophin-3 results in death of spinal sensory neurons and premature differentiation of their precursors.

To understand mechanisms resulting in the absence of two-thirds of spinal sensory neurons in mice lacking NT-3, we have compared dorsal root ganglia development in normal and mutant embryos. The reduction in neurons, achieved by E13, results from several deficits: first, elevated neuronal apoptosis significantly reduces neuronal numbers; second, elevated neurogenesis between E11 and E12, without changes in rates of precursor proliferation or apoptosis, depletes the precursor pool; consequently, the reduced precursor pool prevents increases in neuronal numbers between E12 and E13, when most neurons are born in normal animals. Although deficits occur before final target innervation, we show that NT-3 is expressed at all stages in regions accessible to these neurons or their axons and is only restricted to final targets after innervation.

Animals↗
PubMed1996

Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex.

Cadherins are calcium-dependent cell adhesion molecules that play fundamental roles in embryonic development, tissue morphogenesis, and cancer. A prerequisite for their function is association with the actin cytoskeleton via the catenins. Tyrosine phosphorylation of beta-catenin, which correlates with a reduction in cadherin-dependent cell adhesion, may provide cells with a mechanism to regulate cadherin activity. Here we report that beta-catenin immune precipitates from PC12 cells contain tyrosine phosphatase activity which dephosphorylates beta-catenin in vitro. In addition, we show that a member of the leukocyte antigen-related protein (LAR)-related transmembrane tyrosine phosphatase family (LAR-PTP) associates with the cadherin-catenin complex. This association required the amino-terminal domain of beta-catenin but does not require the armadillo repeats, which mediate association with cadherins. The interaction also is detected in PC9 cells, which lack alpha-catenin. Thus, the association is not mediated by alpha-catenin or by cadherins. Interestingly, LAR-PTPs are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. We propose that changes in tyrosine phosphorylation of beta-catenin mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth.

Amino Acid Sequence↗
PubMed1996

Heterologous expression of alpha 1-integrin cDNA generates variable ligand specificities and alterations in cell shape.

Integrins can mediate a diverse variety of functions that are regulated by unknown mechanisms. Integrin alpha 1 beta 1 can serve as a receptor for laminin-1 and collagen in certain cell types, but is a receptor for only collagen in others. To examine the molecular basis of this difference in specificity, three cell types were transfected with cDNA for the rat alpha 1 subunit. Following transfection with rat alpha 1, pluripotential hematopoietic human K562 cells exhibited alpha 1 beta 1-dependent attachment to collagen IV, but not laminin-1, unless activating antibody TS2/16 was added. The attachment to collagen IV stimulated the elaboration of a spread morphology resembling a differentiated megakarocyte with extensive processes which were absent in response to all other substrates. When MRC-5 cells, a human fibroblastic cell, or RD cells, a human rhabdomyosarcoma line, were transfected with the identical alpha 1-integrin construct, rat alpha 1 beta 1-dependent attachment to both collagen IV and laminin-1 was seen. Therefore differences in ligand specificity can be generated by translation of an identical integrin alpha 1 beta 1 mRNA in different cell types. Despite differences in ligand binding, alpha 1 cDNA-transfected K562 and RD cells express an alpha 1 subunit that appears to be antigenically and electrophoretically similar. Small differences in glycosylation were apparent, and correlated with changes in ligand specificity. Together these results show for the first time that identical cDNAs, absent activating antibodies or other manipulations, can change ligand selectivity and better establish the importance of cellular context in determining integrin function. Moreover they show that select integrins can shift the differentiated state of pluripotential cells.

Animals↗
PubMed1995

Disruption of retinal axon ingrowth by ablation of embryonic mouse optic chiasm neurons.

Mouse retinal ganglion cell axons growing from the eye encounter embryonic neurons at the future site of the optic chiasm. After in vivo ablation of these chiasm neurons with a monoclonal antibody and complement, retinal axons did not cross the midline and stalled at approximately the entry site into the chiasm region. Thus, in the mouse, the presence of early-generated neurons that reside at the site of the future chiasm is required for formation of the optic chiasm by retinal ganglion cell axons.

Animals↗
PubMed1995

Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons.

Immunocytochemistry has been used to examine the location of trkA, the high-affinity receptor for nerve growth factor, in adult rat dorsal root ganglia, trigeminal ganglia and spinal cord. TrkA immunoreactivity was observed in small and medium sized ganglion cells and in the dorsal horn of the spinal cord. In lumbar L4 and L5 ganglia trkA-immunoreactive cells constitute 40% of dorsal root ganglion cells and range in size from 15 to 45 microns in diameter. Double labelling using markers for various dorsal root ganglion subpopulations revealed that virtually all (92%) trkA-immunoreactive cells express calcitonin gene-related peptide (CGRP) immunoreactivity. In contrast only 4 and 13% of trkA-immunoreactive cells are labelled by the monoclonal antibody LA4 or the lectin Griffonia simplicifolia IB4, markers for small non-peptide-containing cells. Eighteen percent of trkA-immunoreactive cells belong to the 'large light' subpopulation, identified by their strong immunostaining by the neurofilament antibody RT97. TrkA immunoreactivity in the dorsal horn is heaviest in laminae I and II outer, has a similar distribution to CGRP, and is depleted by dorsal rhizotomy. Our results show that trkA-expressing cells in dorsal root ganglia correspond almost exactly with the CGRP, peptide-producing population. The receptor is present not only on cell bodies but also on central terminals. Non-peptide-containing small cells, which constitute 30% of dorsal root ganglion cells, are not trkA-immunoreactive and therefore most probably are functionally independent of nerve growth factor.

Animals↗
PubMed1995

Brain-derived neurotrophic factor-transduced fibroblasts: production of BDNF and effects of grafting to the adult rat brain.

Local delivery of brain-derived neurotrophic factor (BDNF) by genetically modified cells provides the unique opportunity to examine the effects of BDNF on adult dopaminergic and cholinergic neurons in vivo. Primary rat fibroblasts were genetically engineered to produce BDNF. Conditioned media from BDNF-transduced fibroblasts supported embryonic chick dorsal root ganglion neurons as well as rat fetal mesencephalic neurons. BDNF-transduced fibroblasts grafted to the rat brain survived and showed continued mRNA production for at least 2 weeks. The effects of BDNF-transduced fibroblast grafts on the dopaminergic and cholinergic systems were then assessed. BDNF-transduced fibroblasts grafted into the normal intact substantia nigra induced sprouting of tyrosine hydroxylase- and neurofilament-immunoreactive fibers into the graft. Fibroblast grafts implanted into the normal intact striatum and midbrain as well as the 6-hydroxydopamine-lesioned brain did not induce sprouting of dopaminergic fibers; neither did they affect drug-induced rotational behavior. BDNF-transduced fibroblasts did, however, significantly increase the homovanillic acid/dopamine ratio when grafted into the normal midbrain. Following transection of the fimbriafornix, BDNF-transduced fibroblasts grafted into the septum were unable to rescue the septal cholinergic population, as did nerve growth factor-producing fibroblast grafts. Genetically modified fibroblast grafts may provide an effective, localized method of BDNF delivery in vivo to test biological effects of this factor on the central nervous system.

Animals↗
PubMed1995

Regional differences in responsiveness of adult CNS axons to grafts of cells expressing human neurotrophin 3.

Neurotrophin 3 (NT3) belongs to the neurotrophin family, which also includes nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 4/5. NT3 mRNA is widely expressed in the rodent nervous system, but the physiological function of the native protein is still unclear. Genetically modified cell lines that produce physiological amounts of NT3 can provide a useful tool in the elucidation of the NT3 effects in the adult central nervous system (CNS). Genetically modified rat primary skin fibroblasts expressing and secreting human NT3 (hNT3) were prepared and characterized. In vitro, cell lines derived from different retroviral constructs expressed hNT3 mRNA, as determined by PCR and RNA blot analysis. Secretion of biologically active hNT3 was confirmed by specific elicitation of neurite outgrowth from cultured chick primary sympathetic and sensory neurons and from rat fetal locus coeruleus neurons in the presence of hNT3-producing cell conditioned media. In vivo, implanted fibroblasts survived well up to the maximal experimental time points of 6 weeks (brain) and 4 weeks (spinal cord) and continued to express hNT3 mRNA in vivo. As early as 2 weeks postgrafting, specific sprouting of host sensory neurites in response to hNT3-producing grafts was observed in the spinal cord. In contrast, hNT3-producing cerebral grafts did not induce a sprouting response different from that observed with control grafts. These findings establish the existence of a regionally different responsiveness of the CNS axons to local hNT3 overexpression.

Animals↗
PubMed1995

Identification of integrin alpha 3 beta 1 as a neuronal thrombospondin receptor mediating neurite outgrowth.

Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these interactions have not previously been identified. In this study, monoclonal antibodies raised to the integrin alpha 3 beta 1 heterodimer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondin-1. Alpha 3 beta 1 is found to be expressed on the cell body, neurites, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-ligand binding assay is used to demonstrate the direct binding of immunopurified alpha 3 beta 1 to thrombospondin-1. These results demonstrate a direct interaction between the integrin alpha 3 beta 1 and thrombospondin-1, which mediates neurite outgrowth in vitro and is likely to mediate the same interactions in vivo.

Adrenergic Fibers↗