SUMMARYEstablishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. Here, we show that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. These chamber deformities emerge gradually during development, with a severe collapse in the number of ventricular cardiomyocytes and an accumulation of excess atrial cardiomyocytes as the heart matures. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, our data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium. Thus, our results are relevant to the etiologies of fetal and neonatal cardiac pathology and could direct future innovations in cardiac regenerative medicine.

Temporally controlled mechanisms that define the unique features of ventricular and atrial cardiomyocyte identity are essential for the construction of a coordinated, morphologically intact heart. We have previously demonstrated an important role for nkx genes in maintaining ventricular identity, however, the specific timing of nkx2.5 function in distinct cardiomyocyte populations has yet to be elucidated. Here, we show that heat-shock induction of a novel transgenic line, Tg(hsp70l:nkx2.5-EGFP), during the initial stages of cardiomyocyte differentiation leads to rescue of chamber shape and identity in nkx2.5−/− embryos as chambers emerge. Intriguingly, our findings link an early role of this essential cardiac transcription factor with a later function. Moreover, these data reveal that nkx2.5 is also required in the second heart field as the heart tube forms, reflecting the temporal delay in differentiation of this population. Thus, our results support a model in which nkx genes induce downstream targets that are necessary to maintain chamber-specific identity in both early- and late-differentiating cardiomyocytes at discrete stages in cardiac morphogenesis. Furthermore, we show that overexpression of nkx2.5 during first and second heart field development not only rescues the mutant phenotype, but also is sufficient for proper function of the adult heart. Taken together, these results shed new light on the stage-dependent mechanisms that sculpt chamber-specific cardiomyocytes and, therefore, have the potential to improve in vitro generation of ventricular cells to treat myocardial infarction and congenital heart disease.

Atrial and ventricular cardiac chambers behave as distinct subunits with unique morphological, electrophysiological and contractile properties. Despite the importance of chamber-specific features, chamber fate assignments remain relatively plastic, even after differentiation is underway. In zebrafish, Nkx transcription factors are essential for the maintenance of ventricular characteristics, but the signaling pathways that operate upstream of Nkx factors in this context are not well understood. Here, we show that FGF signaling plays an essential part in enforcing ventricular identity. Loss of FGF signaling results in a gradual accumulation of atrial cells, a corresponding loss of ventricular cells, and the appearance of ectopic atrial gene expression within the ventricle. These phenotypes reflect important roles for FGF signaling in promoting ventricular traits, both in early-differentiating cells that form the initial ventricle and in late-differentiating cells that append to its arterial pole. Moreover, we find that FGF signaling functions upstream of Nkx genes to inhibit ectopic atrial gene expression. Together, our data suggest a model in which sustained FGF signaling acts to suppress cardiomyocyte plasticity and to preserve the integrity of the ventricular chamber.

Telomeres are repetitive nucleoprotein structures that cap the ends of chromosomes. Without telomerase, telomeres shorten with replication and eventually signal cell cycle arrest (cell senescence). Homologous recombination (HR)-based mechanisms slow senescence, and distinct HR mechanisms support the growth of the rare survivors of senescence. Here, we report novel roles for the post-translational modification of small ubiquitin-like modifier (SUMO) in regulating the rate of senescence in Saccharomyces cerevisiae telomerase mutants. We identify Mms21 as the relevant SUMO E3 ligase and demonstrate that cells lacking Mms21-dependent sumoylation accumulate HR intermediates selectively at telomeres during senescence. One target of Mms21-dependent sumoylation is the cohesin-and condensin-related Smc5-Smc6 complex (Smc5/6). We show that hypomorphic smc5 or smc6 alleles exhibit phenotypes similar to mms21 sumoylation-deficient mutants with regard to senescence and the accumulation of unresolved HR intermediates. Further, we provide evidence that Mms21 and Smc5/6 prevent aberrant recombination between sister telomeres and also globally facilitate resolution of sister chromatid HR intermediates.Telomeres comprise the repeated DNA sequences and associated proteins that protect the ends of linear chromosomes. Telomeres can be elongated via the ribonucleoprotein telomerase, which promotes de novo telomere addition and, by doing so, allows cells to combat the loss of terminal sequences that occur during telomere replication and processing. If telomerase is down-regulated or genetically inactivated, telomere length decreases with each cell division until a critical length is reached that signals cell cycle arrest, termed replicative senescence (1, 2). The yeast Saccharomyces cerevisiae expresses telomerase constitutively, but the genetic inactivation of telomerase (e.g. tlc1⌬ mutants, which lack the telomerase RNA template) causes telomere loss leading to senescence (3, 4). Homologous recombination (HR)2 -dependent mechanisms slow senescence and also enable the emergence of rare telomerase-independent "survivors" of senescence (4 -7). A mechanism of telomere maintenance similar to that used by yeast survivors is seen in a small but significant subset of human cancers termed ALT (for alternative lengthening of telomeres) (8 -10). Examination of ALT cells has revealed that the mammalian homologues of several of the proteins first identified as important in S. cerevisiae telomerase-independent survivors appear to function in the mammalian ALT mechanism, making S. cerevisiae an attractive model for studying HR-based telomere maintenance (5, 11-15). In yeast, although the HR mechanisms that slow senescence and those that support survivor growth use shared factors (e.g. Rad52), the mechanisms are nonetheless distinct. For example, survivors use a breakinduced replication mechanism that depends on Pol32 (a nonessential subunit of DNA polymerase ␦) and that involves intertelomere recombination, but pol32 deletion does not affect the r...

is the most commonly mutated gene associated with human congenital heart defects (CHDs), with a predilection for cardiac pole abnormalities. This homeodomain transcription factor is a central regulator of cardiac development and is expressed in both the first and second heart fields (FHF and SHF). We have previously revealed essential functions of and, two homologs expressed in zebrafish cardiomyocytes, in maintaining ventricular identity. However, the differential roles of these genes in the specific subpopulations of the anterior (aSHF) and posterior (pSHF) SHFs have yet to be fully defined. Here, we show that Nkx genes regulate aSHF and pSHF progenitors through independent mechanisms. We demonstrate that Nkx genes restrict proliferation of aSHF progenitors in the outflow tract, delimit the number of pSHF progenitors at the venous pole and pattern the sinoatrial node acting through Isl1 repression. Moreover, optical mapping highlights the requirement for Nkx gene dose in establishing electrophysiological chamber identity and in integrating the physiological connectivity of FHF and SHF cardiomyocytes. Ultimately, our results may shed light on the discrete errors responsible for-dependent human CHDs of the cardiac outflow and inflow tracts.

In A. frigida Willd., B-chromosomes are reported for the first time. Their number was limited to 2 in all the cells studied. Analysis of chromosome behaviour at meiosis showed that B-chromosomes reduced the mean chiasma frequency and modified the phenotypic and biochemical characteristics of the plants.

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