Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples — including Xist, which orchestrates X chromosome inactivation — has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes

The study of Priapulus caudatus reveals conserved molecular patterning underlying different gut morphogenesis in the Ecdysozoa

Background The digestive systems of animals can become highly specialized in response to their exploration and occupation of new ecological niches. Although studies on different animals have revealed commonalities in gut formation, the model systems Caenorhabditis elegans and Drosophila melanogaster, which belong to the invertebrate group Ecdysozoa, exhibit remarkable deviations in how their intestines develop. Their morphological and developmental idiosyncrasies have hindered reconstructions of ancestral gut characters for the Ecdysozoa, and limit comparisons with vertebrate models. In this respect, the phylogenetic position, and slow evolving morphological and molecular characters of marine priapulid worms advance them as a key group to decipher evolutionary events that occurred in the lineages leading to C. elegans and D. melanogaster. Results In the priapulid Priapulus caudatus, the gut consists of an ectodermal foregut and anus, and a mid region of at least partial endodermal origin. The inner gut develops into a 16-cell primordium devoid of visceral musculature, arranged in three mid tetrads and two posterior duplets. The mouth invaginates ventrally and shifts to a terminal anterior position as the ventral anterior ectoderm differentially proliferates. Contraction of the musculature occurs as the head region retracts into the trunk and resolves the definitive larval body plan. Despite obvious developmental differences with C. elegans and D. melanogaster, the expression in P. caudatus of the gut-related candidate genes NK2.1, foxQ2, FGF8/17/18, GATA456, HNF4, wnt1, and evx demonstrate three distinct evolutionarily conserved molecular profiles that correlate with morphologically identified sub-regions of the gut. Conclusions The comparative analysis of priapulid development suggests that a midgut formed by a single endodermal population of vegetal cells, a ventral mouth, and the blastoporal origin of the anus are ancestral features in the Ecdysozoa. Our molecular data on P. caudatus reveal a conserved ecdysozoan gut-patterning program and demonstrates that extreme morphological divergence has not been accompanied by major molecular innovations in transcriptional regulators during digestive system evolution in the Ecdysozoa. Our data help us understand the origins of the ecdysozoan body plan, including those of C. elegans and D. melanogaster, and this is critical for comparisons between these two prominent model systems and their vertebrate counterparts

Coupling multielectrode array recordings with silver labeling of recording sites to study cervical spinal network connectivity

Midcervical spinal interneurons form a complex and diffuse network and may be involved in modulating phrenic motor output. The intent of the current work was to enable a better understanding of midcervical “network-level” connectivity by pairing the neurophysiological multielectrode array (MEA) data with histological verification of the recording locations. We first developed a method to deliver 100-nA currents to electroplate silver onto and subsequently deposit silver from electrode tips after obtaining midcervical (C3–C5) recordings using an MEA in anesthetized and ventilated adult rats. Spinal tissue was then fixed, harvested, and histologically processed to “develop” the deposited silver. Histological studies verified that the silver deposition method discretely labeled (50-μm resolution) spinal recording locations between laminae IV and X in cervical segments C3-C5. Using correlative techniques, we next tested the hypothesis that midcervical neuronal discharge patterns are temporally linked. Cross-correlation histograms produced few positive peaks (5.3%) in the range of 0–0.4 ms, but 21.4% of neuronal pairs had correlogram peaks with a lag of ≥0.6 ms. These results are consistent with synchronous discharge involving mono- and polysynaptic connections among midcervical neurons. We conclude that there is a high degree of synaptic connectivity in the midcervical spinal cord and that the silver-labeling method can reliably mark metal electrode recording sites and “map” interneuron populations, thereby providing a low-cost and effective tool for use in MEA experiments. We suggest that this method will be useful for further exploration of midcervical network connectivity. NEW &amp; NOTEWORTHY We describe a method that reliably identifies the locations of multielectrode array (MEA) recording sites while preserving the surrounding tissue for immunohistochemistry. To our knowledge, this is the first cost-effective method to identify the anatomic locations of neuronal ensembles recorded with a MEA during acute preparations without the requirement of specialized array electrodes. In addition, evaluation of activity recorded from silver-labeled sites revealed a previously unappreciated degree of connectivity between midcervical interneurons. </jats:p

Immunogenicity of Heterologous Recombinant Adenovirus Prime-Boost Vaccine Regimens Is Enhanced by Circumventing Vector Cross-Reactivity

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have been constructed from Ad subgroup B, including rAd11 and rAd35, as well as from Ad subgroup D, including rAd49. However, the optimal combination of vectors for heterologous rAd prime-boost vaccine regimens and the extent of cross-reactive vector-specific neutralizing antibodies (NAbs) remain poorly defined. We have shown previously that the closely related vectors rAd11 and rAd35 elicited low levels of cross-reactive NAbs. Here we show that these cross-reactive NAbs correlated with substantial sequence homology in the hexon hypervariable regions (HVRs) and suppressed the immunogenicity of heterologous rAd prime-boost regimens. In contrast, vectors with lower hexon HVR homology, such as rAd35 and rAd49, did not elicit detectable cross-reactive vector-specific NAbs. Consistent with these findings, rAd35-rAd49 vaccine regimens proved more immunogenic than both rAd35-rAd5 and rAd35-rAd11 regimens in mice with anti-Ad5 immunity. These data suggest that optimal heterologous rAd prime-boost regimens should include two vectors that are both rare in human populations to circumvent preexisting antivector immunity as well as sufficiently immunologically distinct to avoid cross-reactive antivector immunity

Magnitude and Phenotype of Cellular Immune Responses Elicited by Recombinant Adenovirus Vectors and Heterologous Prime-Boost Regimens in Rhesus Monkeys▿

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for human immunodeficiency virus type 1 (HIV-1) and other pathogens have been shown to elicit antigen-specific cellular immune responses. Rare serotype rAd vectors have also been constructed to circumvent preexisting anti-Ad5 immunity and to facilitate the development of novel heterologous rAd prime-boost regimens. Here we show that rAd5, rAd26, and rAd48 vectors elicit qualitatively distinct phenotypes of cellular immune responses in rhesus monkeys and can be combined as potent heterologous prime-boost vaccine regimens. While rAd5-Gag induced primarily gamma interferon-positive (IFN-γ+) and IFN-γ+/tumor necrosis factor alpha+ (TNF-α+) T-lymphocyte responses, rAd26-Gag and rAd48-Gag induced higher proportions of interleukin-2+ (IL-2+) and polyfunctional IFN-γ+/TNF-α+/IL-2+ T-lymphocyte responses. Priming with the rare serotype rAd vectors proved remarkably effective for subsequent boosting with rAd5 vectors. These data demonstrate that the rare serotype rAd vectors elicited T-lymphocyte responses that were phenotypically distinct from those elicited by rAd5 vectors and suggest the functional relevance of polyfunctional CD8+ and CD4+ T-lymphocyte responses. Moreover, qualitative differences in cellular immune responses may prove critical in determining the overall potency of heterologous rAd prime-boost regimens