Yumiko Saga

An Nkx2-5/Bmp2/Smad1 Negative Feedback Loop Controls Heart Progenitor Specification and Proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice

We previously reported that primordial germ cells (PGCs) in mice erase genome-wide DNA methylation and histone H3 lysine9 dimethylation (H3K9me2), and instead acquire high levels of tri-methylation of H3K27 (H3K27me3) during their migration, a process that might be crucial for the re-establishment of potential totipotency in the germline. We here explored a cellular dynamics associated with this epigenetic reprogramming. We found that PGCs undergo erasure of H3K9me2 and upregulation of H3K27me3 in a progressive, cell-by-cell manner, presumably depending on their developmental maturation. Before or concomitant with the onset of H3K9 demethylation, PGCs entered the G2 arrest of the cell cycle, which apparently persisted until they acquired high H3K27me3 levels. Interestingly, PGCs exhibited repression of RNA polymerase II-dependent transcription, which began after the onset of H3K9me2 reduction in the G2 phase and tapered off after the acquisition of high-level H3K27me3. The epigenetic reprogramming and transcriptional quiescence were independent from the function of Nanos3. We found that before H3K9 demethylation, PGCs exclusively repress an essential histone methyltransferase, GLP, without specifically upregulating histone demethylases. We suggest the possibility that active repression of an essential enzyme and subsequent unique cellular dynamics ensures successful implementation of genome-wide epigenetic reprogramming in migrating PGCs.

The making of the somite: molecular events in vertebrate segmentation

The reiterated structures of the vertebrate axial skeleton, spinal nervous system and body muscle are based on the metameric structure of somites, which are formed in a dynamic morphogenetic process. Somite segmentation requires the activity of a biochemical oscillator known as the somite-segmentation clock. Although the molecular identity of the clock remains unknown, genetic and experimental evidence has accumulated that indicates how the periodicity of somite formation is generated, how the positions of segment borders are determined, and how the rostrocaudal polarity within somite primordia is generated.

The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity

The serially segmented (metameric) structures of vertebrates are based on somites that are periodically formed during embryogenesis. A ‘clock and wavefront’ model has been proposed to explain the underlying mechanism of somite formation, in which the periodicity is generated by oscillation of Notch components (the clock) in the posterior pre-somitic mesoderm (PSM). This temporal periodicity is then translated into the segmental units in the ‘wavefront’. The wavefront is thought to exist in the anterior PSM and progress backwards at a constant rate; however, there has been no direct evidence as to whether the levels of Notch activity really oscillate and how such oscillation is translated into a segmental pattern in the anterior PSM. Here, we have visualized endogenous levels of Notch1 activity in mice, showing that it oscillates in the posterior PSM but is arrested in the anterior PSM. Somite boundaries formed at the interface between Notch1-activated and -repressed domains. Genetic and biochemical studies indicate that this interface is generated by suppression of Notch activity by mesoderm posterior 2 (Mesp2) through induction of the lunatic fringe gene (Lfng). We propose that the oscillation of Notch activity is arrested and translated in the wavefront by Mesp2.

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.

Distinct roles of Wnt/β-catenin and Bmp signaling during early cardiogenesis

Heart formation requires the coordinated recruitment of multiple cardiac progenitor cell populations derived from both the first and second heart fields. In this study, we have ablated the Bmp receptor 1a and the Wnt effector β-catenin in the developing heart of mice by using MesP1-cre, which acts in early mesoderm progenitors that contribute to both first and second heart fields. Remarkably, the entire cardiac crescent and later the primitive ventricle were absent in MesP1-cre; BmpR1alox/lox mutants. Although myocardial progenitor markers such as Nkx2-5 and Isl1 and the differentiation marker MLC2a were detected in the small, remaining cardiac field in these mutants, the first heart field markers, eHand and Tbx-5, were not expressed. We conclude from these results that Bmp receptor signaling is crucial for the specification of the first heart field. In MesP1-cre; β-cateninlox/lox mutants, cardiac crescent formation, as well as first heart field markers, were not affected, although cardiac looping and right ventricle formation were blocked. Expression of Isl1 and Bmp4 in second heart field progenitors was strongly reduced. In contrast, in a gain-of-function mutation of β-catenin using MesP1-cre, we revealed an expansion of Isl1 and Bmp4 expressing cells, although the heart tube was not formed. We conclude from these results that Wnt/β-catenin signaling regulates second heart-field development, and that a precise amount and/or timing of Wnt/β-catenin signaling is required for proper heart tube formation and cardiac looping.

Mesp1 Expression Is the Earliest Sign of Cardiovascular Development

Understanding the molecular mechanism leading to formation of the heart and vasculature during embryogenesis is critically important because malformation of the cardiovascular system is the most frequently occurring type of birth defect. While the hearts of all vertebrates are derived from bilateral paired fields of primary mesodermal cells that are specified to the cardiac lineage during gastrulation, the mechanism for lineage restriction, and the origin of the myocardium and endocardium have not been defined. Recently, we found that a transcription factor, Mesp1, is expressed in almost all precursors of the cardiovascular system and plays an essential role in cardiac morphogenesis. Mesp1 may play a key role in the early specification for cardiac precursor cells.

The RNA-Binding Protein NANOS2 Is Required to Maintain Murine Spermatogonial Stem Cells

Maintaining Germline Stem Cells Spermatogonial stem cell pools in postnatal testes have to be maintained to continuously generate spermatozoa. It has been difficult to identify these stem cells in vivo, because of their small numbers and lack of appropriate molecular markers, but now Sada et al. (p. 1394) show that the RNA-binding protein NANOS2 is expressed in a small subset of spermatogonia that behave as self-renewing stem cells in intact testes. By a combinatorial use of loss- and gain-of-function studies, NANOS2 was found to be essential for the maintenance of the immature state of spermatogonial stem cells by supporting their self-renewing properties and by suppressing differentiation. Cell lineage tracing reveals the factor that preserves stem cells in the undifferentiated state in the mouse male germ line. Stem cells give rise to differentiated cell types but also preserve their undifferentiated state through cell self-renewal. With the use of transgenic mice, we found that the RNA-binding protein NANOS2 is essential for maintaining spermatogonial stem cells. Lineage-tracing analyses revealed that undifferentiated spermatogonia expressing Nanos2 self-renew and generate the entire spermatogenic cell lineage. Conditional disruption of postnatal Nanos2 depleted spermatogonial stem cell reserves, whereas mouse testes in which Nanos2 had been overexpressed accumulated spermatogonia with undifferentiated, stem cell–like properties. Thus, NANOS2 is a key stem cell regulator that is expressed in self-renewing spermatogonial stem cells and maintains the stem cell state during murine spermatogenesis.

Mesp2 initiates somite segmentation through the Notch signalling pathway

The Notch-signalling pathway is important in establishing metameric pattern during somitogenesis. In mice, the lack of either of two molecules involved in the Notch-signalling pathway, Mesp2 or presenilin-1 (Ps1), results in contrasting phenotypes: caudalized versus rostralized vertebra. Here we adopt a genetic approach to analyse the molecular mechanism underlying the establishment of rostro-caudal polarity in somites. By focusing on the fact that expression of a Notch ligand, Dll1, is important for prefiguring somite identity, we found that Mesp2 initiates establishment of rostro-caudal polarity by controlling two Notch-signalling pathways. Initially, Mesp2 activates a Ps1-independent Notch-signalling cascade to suppress Dll1 expression and specify the rostral half of the somite. Ps1-mediated Notch-signalling is required to induce Dll1 expression in the caudal half of the somite. Therefore, Mesp2- and Ps1-dependent activation of Notch-signalling pathways might differentially regulate Dll1 expression, resulting in the establishment of the rostro-caudal polarity of somites.

Nanos2 suppresses meiosis and promotes male germ cell differentiation

In mouse fetal gonads, retinoic acid (RA) induces meiosis in the female germ cells, whereas the male germ cells never enter meiosis due to Cyp26b1-mediated RA metabolism. We show here that Nanos2 plays critical roles in the differentiation of male germ cells. We find that Nanos2 maintains the suppression of meiosis by preventing Stra8 expression, which is required for premeiotic DNA replication, after Cyp26b1 is decreased. We also demonstrate that Nanos2 activates a male-specific genetic program, which is supported by the inhibition of meiosis and the induction of male-type differentiation in female germ cells following the forced expression of Nanos2.