The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAiinjected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.

The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. We developed new genetic tools that target neuroblasts and their diverse descendants, increasing our ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. We successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, we expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. Our transgenic tools can be combined to map individual neurons in specific lineages of various genotypes.

Factors that enhance the intrinsic growth potential of adult neurons are key players in the successful repair and regeneration of neurons following injury. Injury-induced activation of transcription factors has a central role in this process because they regulate expression of regeneration-associated genes. Sox11 is a developmentally expressed transcription factor that is significantly induced in adult neurons in response to injury. Its function in injured neurons is however undefined. Here, we report studies that use herpes simplex virus (HSV)-vector-mediated expression of Sox11 in adult sensory neurons to assess the effect of Sox11 overexpression on neuron regeneration. Cultured mouse dorsal root ganglia (DRG) neurons transfected with HSV-Sox11 exhibited increased neurite elongation and branching relative to naïve and HSV-vector control treated neurons. Neurons from mice injected in foot skin with HSV-Sox11 exhibited accelerated regeneration of crushed saphenous nerves as indicated by faster regrowth of axons and nerve fibers to the skin, increased myelin thickness and faster return of nerve and skin sensitivity. Downstream targets of HSV-Sox11 were examined by analyzing changes in gene expression of known regeneration-associated genes. This analysis in combination with mutational and chromatin immunoprecipitation assays indicates that the ability of Sox11 to accelerate in vivo nerve regeneration is dependent on its transcriptional activation of the regeneration-associated gene, small proline rich protein 1a (Sprr1a). This finding reveals a new functional linkage between Sox11 and Sprr1a in adult peripheral neuron regeneration.

Neurturin (NRTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands that exerts its actions via Ret tyrosine kinase and GFRα2. Expression of the Ret/GFRα2 co-receptor complex is primarily restricted to the peripheral nervous system and is selectively expressed by sensory neurons that bind the isolectin B4 (IB4). To determine how target-derived NRTN affects sensory neuron properties, transgenic mice that overexpress NRTN in keratinocytes (NRTN-OE mice) were analyzed. Overexpression of NRTN increased the density of PGP9.5-positive, but not calcitonin gene-related peptide (CGRP)-positive, free nerve endings in footpad epidermis. GFRα2- immunopositive somata were hypertrophied in NRTN-OE mice. Electron microscopic analysis further revealed hypertrophy of unmyelinated sensory axons and a subset of myelinated axons. Overexpression of NRTN increased the relative level of mRNAs encoding GFRα2 and Ret, the ATP receptor P2X3 (found in IB4-positive, GFRα2-expressing sensory neurons), the acid-sensing ion channel (ASIC) 2a, and transient receptor potential cation channel subfamily member M8 (TRPM8) in sensory ganglia. Behavioral testing of NRTN-OE mice revealed an increased sensitivity to mechanical stimuli in glabrous skin of the hind paw. NRTN-OE mice also displayed increased behavioral sensitivity to cool temperature (17°C-20°C) and oral sensitivity to menthol. The increase in cool and menthol sensitivity correlated with a significant increase in TRPM8 expression and the percentage of menthol-responsive cutaneous sensory neurons. These data indicate that the expression level of NRTN in the skin modulates gene expression in cutaneous sensory afferents and behavioral sensitivity to thermal, chemical and mechanical stimuli.

Sox11 is a high mobility group (HMG) containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity and regeneration. BDNF transcription is elevated in the DRG following nerve injury in parallel with Sox11 allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11 we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5′-AACAAAG-3′ (I, IV, VII and VIII) were increased at 1d or 3d following axotomy. Exons 1 and IV showed the greatest increase and only exon 1 remained elevated at 3d. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription.