Research reportRescue of NGF-deficient mice I: transgenic expression of NGF in skin rescues mice lacking endogenous NGF
Introduction
Nerve growth factor (NGF) is the founding member of the neurotrophin growth factor family and was isolated because of its ability to promote survival and axonal outgrowth in developing sensory and sympathetic neurons [23], [24], [25], [26]. The temporal and spatial expression pattern of NGF was the basis for development of the neurotrophic hypothesis, which proposes that target-derived growth factors modulate neuronal survival and innervation density [22], [35], [38]. Analysis of ngf−/− mice verified the intrinsic role of NGF as a target-derived survival factor by demonstrating loss of small diameter sensory neurons and sympathetic postganglionic neurons [9]. In contrast to mice that lack NGF, mice that overexpress NGF in the skin (NGF-OE) exhibit hypertrophy of both sensory and sympathetic nervous systems [1], [2], [12]. One line of NGF-OE mice demonstrated a two-fold increase in the number of trigeminal and dorsal root ganglion sensory neurons and a 2.5 fold increase in the number of sympathetic neurons in the superior cervical ganglia [1], [2], [21], [32]. In addition, preferential increases were found in unmyelinated and thinly myelinated fibers [13], [43], a finding consistent with the types of axons lost in ngf−/− mice.
Mutant mice that lack NGF typically die within the first postnatal week, greatly limiting study of the impact of NGF withdrawal on neuron maturation and function in peripheral and central systems [9]. A likely cause underlying the premature death of ngf−/− mice is the failure of these animals to properly feed due to disruption of the sensory nervous system. To test whether target-derived NGF can rescue these deficits in the peripheral nervous system (PNS), mice that lack a functional endogenous NGF gene were crossed with mice that overexpress NGF in the periphery driven by the human K14-keratin promoter [1], [45]. Using this approach, hybrid transgenic-knockout mice, or ngfTKO mice, were produced that showed normal growth and viability. Transgenic expression of NGF restored the number of sympathetic neurons to normal levels, whereas the number of sensory neurons was only marginally improved. An increase in the proportion of peptidergic sensory neuron profiles did occur, however, and was accompanied by restoration of peripheral and central projections. These anatomical changes were paralleled by the ability of ngfTKO mice to detect noxious stimuli. The restored sensory function and viability of ngfTKO mice points to the importance of peripheral targets in providing critical trophic influences in the development and function of the PNS.
Section snippets
Isolation of mouse lines
NgfTKO mice were isolated by mating previously isolated ngf+/− mice [9] with NGF-OE mice that express the K14-NGF transgene [1]. Hybrid heterozygous knockout mice (NGF-OE/NGF+/−) obtained in the first round of mating were then bred to isolate hybrid homozygous knockout-NGF-OE mice (NGF-OE/ngf−/−). Hybrid breeding used the 47-8 NGF-OE or medium expresser transgenic line (described in Ref. [1]). This mouse line was used to avoid systemic effects of NGF overexpression that may be seen in mice
Transgenic-NGF rescues viability of ngf−/− mice
The introduction of a transgenic source of NGF driven by the K14 keratin promoter significantly affected survival of ngf−/− mice (Table 1). While no non-transgenic ngf−/− mice were viable at the time of genotype identification, ngf−/− mice carrying the K14-NGF transgene (ngfTKO mice) were identified at roughly 2/3 the predicted frequency (Table 1). The frequency of other genotypes present at the time of identification was not markedly different from the predicted number of births, suggesting
ngfTKO mice exhibit sensitivity to noxious stimuli
Neonatal ngf−/− mice typically do not respond to tail pinch stimuli [9]. To determine whether transgenic-NGF could restore behavioral sensitivity, a tail pinch behavioral assay was conducted on ngfTKO mice. Mice heterozygous for the NGF null mutation (ngf+/−) responded strongly to tail pinch (Table 5). Mice heterozygous for both the NGF transgene and for the NGF null mutation (NGF-OE/ngf+/−) expressed strong responses to tail pinch in 15 of 17 cases. Similarly, 18 out of 20 ngfTKO mice
Discussion
Data from over half a century of research supports a role for NGF in development and function of selected subpopulations of neurons in both the central and peripheral nervous system. The biological importance of NGF is underscored by the observation that ngf−/− mice demonstrate loss of sympathetic and small diameter sensory neurons and typically die within the first postnatal week after exhibiting poor feeding and lack of growth [9]. While these findings confirm the importance of NGF in
Acknowledgements
This work was funded in part by the NIH from NS33730 (KMA), NS31826 (BMD), and by Genentech.
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