We focus this report on the nucleus of the lateral olfactory tract (NLOT), a superficial amygdalar nucleus receiving olfactory input. Mixed with its Tbr1-expressing layer 2 pyramidal cell population (NLOT2), there are Sim1-expressing cells whose embryonic origin and mode of arrival remain unclear. We examined this population with Sim1-ISH and a Sim1-tauLacZ mouse line. An alar hypothalamic origin is apparent at the paraventricular area, which expresses Sim1 precociously. This progenitor area shows at E10.5 a Sim1-expressing dorsal prolongation that crosses the telencephalic stalk and follows the terminal sulcus, reaching the caudomedial end of the pallial amygdala. We conceive this Sim1-expressing hypothalamo-amygdalar corridor (HyA) as an evaginated part of the hypothalamic paraventricular area, which participates in the production of Sim1-expressing cells. From E13.5 onwards, Sim1-expressing cells migrated via the HyA penetrate the posterior pallial amygdalar radial unit and associate therein to the incipient Tbr1-expressing migration stream which swings medially past the amygdalar anterior basolateral nucleus (E15.5), crosses the pallio-subpallial boundary (E16.5), and forms the NLOT2 within the anterior amygdala by E17.5. We conclude that the Tbr1-expressing NLOT2 cells arise strictly within the posterior pallial amygdalar unit, involving a variety of required gene functions we discuss. Our results are consistent with the experimental data on NLOT2 origin reported by Remedios et al. (Nat Neurosci 10:1141-1150, 2007), but we disagree on their implication in this process of the dorsal pallium, observed to be distant from the amygdala.

Clinical scores, molecular markers and cellular phenotypes have been used to predict the clinical outcomes of patients with glioblastoma. However, their clinical use has been hampered by confounders such as patient co-morbidities, by the tumoral heterogeneity of molecular and cellular markers, and by the complexity and cost of high-throughput single-cell analysis. Here, we show that a microfluidic assay for the quantification of cell migration and proliferation can categorize patients with glioblastoma according to progression-free survival. We quantified with a composite score the ability of primary glioblastoma cells to proliferate (via the protein biomarker Ki-67) and to squeeze through microfluidic channels, mimicking aspects of the tight perivascular conduits and white-matter tracts in brain parenchyma. The assay retrospectively categorized 28 patients according to progression-free survival (short-term or long-term) with an accuracy of 86%, predicted time to recurrence and correctly categorized five additional patients on the basis of survival prospectively. RNA sequencing of the highly motile cells revealed differentially expressed genes that correlated with poor prognosis. Our findings suggest that cell-migration and proliferation levels can predict patient-specific clinical outcomes.

Several species of crustose coralline algae (CCA) and their associated microbial biofilms play important roles in determining the settlement location of scleractinian corals on tropical reefs. In recent decades, peyssonnelid algal crusts (PAC) have become spatial dominants across large areas of shallow Caribbean reefs, where they appear to deter the recruitment of scleractinians. Our genetic investigations of PAC in St. John, US Virgin Islands, amplifying the large-subunit ribosomal RNA and psbA protein D1 marker genes, revealed them to be identical to Ramicrusta textilis previously reported overgrowing corals in Jamaica. Specimens of PAC sampled from the Honduras were likewise identical, confirming that this crustose alga inhabits the easternmost and westernmost regions of the Caribbean. We also analysed 16S rDNA tag amplicon libraries of the biofilms associated with PAC and sympatric CCA, which is favoured for coral settlement. Our results show that the microbial communities on PAC (vs. CCA) are characterized by significantly lower numbers of the epibiotic bacterial genus Pseudoalteromonas, which facilitates the recruitment and settlement of marine invertebrates. From these data, we infer that PAC are therefore unlikely to be attractive as settlement sites for coral larvae. Given the significant ecological change anticipated on these reefs due to increasing cover of PAC, there is an urgent need to further investigate competitive interactions between PAC and scleractinian corals, and elucidate the role of PAC and their associated microbiomes in accentuating phase shifts from coral to algae on tropical reefs.

We discovered mechanical compression is able to bring activated MuSCs back to their quiescent stem cell state.

The N-terminal caveolin-binding motif (CBM) in Na/K-ATPase (NKA) alpha 1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human-induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared with the parental wild-type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated induced Skm cells. Another NKA functional mutant, A420P which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells' ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (7) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of beta-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/beta-catenin signaling.

Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNA5 (mRNAs) from mouse somites revealed that the 5' leaders of Hox mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.

Skeletal muscles can regenerate over the lifetime from resident muscle stem cells (MuSCs). Interactions between MuSCs and extracellular matrix (ECM) proteins are essential for muscle regeneration. The best-known receptors for ECM proteins are integrins, a family composed of twenty-some heterodimeric combinations of an alpha- and a beta-subunit. beta 1-integrin (encoded by Itgb1) is required for quiescence, proliferation, migration, and fusion of Pax7(+) MuSCs in the mouse model. beta 3-integrin (encoded by Itgb3) has been reported to be critical for the myogenic differentiation of C2C12 myoblasts, and Itgb3 germline mutant mice were shown to regenerate few if any myofibers after injury. To investigate the autonomous role of Itgb3 in the myogenic lineage in vivo, we conditionally inactivated a floxed Itgb3 allele (Itgb3(F)) by constitutive Pax7-Cre and tamoxifen-inducible Pax7-CreERT2 drivers. Unexpectedly, we found no defects in muscle regeneration in both conditional knockout models. In vitro studies using Itgb3 mutant myoblasts or RNAi knockdown of Itgb3 in myoblasts also did not reveal a role for myogenic differentiation. As beta 1- and beta 3-integrins share ECM ligands and downstream signaling effectors, we further examined Itgb3's role in a Itgb1 haploid background. Still, we found no evidence for an autonomous role of Itgb3 in muscle regeneration in vivo. Thus, while Itgb3 is critical for the differentiation of C2C12 cells, the regenerative defects reported for the Itgb3 germline mutant are not due to its role in the MuSC. We conclude that if beta 3-integrin does have a role in Pax7(+) MuSCs, it is compensated by beta 1- and/or another beta-integrin(s).

Tight control of transposon activity is essential for the integrity of the germline. Recently, a germ-cell-specific organelle, nuage, was proposed to play a role in transposon repression. To test this hypothesis, we disrupted a murine homolog of a Drosophila nuage protein Maelstrom. Effects on male meiotic chromosome synapsis and derepression of transposable elements (TEs) were observed. In the adult Mael(-/-) testes, LINE-1 (L1) derepression occurred at the onset of meiosis. As a result, Mael(-/-) spermatocytes were flooded with L1 ribonucleoproteins (RNPs) that accumulated in large cytoplasmic enclaves and nuclei. Mael(-/-) spermatocytes with nuclear L1 RNPs exhibited massive DNA damage and severe chromosome asynapsis even in the absence of SP011-generated meiotic double-strand breaks. This study demonstrates that MAEL, a nuage component, is indispensable for the silencing of TEs and identifies the initiation of meiosis as an important step in TE control in the male germline.

Postmigratory mouse primordial germ cells (PGCs) undergo extensive epigenetic remodeling that includes DNA methylation (DM) reprogramming of imprinted genes and, surprisingly, of transposable elements (TEs). Given the danger posed by TEs to the integrity of the germline, even a brief derepression of TEs is counterintuitive and puzzling. In the male fetal gonocytes, a sophisticated repressive mechanism that uses DM and TE-targeting piRNAs has evolved to stably silence TEs. A recent study has further increased the complexity of this problem by revealing that TE silencing is alleviated specifically at the onset of meiosis in testes lacking MAEL, a piRNA pathway protein. These observations and prior work of others are consistent with existence of an additional reprogramming event, transient relaxation of transposon silencing (TRTS), at the onset of both male and female meiosis in mice. In this Point of View we propose that TE expression is inherent to mammalian meiosis and discuss potential functional significance of this phenomenon.