quantitative-whole-mount-immunofluorescence-analysis-cardiac

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Icahn School of Medicine at Mount Sinai

Chromatin regulators have recently emerged as key players in the control of tissue development and tumorigenesis. One specific chromatin regulator, the Polycomb complex, has been shown to regulate the identity of embryonic stem cells, but its role in controlling fates of multipotent progenitors in developing tissues is still largely unknown. Recent findings have revealed that this complex plays a critical role in control of skin stem cell renewal and differentiation. Moreover, the expression of Polycomb complex components is often aberrant in skin diseases, including skin cancers. This review will detail recent findings on Polycomb control of skin and highlight critical unknown questions.

Epigenetic regulation of skin: focus on the Polycomb complex.

Chromatin regulatory complexes are well known regulators of stem cell fate; however, the mechanisms regulating their activity are not well understood. In this issue of Cell Stem Cell, Bao et al. (2013) show that ACTL6a inhibits targeting of the SWI/SNF complex to differentiation genes, thereby preserving the epidermal progenitor state.

SWItching on epidermal cell fate.

While the Polycomb complex is known to regulate cell identity in ES cells, its role in controlling tissue-specific stem cells is not well understood. Here we show that removal of Ezh1 and Ezh2, key Polycomb subunits, from mouse skin results in a marked change in fate determination in epidermal progenitor cells, leading to an increase in the number of lineage-committed Merkel cells, a specialized subtype of skin cells involved in mechanotransduction. By dissecting the genetic mechanism, we showed that the Polycomb complex restricts differentiation of epidermal progenitor cells by repressing the transcription factor Sox2. Ablation of Sox2 results in a dramatic loss of Merkel cells, indicating that Sox2 is a critical regulator of Merkel cell specification. We show that Sox2 directly activates Atoh1, the obligate regulator of Merkel cell differentiation. Concordantly, ablation of Sox2 attenuated the Ezh1/2-null phenotype, confirming the importance of Polycomb-mediated repression of Sox2 in maintaining the epidermal progenitor cell state. Together, these findings define a novel regulatory network by which the Polycomb complex maintains the progenitor cell state and governs differentiation in vivo.

Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.

In a cell, the chromatin state is controlled by the highly regulated interplay of epigenetic mechanisms ranging from DNA methylation and incorporation of different histone variants to posttranslational modification of histones and ATP-dependent chromatin remodeling. These changes alter the structure of the chromatin to either facilitate or restrict the access of transcription machinery to DNA. These epigenetic modifications function to exquisitely orchestrate the expression of different genes, and together constitute the epigenome of a cell. In the skin, different epigenetic regulators form a regulatory network that operates to guarantee skin stem cell maintenance while controlling differentiation to multiple skin structures. In this review, we will discuss recent findings on epigenetic mechanisms of skin control and their relationship to skin pathologies.

Epigenetic regulation of epidermal differentiation.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor β (TGF-β) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-β. Mechanistically, we showed that after TGF-β exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-β/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor β in mammary epithelial cells.

Merkel cell-neurite complexes are located in touch-sensitive areas of the mammalian skin and are involved in recognition of the texture and shape of objects. Merkel cells are essential for these tactile discriminations, as they generate action potentials in response to touch stimuli and induce the firing of innervating afferent nerves. It has been shown that Merkel cells originate from epidermal stem cells, but the cellular and molecular mechanisms of their development are largely unknown. In this study, we analyzed Merkel cell differentiation during development and found that it is a temporally regulated maturation process characterized by a sequential activation of Merkel cell-specific genes. We uncovered key transcription factors controlling this process and showed that the transcription factor Atoh1 is required for initial Merkel cell specification. The subsequent maturation steps of Merkel cell differentiation are controlled by cooperative function of the transcription factors Sox2 and Isl1, which physically interact and work to sustain Atoh1 expression. These findings reveal the presence of a robust transcriptional network required to produce functional Merkel cells that are required for tactile discrimination.

Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network.

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning.

RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins.

Scientists have studied organs and their development for centuries and, along that path, described models and mechanisms explaining the developmental principles of organogenesis. In particular, with respect to the heart, new fundamental discoveries are reported continuously that keep changing the way we think about early cardiac development. These discoveries are driven by the need to answer long-standing questions regarding the origin of the earliest cells specified to the cardiac lineage, the differentiation potential of distinct cardiac progenitor cells, and, very importantly, the molecular mechanisms underlying these specification events. As evidenced by numerous examples, the wealth of developmental knowledge collected over the years has had an invaluable impact on establishing efficient strategies to generate cardiovascular cell types ex vivo, from either pluripotent stem cells or via direct reprogramming approaches. The ability to generate functional cardiovascular cells in an efficient and reliable manner will contribute to therapeutic strategies aimed at alleviating the increasing burden of cardiovascular disease and morbidity. Here we will discuss the recent discoveries in the field of cardiac progenitor biology and their translation to the pluripotent stem cell model to illustrate how developmental concepts have instructed regenerative model systems in the past and promise to do so in the future. Developmental Dynamics 245:1130-1144, 2016. © 2016 Wiley Periodicals, Inc.

Probing early heart development to instruct stem cell differentiation strategies.

The recent identification of progenitor populations that contribute to the developing heart in a distinct spatial and temporal manner has fundamentally improved our understanding of cardiac development. However, the mechanisms that direct atrial versus ventricular specification remain largely unknown. Here we report the identification of a progenitor population that gives rise primarily to cardiovascular cells of the ventricles and only to few atrial cells (<5%) of the differentiated heart. These progenitors are specified during gastrulation, when they transiently express Foxa2, a gene not previously implicated in cardiac development. Importantly, Foxa2+ cells contribute to previously identified progenitor populations in a defined pattern and ratio. Lastly, we describe an analogous Foxa2+ population during differentiation of embryonic stem cells. Together, these findings provide insight into the developmental origin of ventricular and atrial cells, and may lead to the establishment of new strategies for generating chamber-specific cell types from pluripotent stem cells.

Article	Year
Epigenetic regulation of skin: focus on the Polycomb complex. Cellular and molecular life sciences : CMLS\| PubMed ID: 22314499	2012
SWItching on epidermal cell fate. Cell stem cell\| PubMed ID: 23395438	2013
Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells. The EMBO journal\| PubMed ID: 23673358	2013
Epigenetic regulation of epidermal differentiation. Cold Spring Harbor perspectives in medicine\| PubMed ID: 24492849	2014
The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor β in mammary epithelial cells. Molecular and cellular biology\| PubMed ID: 25266660	2014
Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network. Development (Cambridge, England)\| PubMed ID: 25468937	2014
RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins. Development (Cambridge, England)\| PubMed ID: 26755701	2016
Probing early heart development to instruct stem cell differentiation strategies. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 27580352	2016
Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential. Nature communications\| PubMed ID: 28195173	2017

Evan Bardot

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