Author Correction: Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals.

The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function. Alterations in developmental pathways have been associated with declines in stem-cell function during ageing, but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing. Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFß, JAK/STAT and senescence signalling. Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

Analyses of fugu hoxa2 genes provide evidence for subfunctionalization of neural crest cell and rhombomere cis-regulatory modules during vertebrate evolution.

Hoxa2 gene is a primary player in regulation of craniofacial programs of head development in vertebrates. Here we investigate the evolution of a Hoxa2 neural crest enhancer identified originally in mouse by comparing and contrasting the fugu hoxa2a and hoxa2b genes with their orthologous teleost and mammalian sequences. Using sequence analyses in combination with transgenic regulatory assays in zebrafish and mouse embryos we demonstrate subfunctionalization of regulatory activity for expression in hindbrain segments and neural crest cells between these two fugu co-orthologs. hoxa2a regulatory sequences have retained the ability to mediate expression in neural crest cells while those of hoxa2b include cis-elements that direct expression in rhombomeres. Functional dissection of the neural crest regulatory potential of the fugu hoxa2a and hoxa2b genes identify the previously unknown cis-element NC5, which is implicated in generating the differential activity of the enhancers from these genes. The NC5 region plays a similar role in the ability of this enhancer to mediate reporter expression in mice, suggesting it is a conserved component involved in control of neural crest expression of Hoxa2 in vertebrate craniofacial development.

no tail integrates two modes of mesoderm induction.

During early zebrafish development the nodal signalling pathway patterns the embryo into three germ layers, in part by inducing the expression of no tail (ntl), which is essential for correct mesoderm formation. When nodal signalling is inhibited ntl fails to be expressed in the dorsal margin, but ventral ntl expression is unaffected. These observations indicate that ntl transcription is under both nodal-dependent and nodal-independent regulation. Consistent with these observations and with a role for ntl in mesoderm formation, some somites form within the tail region of embryos lacking nodal signalling. In an effort to understand how ntl is regulated and thus how mesoderm forms, we have mapped the elements responsible for nodal-dependent and nodal-independent expression of ntl in the margin of the embryo. Our work demonstrates that expression of ntl in the margin is the consequence of two separate enhancers, which act to mediate different mechanisms of mesoderm formation. One of these enhancers responds to nodal signalling, and the other to Wnt and BMP signalling. We demonstrate that the nodal-independent regulation of ntl is essential for tail formation. Misexpression of Wnt and BMP ligands can induce the formation of an ectopic tail, which contains somites, in embryos devoid of nodal signalling, and this tail formation is dependent on ntl function. Similarly, nodal-independent tail somite formation requires ntl. At later stages in development ntl is required for notochord formation, and our analysis has also led to the identification of the enhancer required for ntl expression in the developing notochord.

A regulatory module embedded in the coding region of Hoxa2 controls expression in rhombomere 2.

Here, we define a gene regulatory network for Hoxa2, responsible for temporal and spatial expression in hindbrain development. Hoxa2 plays an important role in regulating the regional identity of rhombomere 2 (r2) and is the only Hox gene expressed in this segment. In this study, we found that a Hoxa2 cis-regulatory module consists of five elements that direct expression in r2 of the developing hindbrain. Surprisingly, the module is imbedded in the second coding exon of Hoxa2 and therefore may be constrained by both protein coding and gene regulatory requirements. This highly conserved enhancer consists of two consensus Sox binding sites and several additional elements that act in concert to direct strong r2 specific expression. Our findings provide important insight into the regulation of segmental identity in the anterior hindbrain. Furthermore, they have broader implications in designing arrays and interpreting data from global analyses of gene regulation because regulatory input from coding regions needs to be considered.

Quiescence: Good and Bad of Stem Cell Aging.

Stem cells are required for lifelong homeostasis and regeneration of tissues and organs in mammals, but the function of stem cells declines during aging. To preserve stem cells during life, they are kept in a quiescent state with low metabolic and low proliferative activity. However, activation of quiescent stem cells - an essential process for organ homeostasis/regeneration - requires concerted and faithful regulation of multiple molecular circuits controlling biosynthetic processes, repair mechanisms, and metabolic activity. Thus, while protecting stem cell maintenance, quiescence comes at the cost of vulnerability during the process of stem cell activation. Here we discuss molecular and biochemical processes regulating stem cells' maintenance in and exit from quiescence and how age-related failures of these circuits can contribute to organism aging.

Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites.

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.

A Boolean network of the crosstalk between IGF and Wnt signaling in aging satellite cells.

Aging is a complex biological process, which determines the life span of an organism. Insulin-like growth factor (IGF) and Wnt signaling pathways govern the process of aging. Both pathways share common downstream targets that allow competitive crosstalk between these branches. Of note, a shift from IGF to Wnt signaling has been observed during aging of satellite cells. Biological regulatory networks necessary to recreate aging have not yet been discovered. Here, we established a mathematical in silico model that robustly recapitulates the crosstalk between IGF and Wnt signaling. Strikingly, it predicts critical nodes following a shift from IGF to Wnt signaling. These findings indicate that this shift might cause age-related diseases.

Aging stem cells: transcriptome meets epigenome meets methylome.

Aging is associated with impairments in hematopoietic stem cell (HSC) function and an increased risk of leukemogenesis. In this issue of Cell Stem Cell, Sun et al. (2014) use highly purified HSCs along with an integrated genomic approach to evaluate aging-associated alterations in the epigenome and transcriptome of HSCs.

The role of telomere shortening in somatic stem cells and tissue aging: lessons from telomerase model systems.

The analysis of model systems has broadened our understanding of telomere-related aging processes. Telomerase-deficient mouse models have demonstrated that telomere dysfunction impairs tissue renewal capacity and shortens lifespan. Telomere shortening limits cell proliferation by activating checkpoints that induce replicative senescence or apoptosis. These checkpoints protect against an accumulation of genomically instable cells and cancer initiation. However, the induction of these checkpoints can also limit organ homeostasis, regeneration, and survival during aging and in the context of diseases. The decline in tissue regeneration in response to telomere shortening has been related to impairments in stem cell function. Telomere dysfunction impairs stem cell function by activation of cell-intrinsic checkpoints and by the induction of alterations in the micro- and macro-environment of stem cells. In this review, we discuss the current knowledge about the impact of telomere shortening on disease stages induced by replicative cell aging as indicated by studies on telomerase model systems.

Protein kinase D2 is an essential regulator of murine myoblast differentiation.

Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC) family. The protein kinase D (PKD) isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12) as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

Hox genes and segmentation of the vertebrate hindbrain.

In the vertebrate central nervous system, the hindbrain is an important center for coordinating motor activity, posture, equilibrium, sleep patterns, and essential unconscious functions, such as breathing rhythms and blood circulation. During development, the vertebrate hindbrain depends upon the process of segmentation or compartmentalization to create and organize regional properties essential for orchestrating its highly conserved functional roles. The process of segmentation in the hindbrain differs from that which functions in the paraxial mesoderm to generate somites and the axial skeleton. In the prospective hindbrain, cells in the neural epithelia transiently alter their ability to interact with their neighbors, resulting in the formation of seven lineage-restricted cellular compartments. These different segments or rhombomeres each go on to adopt unique characters in response to environmental signals. The Hox family of transcription factors is coupled to this process. Overlapping or nested patterns of Hox gene expression correlate with segmental domains and provide a combinatorial code and molecular framework for specifying the unique identities of hindbrain segments. The segmental organization and patterns of Hox expression and function are highly conserved among vertebrates and, as a consequence, comparative studies between different species have greatly enhanced our ability to build a picture of the regulatory cascades that control early hindbrain development. The purpose of this chapter is to review what is known about the regulatory mechanisms which establish and maintain Hox gene expression and function in hindbrain development.

Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1.

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.

Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish (Takifugu rubripes).

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.

Direct crossregulation between retinoic acid receptor {beta} and Hox genes during hindbrain segmentation.

During anteroposterior (AP) patterning of the developing hindbrain, the expression borders of many transcription factors are aligned at interfaces between neural segments called rhombomeres (r). Mechanisms regulating segmental expression have been identified for Hox genes, but for other classes of AP patterning genes there is only limited information. We have analysed the murine retinoic acid receptor beta gene (Rarb) and show that it is induced prior to segmentation, by retinoic-acid (RA) signalling from the mesoderm. Induction establishes a diffuse expression border that regresses until, at later stages, it is stably maintained at the r6/r7 boundary by inputs from Hoxb4 and Hoxd4. Separate RA- and Hox-responsive enhancers mediate the two phases of Rarb expression: a regulatory mechanism remarkably similar to that of Hoxb4. By showing that Rarb is a direct transcriptional target of Hoxb4, this study identifies a new molecular link, completing a feedback circuit between Rarb, Hoxb4 and Hoxd4. We propose that the function of this circuit is to align the initially incongruent expression of multiple RA-induced genes at a single segment boundary.

Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates.

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components.

Regulation of Tbx3 expression by anteroposterior signalling in vertebrate limb development.

Tbx3, a T-box gene family member related to the Drosophila gene optomotor blind (omb) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar-mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarising region which controls anteroposterior pattern. Here we carried out tissue transplantations, grafted beads soaked in Shh, Bmps, and Noggin in chick limb buds, and analysed Tbx3 expression. We also investigated Tbx3 expression in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. We show that Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centred on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. Our results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number.