Generation and Characterization of a Tissue-Specific Centrosome Indicator Mouse Line.

Centrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue-specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP-Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre-mediated excision, EGFP-Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real-time imaging, and showed that EGFP-Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP-Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286-296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc

TBX5 and NuRD Divide the Heart

In this issue of Developmental Cell, Waldron et al. (2016) identify an interaction between a master regulator of heart development, TBX5, and the NuRD complex and describe how mutations affecting the interaction may contribute to congenital heart disease. Furthermore, these interactions may have contributed to the evolution of cardiac septation

Redox Paradox: Can Hypoxia Heal Ischemic Hearts?

Adult cardiomyocytes are largely thought to lack proliferative and therefore regenerative potential. Reporting in Nature, Nakada et al. (2016) find that a hypoxic regime reduces mitochondrial metabolism and promotes proliferation in adult mouse cardiomyocytes, resulting in increased regeneration following myocardial infarction. These findings suggest the potential to transform post-MI care

MicroRNAs in a Cardiac Loop: Progenitor or Myocyte?

Like transcription factors, microRNAs are emerging as regulators of cell fate decisions. In this issue, Wang et al. (2010) identify a critical microRNA pathway under the control of Bmp signaling that promotes outflow tract myocardial differentiation from cardiac progenitors in vivo

Tbx20 Is Required in Mid-Gestation Cardiomyocytes and Plays a Central Role in Atrial Development.

RationaleMutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type-specific requirement for TBX20 in early myocardial development remains to be explored.ObjectiveHere, we investigated roles of TBX20 in midgestation cardiomyocytes for heart development.Methods and resultsAblation of Tbx20 from developing cardiomyocytes using a doxycycline inducible cTnTCre transgene led to embryonic lethality. The circumference of developing ventricular and atrial chambers, and in particular that of prospective left atrium, was significantly reduced in Tbx20 conditional knockout mutants. Cell cycle analysis demonstrated reduced proliferation of Tbx20 mutant cardiomyocytes and their arrest at the G1-S phase transition. Genome-wide transcriptome analysis of mutant cardiomyocytes revealed differential expression of multiple genes critical for cell cycle regulation. Moreover, atrial and ventricular gene programs seemed to be aberrantly regulated. Putative direct TBX20 targets were identified using TBX20 ChIP-Seq (chromatin immunoprecipitation with high throughput sequencing) from embryonic heart and included key cell cycle genes and atrial and ventricular specific genes. Notably, TBX20 bound a conserved enhancer for a gene key to atrial development and identity, COUP-TFII/Nr2f2 (chicken ovalbumin upstream promoter transcription factor 2/nuclear receptor subfamily 2, group F, member 2). This enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20-dependent manner.ConclusionsMyocardial TBX20 directly regulates a subset of genes required for fetal cardiomyocyte proliferation, including those required for the G1-S transition. TBX20 also directly downregulates progenitor-specific genes and, in addition to regulating genes that specify chamber versus nonchamber myocardium, directly activates genes required for establishment or maintenance of atrial and ventricular identity. TBX20 plays a previously unappreciated key role in atrial development through direct regulation of an evolutionarily conserved COUPT-FII enhancer

Myocardial Lineage Development

The myocardium of the heart is composed of multiple highly specialized myocardial lineages, including those of the ventricular and atrial myocardium, and the specialized conduction system. Specification and maturation of each of these lineages during heart development is a highly ordered, ongoing process involving multiple signaling pathways and their intersection with transcriptional regulatory networks. Here, we attempt to summarize and compare much of what we know about specification and maturation of myocardial lineages from studies in several different vertebrate model systems. To date, most research has focused on early specification, and while there is still more to learn, less is known about factors that promote subsequent maturation of myocardial lineages required to build the functioning adult heart

Repeated games for eikonal equations, integral curvature flows and non-linear parabolic integro-differential equations

The main purpose of this paper is to approximate several non-local evolution equations by zero-sum repeated games in the spirit of the previous works of Kohn and the second author (2006 and 2009): general fully non-linear parabolic integro-differential equations on the one hand, and the integral curvature flow of an interface (Imbert, 2008) on the other hand. In order to do so, we start by constructing such a game for eikonal equations whose speed has a non-constant sign. This provides a (discrete) deterministic control interpretation of these evolution equations. In all our games, two players choose positions successively, and their final payoff is determined by their positions and additional parameters of choice. Because of the non-locality of the problems approximated, by contrast with local problems, their choices have to "collect" information far from their current position. For integral curvature flows, players choose hypersurfaces in the whole space and positions on these hypersurfaces. For parabolic integro-differential equations, players choose smooth functions on the whole space

Experiences of Kenyan Healthcare Workers Providing Services to Men Who Have Sex With Men: Qualitative Findings from a Sensitivity Training Programme

Introduction Men who have sex with men (MSM) in Kenya are at high risk for HIV and may experience prejudiced treatment in health settings due to stigma. An on-line computer-facilitated MSM sensitivity programme was conducted to educate healthcare workers (HCWs) about the health issues and needs of MSM patients. Methods Seventy-four HCWs from 49 ART-providing health facilities in the Kenyan Coast were recruited through purposive sampling to undergo a two-day MSM sensitivity training. We conducted eight focus group discussions (FGDs) with programme participants prior to and three months after completing the training programme. Discussions aimed to characterize HCWs’ challenges in serving MSM patients and impacts of programme participation on HCWs’ personal attitudes and professional capacities. Results Before participating in the training programme, HCWs described secondary stigma, lack of professional education about MSM, and personal and social prejudices as barriers to serving MSM clients. After completing the programme, HCWs expressed greater acknowledgement of MSM patients in their clinics, endorsed the need to treat MSM patients with high professional standards and demonstrated sophisticated awareness of the social and behavioural risks for HIV among MSM. Conclusions Findings provide support for this approach to improving health services for MSM patients. Further efforts are needed to broaden the reach of this training in other areas, address identified barriers to HCW participation and evaluate programme effects on patient and HCW outcomes using rigorous methodology

Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells.

Chromatin architecture has been implicated in cell type-specific gene regulatory programs, yet how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (hPSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon human endogenous retrovirus subfamily H (HERV-H) in creating topologically associating domains (TADs) in hPSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces the transcription of upstream genes, while de novo insertion of HERV-H elements can introduce new TAD boundaries. The ability of HERV-H to create TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents the formation of boundaries. This ability is not limited to hPSCs, as these actively transcribed HERV-H elements and their corresponding TAD boundaries also appear in pluripotent stem cells from other hominids but not in more distantly related species lacking HERV-H elements. Overall, our results provide direct evidence for retrotransposons in actively shaping cell type- and species-specific chromatin architecture