Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape.

The hindlimb and external genitalia of present-day tetrapods are thought to derive from an ancestral common primordium that evolved to generate a wide diversity of structures adapted for efficient locomotion and mating in the ecological niche occupied by the species. We show that despite long evolutionary distance from the ancestral condition, the early primordium of the mouse external genitalia preserved the capacity to take hindlimb fates. In the absence of Tgfbr1, the pericloacal mesoderm generates an extra pair of hindlimbs at the expense of the external genitalia. It has been shown that the hindlimb and the genital primordia share many of their key regulatory factors. Tgfbr1 controls the response to those factors by modulating the accessibility status of regulatory elements that control the gene regulatory networks leading to the formation of genital or hindlimb structures. Our work uncovers a remarkable tissue plasticity with potential implications in the evolution of the hindlimb/genital area of tetrapods, and identifies an additional mechanism for Tgfbr1 activity that might also contribute to the control of other physiological or pathological processes.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart.

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Expansion Microscopy on Saccharomyces cerevisiae.

The unicellular eukaryote Saccharomyces cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross-linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi - including yeast - and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts is a crucial step to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae , with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae .

Normal Mouse Brain Proteome II: Analysis of Brain Regions by High-resolution Mass Spectrometry.

BACKGROUND/AIM: Proteomics technologies provide fundamental insights into the high organizational complexity and diversity of the central nervous system. In the present study, high-resolution mass spectrometry (MS) was applied in order to identify whole-proteome content of anatomically distinct and functionally specific mouse brain regions. MATERIALS AND METHODS: Brains from eight 8-week-old C57BL/6N normal male mice were separated into seven anatomically district regions. The protein content of each region was analyzed by high-throughput nano-liquid chromatography-MS/MS Orbitrap elite technology. RESULTS: A total of 16,574 proteins were identified: 2,795 in cerebral cortex, 2,311 in olfactory bulb, 2,246 in hippocampus, 2,247 in hypothalamus, 2,250 in mid brain, 2,334 in cerebellum and 2,391 in medulla. Of these proteins, 534 were uniquely expressed in cerebral cortex, 323 in olfactory bulb, 230 in hippocampus, 272 in hypothalamus, 1,326 in mid brain, 320 in cerebellum and 268 in medulla. CONCLUSION: These data represent the most comprehensive proteomic map of the normal mouse brain and they might further be used in studies related to brain diseases, including cancer and neurodegenerative diseases.

The mitochondrial intergenic regions nad1-cob and cob-rps3 as molecular identification tools for pathogenic members of the genus Cryptococcus.

Cryptococcus spp. are fungal species belonging to Tremellomycetes, Agaricomycotina, Basidiomycota, and several members are responsible for cryptococcosis, one of the most ubiquitous human mycoses. Affecting mainly immunosuppressed patients, but also immunocompetent ones, the members of this genus present a high level of genetic diversity. In this study, two mitochondrial intergenic regions, i.e. nad1-cob and cob-rps3, were tested for the intra- or interspecies discrimination and identification of strains and species of the genus Cryptococcus. Phylogenetic trees were constructed based on individual and concatenated sequences from representative pathogenic strains of the Cryptococcus neoformans/Cryptococcus gattii complex, representing serotypes and AFLP genotypes of all newly introduced species of this complex. Using both intergenic regions, as well as the concatenated dataset, the strains clustered in accordance with the new taxonomy. These results suggest that identification of Cryptococcus strains is possible by employing these mitochondrial intergenic regions using PCR amplification as a quick and effective method to elucidate genotypic and taxonomic differences. Thus, these regions may be applicable to a broad range of clinical studies, leading to a rapid recognition of the clinical profiles of patients.

Mt-rps3 is an ancient gene which provides insight into the evolution of fungal mitochondrial genomes.

The nuclear ribosomal protein S3 (Rps3) is implicated in the assembly of the ribosomal small subunit. Fungi and plants present a gene copy in their mitochondrial (mt) genomes. An analysis of 303 complete fungal mt genomes showed that, when rps3 is found, it is either a free-standing gene or an anchored gene within the omega intron of the rnl gene. Early divergent fungi, Basidiomycota and all yeasts but the CTG group belong to the first case, and Pezizomycotina to the second. Its position, size and genetic code employed are conserved within species of the same Order. Size variability is attributed to different number of repeats. These repeats consist of AT-rich sequences. MtRps3 proteins lack the KH domain, necessary for binding to rRNA, in their N-terminal region. Their C-terminal region is conserved in all Domains of life. Phylogenetic analysis showed that nuclear and mtRps3 proteins are descendants of archaeal and a-proteobacterial homologues, respectively. Thus, fungal mt-rps3 gene is an ancient gene which evolved within the endosymbiotic model and presents different evolutionary routes: (a) coming from a-proteobacteria, it was relocated to another region of the mt genome, (b) via its insertion to the omega intron, it was transferred to the nucleus and/or got lost, and (c) it was re-routed to the mt genome again. Today, Basidiomycota and Saccharomycetales seem to follow the first evolutionary route and almost all Pezizomycotina support the second scenario with their exceptions being the result of the third scenario, i.e., the gene's re-entry to the mt genome.