Egr3-dependent muscle spindle stretch receptor intrafusal muscle fiber differentiation and fusimotor innervation homeostasis.

Muscle stretch proprioceptors (muscle spindles) are required for stretch reflexes and locomotor control. Proprioception abnormalities are observed in many human neuropathies, but the mechanisms involved in establishing and maintaining muscle spindle innervation and function are still poorly understood. During skeletal muscle development, sensory (Ia-afferent) innervation induces contacted myotubes to transform into intrafusal muscle fibers that form the stretch receptor core. The transcriptional regulator Egr3 is induced in Ia-afferent contacted myotubes by Neuregulin1 (Nrg1)/ErbB receptor signaling and it has an essential role in spindle morphogenesis and function. Because Egr3 is widely expressed during development and has a pleiotropic function, whether Egr3 functions primarily in skeletal muscle, Ia-afferent neurons, or in Schwann cells that myelinate Ia-afferent axons remains unresolved. In the present studies, cell-specific ablation of Egr3 in mice showed that it has a skeletal muscle autonomous function in stretch receptor development. Moreover, using genetic tracing, we found that Ia-afferent contacted Egr3-deficient myotubes were induced in normal numbers, but their development was blocked to generate one to two shortened fibers that failed to express some characteristic myosin heavy chain (MyHC) proteins. These "spindle remnants" persisted into adulthood, remained innervated by Ia-afferents, and expressed neurotrophin3 (NT3), which is required for Ia-afferent neuron survival. However, they were not innervated by fusimotor axons and they did not express glial derived neurotrophic factor (GDNF), which is essential for fusimotor neuron survival. These results demonstrate that Egr3 has an essential role in regulating gene expression that promotes normal intrafusal muscle fiber differentiation and fusimotor innervation homeostasis.

A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation.

Egr3 is a nerve growth factor (NGF)-induced transcriptional regulator that is essential for normal sympathetic nervous system development. Mice lacking Egr3 in the germline have sympathetic target tissue innervation abnormalities and physiologic sympathetic dysfunction similar to humans with dysautonomia. However, since Egr3 is widely expressed and has pleiotropic function, it has not been clear whether it has a role within sympathetic neurons and if so, what target genes it regulates to facilitate target tissue innervation. Here, we show that Egr3 expression within sympathetic neurons is required for their normal innervation since isolated sympathetic neurons lacking Egr3 have neurite outgrowth abnormalities when treated with NGF and mice with sympathetic neuron-restricted Egr3 ablation have target tissue innervation abnormalities similar to mice lacking Egr3 in all tissues. Microarray analysis performed on sympathetic neurons identified many target genes deregulated in the absence of Egr3, with some of the most significantly deregulated genes having roles in axonogenesis, dendritogenesis, and axon guidance. Using a novel genetic technique to visualize axons and dendrites in a subpopulation of randomly labeled sympathetic neurons, we found that Egr3 has an essential role in regulating sympathetic neuron dendrite morphology and terminal axon branching, but not in regulating sympathetic axon guidance to their targets. Together, these results indicate that Egr3 has a sympathetic neuron autonomous role in sympathetic nervous system development that involves modulating downstream target genes affecting the outgrowth and branching of sympathetic neuron dendrites and axons.

MicroRNA-184 antagonizes microRNA-205 to maintain SHIP2 levels in epithelia.

Despite their potential to regulate approximately one-third of the whole genome, relatively few microRNA (miRNA) targets have been experimentally validated, particularly in stratified squamous epithelia. Here we demonstrate not only that the lipid phosphatase SHIP2 is a target of miRNA-205 (miR-205) in epithelial cells, but, more importantly, that the corneal epithelial-specific miR-184 can interfere with the ability of miR-205 to suppress SHIP2 levels. This is the first example of a miRNA negatively regulating another to maintain levels of a target protein. Interfering with miR-205 function by using a synthetic antagomir, or by the ectopic expression of miR-184, leads to a coordinated damping of the Akt signaling pathway via SHIP2 induction. This was associated with a marked increase in keratinocyte apoptosis and cell death. Aggressive squamous cell carcinoma (SCC) cells exhibited elevated levels of miR-205. This was associated with a concomitant reduction in SHIP2 levels. Partial knockdown of endogenous miR-205 in SCCs markedly decreased phosphorylated Akt and phosphorylated BAD levels and increased apoptosis. We were able to increase SHIP2 levels in SCC cells after inhibition of miR-205. Therefore, miR-205 might have diagnostic value in determining the aggressivity of SCCs. Blockage of miR-205 activity with an antagomir or via ectopic expression of miR-184 could be novel therapeutic approaches for treating aggressive SCCs.

MicroRNAs of the mammalian eye display distinct and overlapping tissue specificity.

PURPOSE: In mammals, endogenous, noncoding RNAs, designated as microRNAs (miRNAs), inhibit the translation of a target messenger RNA, thereby silencing protein production. MiRNAs have been shown to regulate many aspects of development and differentiation in a wide range of tissues. Surprisingly, little consideration has been directed towards characterizing the expression of miRNAs in mammalian ocular tissues. METHODS: Low molecular weight (LMW) RNA isolated from the adult mouse corneal epithelium, lens/ciliary body, and a retina fractions of the eye was analyzed by miRNA arrays. The validity of the miRNA expression profiles were confirmed by northern blots and the tissue distribution of selected miRNAs was determined by in situ hybridization. RESULTS: MiRNAs exhibited distinct tissue and cell-type specificity in the ocular regions studied. MiRNA (mir)-184 had the highest hybridization signal in the corneal and lens arrays. In situ hybridization analysis revealed that mir-184 was expressed in the basal and immediately suprabasal cells of the corneal epithelium. In contrast, expression of mir-205 was detected throughout the anterior segmental epithelia as well as in the epidermis. Within the lens, expression of mir-184 was more strongly expressed in the epithelial cells of the germinative zone, whereas expression of mir-204 was uniformly expressed in all lens epithelial cells. Mir-181, -182, and -183 were detected in retinal and brain tissues, and their distribution patterns within the retina were both distinct and overlapping. CONCLUSIONS: The tissue and cell specificity of ocular miRNAs suggests that these noncoding RNAs may be regulating aspects of development and differentiation.