Hoxa2 Selects Barrelette Neuron Identity and Connectivity in the Mouse Somatosensory Brainstem.

Mouse whiskers are somatotopically mapped in brainstem trigeminal nuclei as neuronal modules known as barrelettes. Whisker-related afferents form barrelettes in ventral principal sensory (vPrV) nucleus, whereas mandibular input targets dorsal PrV (dPrV). How barrelette neuron identity and circuitry is established is poorly understood. We found that ectopic Hoxa2 expression in dPrV neurons is sufficient to attract whisker-related afferents, induce asymmetrical dendrite arbors, and allow ectopic barrelette map formation. Moreover, the thalamic area forming whisker-related barreloids is prenatally targeted by both vPrV and dPrV axons followed by perinatal large-scale pruning of dPrV axons and refinement of vPrV barrelette input. Ectopic Hoxa2 expression allows topographically directed targeting and refinement of dPrV axons with vPrV axons into a single whisker-related barreloid map. Thus, a single HOX transcription factor is sufficient to switch dPrV into a vPrV barrelette neuron program and coordinate input-output topographic connectivity of a dermatome-specific circuit module.

H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein.

Proteins of the conserved HP1 family are elementary components of heterochromatin and are generally assumed to play a central role in the creation of a rigid, densely packed heterochromatic network that is inaccessible to the transcription machinery. Here, we demonstrate that the fission yeast HP1 protein Swi6 exists as a single highly dynamic population that rapidly exchanges in cis and in trans between different heterochromatic regions. Binding to methylated H3K9 or to heterochromatic RNA decelerates Swi6 mobility. We further show that Swi6 is largely dispensable to the maintenance of heterochromatin domains. In the absence of Swi6, H3K9 methylation levels are maintained by a mechanism that depends on polymeric self-association properties of Tas3, a subunit of the RNA-induced transcriptional silencing complex. Our results disclose a surprising role for Swi6 dimerization in demarcating constitutive heterochromatin from neighboring euchromatin. Thus, rather than promoting maintenance and spreading of heterochromatin, Swi6 appears to limit these processes and appropriately confine heterochromatin.

Projection neuron circuits resolved using correlative array tomography.

Assessment of three-dimensional morphological structure and synaptic connectivity is essential for a comprehensive understanding of neural processes controlling behavior. Different microscopy approaches have been proposed based on light microcopy (LM), electron microscopy (EM), or a combination of both. Correlative array tomography (CAT) is a technique in which arrays of ultrathin serial sections are repeatedly stained with fluorescent antibodies against synaptic molecules and neurotransmitters and imaged with LM and EM (Micheva and Smith, 2007). The utility of this correlative approach is limited by the ability to preserve fluorescence and antigenicity on the one hand, and EM tissue ultrastructure on the other. We demonstrate tissue staining and fixation protocols and a workflow that yield an excellent compromise between these multimodal imaging constraints. We adapt CAT for the study of projection neurons between different vocal brain regions in the songbird. We inject fluorescent tracers of different colors into afferent and efferent areas of HVC in zebra finches. Fluorescence of some tracers is lost during tissue preparation but recovered using anti-dye antibodies. Synapses are identified in EM imagery based on their morphology and ultrastructure and classified into projection neuron type based on fluorescence signal. Our adaptation of array tomography, involving the use of fluorescent tracers and heavy-metal rich staining and embedding protocols for high membrane contrast in EM will be useful for research aimed at statistically describing connectivity between different projection neuron types and for elucidating how sensory signals are routed in the brain and transformed into a meaningful motor output.

Correlative microscopy of densely labeled projection neurons using neural tracers.

Three-dimensional morphological information about neural microcircuits is of high interest in neuroscience, but acquiring this information remains challenging. A promising new correlative technique for brain imaging is array tomography (Micheva and Smith, 2007), in which series of ultrathin brain sections are treated with fluorescent antibodies against neurotransmitters and synaptic proteins. Treated sections are repeatedly imaged in the fluorescence light microscope (FLM) and then in the electron microscope (EM). We explore a similar correlative imaging technique in which we differentially label distinct populations of projection neurons, the key routers of electrical signals in the brain. In songbirds, projection neurons can easily be labeled using neural tracers, because the vocal control areas are segregated into separate nuclei. We inject tracers into areas afferent and efferent to the main premotor area for vocal production, HVC, to retrogradely and anterogradely label different classes of projection neurons. We optimize tissue preparation protocols to achieve high fluorescence contrast in the FLM and good ultrastructure in the EM (using osmium tetroxide). Although tracer fluorescence is lost during EM preparation, we localize the tracer molecules after fixation and embedding by using fluorescent antibodies against them. We detect signals mainly in somata and dendrites, allowing us to classify synapses within a single ultrathin section as belonging to a particular type of projection neuron. The use of our method will be to provide statistical information about connectivity among different neuron classes, and to elucidate how signals in the brain are processed and routed among different areas.