Project description:Sea urchins (echinoids) are common model organisms for research in developmental biology and for their transition from a bilateral larva into their post-metamorphic adult with pentaradial body symmetry. The adult also has a calcareous endoskeleton with a multimetameric pattern of continuously added elements, among them the namesake of this phylum, spines. Nearly all echinoids have both large primary spines, and an associated set of smaller secondary spines.We hypothesize that the secondary spines of the tropical variegated urchin species, Lytechinus variegatus, are morphologically and molecularly distinct structures from primary spines and not just small spines. To test this premise, we examined both spine types using light microscopy, micro-CT imaging, lectin labeling, transcriptomics, and fluorescent in situ hybridization (FISH). Our findings reveal basic similarities between the two types in mineral and cellular anatomy, but with clear differences in growth patterns, genes expressed, and in the location of gene expression within the two types of spines. In particular, secondary spines have non-overlapping, longitudinally concentrated growth bands that lead to a blunt and straight profile, and a distinct transcriptome involving the upregulation in many genes in comparison to the primary spines. Neural, ciliary, and extracellular matrix interacting factors are implicated in the differentially expressed gene (DEG) dataset, including two genes - ONECUT2 and an uncharacterized discoidin- and thrombospondin-containing protein - that show spine type- specific localizations in FISH, and may be of interest to ongoing work in urchin spine patterning.These results demonstrate that primary and secondary spines have overlapping but distinct molecular and biomineralized characteristics, suggesting unique developmental and regenerative mechanisms, and devotion to this spiny dermal phylum.
Project description:Excitatory synapses occur mainly on dendritic spines, and spine density is usually correlated with the strength of excitatory synaptic transmission. We report that Nr4a1, an activity-inducible gene encoding a nuclear receptor, regulates the density and distribution of dendritic spines in CA1 pyramidal neurons. Nr4a1 overexpression resulted in elimination of the majority of spines; however, postsynaptic densities were preserved on dendritic shafts, and the strength of excitatory synaptic transmission was unaffected, showing that excitatory synapses can be dissociated from spines. mRNA expression profiling studies suggest that Nr4a1-mediated transcriptional regulation of the actin cytoskeleton contributes to this effect. Under conditions of chronically elevated activity, when Nr4a1 was induced, Nr4a1 knockdown increased the density of spines and PSDs specifically at the distal ends of dendrites. Thus, Nr4a1 is a key component of an activity-induced transcriptional program that regulates the density and distribution of spines and synapses.
Project description:Excitatory synapses occur mainly on dendritic spines, and spine density is usually correlated with the strength of excitatory synaptic transmission. We report that Nr4a1, an activity-inducible gene encoding a nuclear receptor, regulates the density and distribution of dendritic spines in CA1 pyramidal neurons. Nr4a1 overexpression resulted in elimination of the majority of spines; however, postsynaptic densities were preserved on dendritic shafts, and the strength of excitatory synaptic transmission was unaffected, showing that excitatory synapses can be dissociated from spines. mRNA expression profiling studies suggest that Nr4a1-mediated transcriptional regulation of the actin cytoskeleton contributes to this effect. Under conditions of chronically elevated activity, when Nr4a1 was induced, Nr4a1 knockdown increased the density of spines and PSDs specifically at the distal ends of dendrites. Thus, Nr4a1 is a key component of an activity-induced transcriptional program that regulates the density and distribution of spines and synapses. After 10 days in culture, dissociated mouse hippocampal neurons in 6-well plates were infected with lentivirus expressing either Flag-Nr4a1 or GFP and incubated for 6 days to allow for transgene expression. Total RNA was then isolated using RNeasy Plus kit (QIAGEN). Samples passing an mRNA quality check proceeded to quantitative analysis on Agilent-026655 4x44 Mouse Microarrays.
Project description:Papillomaviruses (PVs) are able to induce papillomas, premalignant lesions, and carcinomas in a wide variety of species. PVs are classified first based on their host and tissue tropism and then their genomic diversities. A laboratory mouse papillomavirus, MmuPV1 (formerly MusPV), naturally infects NMRI-Foxn1nu/Foxn1nu (nude; T cell deficient) mice. C57BL/6J wild-type mice were not susceptible to MmuPV1 infection; however, immunocompetent, alopecic, S/RV/Cri-ba/ba (bare) mice developed small papillomas at injection sites that regressed. NMRI-Foxn1nu and B6.Cg-Foxn1nu but not NU/J-Foxn1nu mice were susceptible to MmuPV1 infection. B6 congenic strains, but not other congenic strains carrying the same allelic mutations, that lack B- and T-cells, but not B-cells alone, were susceptible to infection, indicating that mouse strain and T-cell deficiency are critical to tumor formation. Although lesions initially observed were exophytic papillomas around the muzzle, exophytic papillomas on the tail and condylomas of the vaginal lining could be induced by experimental infections. On the dorsal skin, locally invasive, poorly differentiated tumors developed with features similar to human trichoblastomas. Transcriptome analysis revealed significant differences between the normal skin in these anatomic sites and in papillomas versus trichoblastomas. The primarily dysregulated genes involved molecular pathways associated with cancer, cellular development, cellular growth and proliferation, cell morphology, and connective tissue development and function. Surprisingly, few of the genes commonly associated with basal cell carcinoma or squamous cells carcinoma were dramatically dysregulated. To determine if there were transcriptome differences between papillomas on the tail skin compared to invasive trichoblastomas on the dorsal skin, tumors from the tail and dorsal skin from 3 B6.Cg-Foxn1nu/Foxn1nu mice and unaffected skin from the tail or contra-lateral (dorsal skin) were compared using the Affymetrix GeneChip Mouse Genome 1.0 ST Array. Concurrently, a matched study was done at the University of Louisville comparing facial (muzzle) papillomas and dorsal skin trichoblastomas to respective unaffected contralateral skin.