The POU-domain transcription POU4F3 is expressed in the sensory cells of the inner ear. Expression begins shortly after commitment to the hair cell (HC) fate, and continues throughout life. It is required for terminal HC differentiation and survival. To explore regulation of the murine Pou4f3 gene, we linked enhanced green fluorescent protein (eGFP) to 8.5 kb of genomic sequence 5’ to the start codon in transgenic mice. eGFP was uniformly present in all embryonic and neonatal HCs. Expression of eGFP was also observed in developing Merkel cells and olfactory neurons as well as adult inner and vestibular HCs, mimicking the normal expression pattern of POU4F3 protein, with the exception of adult outer HCs. Apparently ectopic expression was observed in developing inner ear neurons. On a Pou4f3 null background, the transgene produced expression in embryonic HCs which faded soon after birth both in vivo and in vitro. Pou4f3 null HCs treated with caspase 3 and 9 inhibitors survived longer than untreated HCs, but still showed reduced expression of eGFP. The results suggest the existence of separate enhancers for different HC types, as well as strong autoregulation of the Pou4f3 gene. Bioinformatic analysis of four divergent mammalian species revealed three highly-conserved regions within the transgene: 400 bp immediately 5’ to the Pou4f3 ATG, a short sequence at -1.3 kb, and a longer region at -8.2 to -8.5 kb. The latter contained E-box motifs that bind bHLH transcription factors, including motifs activated by ATOH1. Co-transfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared to the transgene alone. Chromatin immunoprecipitation of the three highly conserved regions revealed binding of ATOH1 to the distal-most conserved region. The results are consistent with regulation of Pou4f3 in HCs by ATOH1 at a distal enhancer.
Neurotrophins participate in regulating the survival, differentiation, and target innervation of many neurons, mediated by high-affinity Trk and low-affinity p75 receptors. In the cochlea, spiral ganglion (SG) neuron survival is strongly dependent upon neurotrophic input, including brain-derived neurotrophic factor (BDNF), which increases the number of neurite outgrowth in neonatal rat SG in vitro. Less is known about signal transduction pathways linking the activation of neurotrophin receptors to SG neuron nuclei. In particular, the p38 and cJUN Kinase (JNK), mitogen-activated protein kinase (MAPK) pathways, which participate in JNK signaling in other neurons, have not been studied. We found that inhibition of Ras, p38, phosphatidyl inositol 3 kinase (PI3K) or Akt signaling reduced or eliminated BDNF mediated increase in number of neurite outgrowth, while inhibition of Mek/Erk had no influence. Inhibition of Rac/cdc42, which lies upstream of JNK, modestly enhanced BDNF induced formation of neurites. Western blotting implicated p38 and Akt signaling, but not Mek/Erk. The results suggest that the Ras/p38 and PI3K/Akt are the primary pathways by which BDNF promotes its effects. Activation of Rac/cdc42/JNK signaling by BDNF may reduce the formation of neurites. This is in contrast to our previous results on NT-3, in which Mek/Erk signaling was the primary mediator of SG neurite outgrowth in vitro. Our data on BDNF agree with prior results from others that have implicated PI3K/Akt involvement in mediating the effects of BDNF on SG neurons in vitro, including neuronal survival and neurite extension. However, the identification of p38 and JNK involvement is entirely novel. The results suggest that neurotrophins can exert opposing effects on SG neurons, the balance of competing signals influencing the generation of neurites. This competition could provide a potential mechanism for the control of neurite number during development.
Extracellular matrix (ECM) molecules have been shown to function as cues for neurite guidance in various populations of neurons. Here we show that laminin (LN) and fibronectin (FN) presented in stripe micro-patterns can provide guidance cues to neonatal (P5) inner ear spiral ganglion (SG) neurites. The response to both ECM molecules was dose-dependent. In a LN versus poly-L-lysine (PLL) assay, neurites were more often observed on PLL at low coating concentrations (5 and 10 microg/mL), while they were more often on LN at a high concentration (80 microg/mL). In a FN versus PLL assay, neurites were more often on PLL than on FN stripes at high coating concentrations (40 and 80 microg/mL). In a direct competition between LN and FN, neurites were observed on LN significantly more often than on FN at both 10 and 40 microg/mL. The data suggest a preference by SG neurites for LN at high concentrations, as well as avoidance of both LN at low and FN at high concentrations. The results also support a potential model for neurite guidance in the developing inner ear in vivo. LN, in the SG and osseus spiral lamina may promote SG dendrite growth toward the organ of Corti. Within the organ of Corti, lower concentrations of LN may slow neurite growth, with FN beneath each row of hair cells providing a stop or avoidance signal. This could allow growth cone filopodia increased time to sample their cellular targets, or direct the fibers upward toward the hair cells.
Dlx-3, a homolog of Drosophila Dll, has been isolated from an axolotl blastema cDNA library, and its expression in developing and regenerating limbs characterized. The normal expression pattern, and the changes that occur during experimental treatments, indicate a correlation between Dlx-3 expression and the establishment of the outgrowth-permitting epidermis. Dlx-3 is expressed at high levels in a distal-to-proximal gradient in the epidermis of developing limb buds, and is upregulated in the apical ectodermal cap (AEC) during limb regeneration. Expression is maximal at the late bud stage of regeneration, coincident with the transition from the early phase of nerve dependency to the later phase of nerve independence. Dlx-3 expression in the epidermis is rapidly downregulated by denervation during the nerve-dependent phase and is unaffected by denervation during the nerve-independent phase. We investigated this relationship between nerves and Dlx-3 expression by implanting FGF-2 beads into regenerates that had been denervated at a nerve-dependent stage. Dlx-3 expression was maintained by FGF-2 after denervation, and regeneration progressed to completion. In addition, we detected FGF-2 protein in the AEC and in nerves, and observed that the level of expression in both tissues decreases dramatically in response to denervation. We conclude that both limb development and regeneration require a permissive epidermis, characterized by Dlx-3 and FGF expression, both of which are maintained by FGF through an autocrine loop. The transformation of the limb epidermis into a functional AEC that produces and responds to FGF autocatalytically, is presumed to be induced by FGF. Since nerves appear to be a source of this priming FGF, it is possible that a member of the FGF family of growth factors is the elusive neurotrophic factor of limb regeneration.
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