Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Aug 18.
Published in final edited form as: J Invest Dermatol. 2013 Feb 13;133(9):2191–2201. doi: 10.1038/jid.2013.66

Remodelling of three-dimensional organization of the nucleus during terminal keratinocyte differentiation in the epidermis

Michal R Gdula 1,3, Krzysztof Poterlowicz 1, Andrei N Mardaryev 1, Andrey A Sharov 3, Y Peng 1,2, Michael Y Fessing 1, Vladimir A Botchkarev 1,3
PMCID: PMC4135477  NIHMSID: NIHMS480976  PMID: 23407401

Abstract

The nucleus of epidermal keratinocytes is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multi-color confocal microscopy, followed by computational image analysis and mathematical modelling, we demonstrate that in normal mouse foot-pad epidermis transition of keratinocytes from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and micro-environment including: i) decrease of the nuclear volume, ii) decrease in expression of the markers of transcriptionally-active chromatin; iii) internalization and decrease in the number of nucleoli; iv) increase in the number of pericentromeric heterochromatic clusters; v) increase in the frequency of associations between pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear micro-environment required for proper execution of gene expression programs in differentiating keratinocytes and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions including disorders of epidermal differentiation and epidermal tumors.

Introduction

The cell nucleus is a highly complex organelle that consists of the nuclear membrane, individual chromosomes occupying distinct territories, as well as nuclear bodies (nucleoli, Cajal bodies, promyelocytic leukaemia (PML) bodies, nuclear speckles, Polycomb bodies, etc.) located in inter- and intra-chromosomal compartments (Hubner and Spector, 2010; Lanctot et al., 2007). Genetic material (DNA) in the nucleus is compacted up to several thousand fold and organized into chromosomes as a complex with histones and non-histone proteins that allows the genome to be replicated, transcribed and repaired (Hemberger et al., 2009; Ho and Crabtree, 2010). The nucleus also involves in a number of other functions, including the RNA processing, ribosome assembly, and maintenance of the mechanical integrity of the cell (Rippe, 2007; Rowat et al., 2008).

Three-dimensional organization of the nucleus and spatial compartmentalization of the distinct chromatin domains, nuclear bodies and macro-molecular protein complexes are dynamic and show remarkable changes during cell differentiation (reviewed in Hubner and Spector, 2010; Joffe et al., 2010; Schoenfelder et al., 2010). Activation and silencing of distinct genomic loci during cell differentiation is often accompanied by changes in their positioning relatively to the corresponding chromosomal territories and other nuclear compartments, as well as by changes in the morphology and number of nuclear bodies (Deniaud and Bickmore, 2009). Despite a certain similarity in the nuclear architecture between differentiated versus un-differentiated cells, spatial organization of the genome in the nucleus appears to be unique for each cell type and, together with distinct patterns of nucleosome positioning, DNA methylation and histone modifications, constitute an “epigenetic signature” of the cell (Naumova and Dekker, 2010).

In the epidermis, lineage-committed progenitor cells reside in the basal layer, where they proliferate and differentiate into cells of the suprabasal layers, forming the stratified epithelium (Blanpain and Fuchs, 2009; Fuchs, 2007; Koster and Roop, 2007). Transition of the progenitor cells into keratinocytes of suprabasal layers occurs via asymmetric cell division (Lechler and Fuchs, 2005; Poulson and Lechler, 2010). The process of terminal differentiation in epidermal keratinocytes is accompanied by coordinated activation and silencing of the sets of genes including those that constitute several lineage-specific loci (keratin type I and II loci, the epidermal differentiation complex or EDC, etc.), and encode essential structural components of the epidermal barrier (Bazzi et al., 2007; Segre, 2006).

During terminal differentiation of the epidermal keratinocytes, the nucleus undergoes programmed structural and biochemical changes during transition from a highly active status, associated with execution of the genetic programs of epidermal barrier formation, to a fully inactive condition and finally becomes a part of the keratinized cells of the cornified epidermal layer (Botchkarev et al., 2012). Earlier studies demonstrated that keratinocyte transition between the distinct epidermal layers is accompanied by marked morphological and ultrastructural changes in the nucleus including its size, shape, structure of nucleoli, etc. (Breathnach, 1971; Karasek et al., 1972; Tsuji and Cox, 1977). Because spatial organization of the distinct genomic loci and nuclear bodies are critical for the proper regulation of gene expression (Dundr and Misteli, 2010; Schoenfelder et al., 2010), changes in the size and shape of the keratinocyte nucleus associated with terminal differentiation might, on the one hand, influence 3D-genome structure and gene expression programs in differentiating cells, and, on the other hand, reflect re-organization of these programs accompanied by the differentiation-associated accumulation or loss of the distinct cytoplasmic components, such as keratin 14, interacting with nuclear envelope and regulating nuclear shape (Lee et al., 2012).

Recent studies demonstrate that spatial genome organization in keratinocytes is intimately linked to the regulation of gene expression, and that the higher–order chromatin remodeler Satb1, serving as a direct target for p63 transcription factor, controls a chromatin folding of the EDC and gene expression during epidermal keratinocyte differentiation in vivo (Fessing et al., 2011). Furthermore, 3D-genome organization also changes during keratinocyte differentiation in vitro, including the position of selected chromosomes and EDC locus relatively to the corresponding chromosome territory (Marella et al., 2009; Williams et al., 2002).

However, despite an increasing amount of information on epigenetic mechanisms controlling gene expression in keratinocytes (reviewed in Botchkarev et al., 2012; Eckert et al., 2011; Wang and Chang, 2011; Zhang et al., 2012), a systematic analysis of the remodelling of 3D organization of the nucleus during terminal keratinocyte differentiation in the epidermis in situ have not been done yet. Also, the extent to which nuclear architecture shows preferential changes at defined stages of cell differentiation process in the epidermis in vivo still remains unclear.

Using multi-colour confocal microscopy, 3D image analysis and mathematical modelling of the nucleus, we describe here the remodelling of the nuclear architecture during terminal differentiation of the normal mouse epidermal keratinocytes in vivo. Our results reveal significant changes in multiple parameters of 3D genome organization in keratinocytes during transition from the basal to the spinous and granular epidermal layers, including changes in spatial associations between the pericentromeric heterochromatin domains, nucleoli and chromosomal territory 3 bearing the EDC locus. We summarize these data as a model suggesting that the establishment and silencing of differentiation-associated gene expression programs in epidermal keratinocytes in vivo involves marked changes in their nuclear architecture and 3D genome organization.

Results

Terminal keratinocyte differentiation in the epidermis is accompanied by changes in the nuclear volume and shape

To define the changes in three-dimensional organization of the keratinocyte nucleus during terminal differentiation in the epidermis in situ, cryosections of the 10 day-old mouse foot pads were stained with DAPI and analyzed using confocal microscopy followed by the image analysis and 3D computational reconstruction (Figure S1). Nuclei of the spinous and granular layer cells were determined after immunostaining of the skin cryo-sections with antibodies against Keratin 10 or Loricrin, respectively (Figure S2). Murine foot pad epidermis was purposely selected for this study, because it, at least in part, resembles morphologically the human palmo-plantar epidermis and, in contrast to other post-natal mouse skin areas, consists of several well-defined layers of keratinocytes (Loomis et al., 1996). Moreover, in newborn mice, basal layer of the foot pad epidermis does not contain melanocytes and represents a more homogenous population of epidermal progenitor cells in comparison to the trunk skin (Kunisada et al., 1998; Plikus et al., 2004).

3D reconstructions based on the image stack analyses, collected using confocal microscopy of 80 μm-thick cryosections of the foot pad epidermis, confirmed the previously reported observations on changes in the orientation of the long axis of the nucleus from vertical to horizontal during transition of keratinocytes from the basal to suprabasal layer of the epidermis (Figure 1a) (Lechler and Fuchs, 2005; Lee et al., 2012; Loomis, 2001; Rowden, 1975). Analysis of the ratios between the long axis and the average of two shorter axes of the ellipsoid nuclei showed that keratinocyte nuclei in the basal and granular epidermal layers were significantly more elongated compared to those in the spinous layer, where nuclei were more round-shaped (Figure 1b).

Figure 1. Remodelling of the nuclei during terminal keratinocyte differentiation.

Figure 1

Open in a new tab

(a) 3D reconstruction of nuclei in the mouse footpad based on confocal microscopy of the tissue samples stained with marker of proliferating cells (antibody against Ki67) and DAPI. (b) Changes in nuclear shape measured as the ratio of long axis to the average of two shorter axes during keratinocyte differentiation. (c) Keratinocyte nuclear volume in the different epidermal layers. Data in b and c represent means with 95% confidence interval; 30 nuclei in every group were measured; p-values: * <0.05, **<0.01, ***<0.001.

To assess changes in the nuclear volume among keratinocytes of different epidermal layers, skin was immuno-stained with antibody against proliferative marker Ki-67, and nuclear volume was assessed separately in proliferating and non-proliferating cells. Ki-67 positive cells were seen almost exclusively in the basal epidermal layer (Botchkarev et al., 1999; Lechler and Fuchs, 2005; Sharov et al., 2003), and showed significantly larger nuclear volume compared to nuclei of non-proliferative cells (Figure 1a,c). Both proliferative and non-proliferative basal cells showed significantly larger nuclear volume compared to cells of the spinous or the granular epidermal layers (Figure 1c). These data demonstrate that, in addition to the changes in the orientation and shape of the nucleus, terminal differentiation of the epidermal keratinocytes was accompanied by significant decrease of the nuclear volume.

Markers of transcriptionally-active chromatin are decreased during epidermal keratinocyte differentiation

To check whether changes in the nuclear volume and shape during epidermal keratinocyte differentiation are accompanied by the remodelling of nuclear architecture and re-distribution of the transcriptionally-active chromatin domains, we performed immuno-stainings with antibodies against the corresponding markers, such as the elongating form of RNA polymerase II phosphorylated at serine 2 of the C-terminal domain of its large subunit (pSer2-Pol II), histone H3 trimethylated at lysine 4 (H3K4me3), and histone H3 acetylated at lysine 56 (H3K56ac) (Zhou et al., 2011).

In basal and spinous epidermal cells, all three markers showed highest abundance (Figure 2a-c, Figure S3). However, levels of the pSer2-Pol II, H3K4me3 and H3K56ac were significantly decreased in the granular layer compared to keratinocytes of the basal and spinous layers (Figure 2a-c, Figure S3). These data suggest that global transcription levels are markedly decreased in terminally differentiated keratinocytes of granular layer compared to keratinocytes of the basal and spinous epidermal layers.

Figure 2. Remodelling of transcriptionally active chromatin domains and nucleoli during terminal keratinocyte differentiation.

Figure 2

Open in a new tab

Enrichment of the markers of actively transcribed chromatin in the basal and spinous layers and decrease of their level in the granular layer: phospho-serine-2 RNA Pol II (elongating form) (a), H3K4me3 (b), H3K56ac (c). (d) Representative 200 nm single optical section of the epidermis stained with antibody against nucleolar marker nucleophosmin/B23. Epidermal/dermal border is indicated by dotted line. (e) Representative nuclei from the distinct epidermal layers (left) and geometrical models of the average keratinocyte nuclei form distinct epidermal layers generated on the basis of 3D-reconstruction of the 70-80 confocal stacks of the whole nuclei (right). Decrease in number of nucleoli (f), change in individual and total nucleolar volume (g) and in radial positions of the nucleoli (h) during terminal keratinocyte differentiation. Data in c, d and e represent means with 95% confidence interval; 30 nuclei in every group were measured, p-values: * <0.05, **<0.01, ***<0.001.Note that in geometrical models of the average keratinocyte nuclei (e), orientations of the nuclei from distinct epidermal layers were purportedly changed to provide a better visual appreciation of the topological differences between them. Scale bars = 10 um.

Nucleoli are repositioned towards the nuclear interior and decreased in number in terminally differentiated keratinocytes

Nucleoli are the largest nuclear bodies serving as sites of the ribosomal DNA (rDNA) transcription, rRNA maturation and ribosome production, as well as fulfilling non-canonical functions, such as storage of numerous proteins, etc. (Bartova et al., 2010; Boulon et al., 2010; McKeown and Shaw, 2009). Nucleoli are assembled only around the active nucleolus organization regions (NORs) of selected chromosomes, in which ribosomal RNA genes are clustered (Hernandez-Verdun, 2006; Lam et al., 2005; Smirnov et al., 2006). Because nucleoli are surrounded by the perinucleolar heterochromatin, which represents the sites of gene silencing for many genomic regions, their positioning and number are important not only for maintenance of metabolic activity of the cell, but also for gene silencing during execution of cell differentiation-associated gene expression programs (Mao et al., 2011).

To analyze the number, volume and positioning of nucleoli during epidermal keratinocyte differentiation, we used immuno-staining with antibodies against the nucleolar-specific marker nucleophosmin/B23 (Scherl et al., 2002). Progenitor cells residing in the basal epidermal layer showed significantly more nucleoli compared to keratinocytes of the spinous (p=0.0021) and granular layers (p=0.0186) (Figure 2d-f).

Despite the decrease in number of nucleoli in the spinous layer compared to basal epidermal keratinocytes, the total volume on nucleoli per nucleus was significantly increased in the spinous layer cells (11.3 % of the volume of the nucleus) versus the basal cells (7% of the nuclear volume). In the granular layer, the volume of the nucleoli decreased compared to the spinous layer (9.6% of nuclear volume), but it was still larger than in the basal layer (Figure 2g). Moreover, an average volume of the nucleoli increased during keratinocyte differentiation: the smallest nucleoli were seen in the basal layer, whereas volume of nucleoli in the spinous layer increased more than 2-fold and showed further volume increase in the granular layer (Figure 2g).

To analyse intra-nuclear positions of nucleoli, we calculated their radial positions (distance from the nuclear centre normalized to the length of the nuclear radius coming through the centre of the nucleolus), according to the recommendations published previously (Ronneberger et al., 2008). Radial positions of nucleoli showed significant decrease in keratinocytes of the spinous and granular layers, compared to basal cells (Figure 2h). These data suggest that the decrease in the number of the nucleoli and parallel increase of their volume from lower to upper layers of the epidermis are likely to be associated with their fusion and repositioning towards the nuclear interior.

The changes in the spatial distribution of nucleoli could be the result either of the active remodelling of nuclear architecture associated with keratinocyte differentiation, or of purely geometrical constraints imposed by the changes in nuclear shape and size in the different cell populations. To distinguish between these two possibilities, we generated the mathematical prediction models of nucleoli distribution within the keratinocyte nuclei from different epidermal layers, as described previously (Bolzer et al., 2005; Cremer et al., 2001). For each model, the corresponding experimentally-assessed parameters (size and shape of the nucleus, number and volume of the nucleoli in each epidermal layer) were used. The conditions applied were that the mathematically-predicted nucleoli distribution was random and restricted by only two parameters: nucleoli have to fit within the nucleus and cannot overlap.

The mathematical modelling showed that, despite changes in the nuclear size and shape, predicted radial positions of nucleoli within the keratinocyte nuclei from different epidermal layers should show no significant changes and remain within the range of 55%-57% of average nuclear radius (Figure 2h). However, radial positions of the nucleoli in the spinous and granular layers measured experimentally, differed significantly compared to the basal layer (42.2±5.6% and 43.4±4.2% versus 54.3±3.5%, respectively) or to mathematically predicted values (see above) (Figure 2h). This indicates that internalization (or centripetal repositioning) of the nucleoli seen in the spinous and granular epidermal layers is not a mere consequence of the geometrical changes in nuclear shape and size, but rather represents a result of active remodelling of the nuclear architecture associated with terminal keratinocyte differentiation.

Pericentromeric heterochromatin clusters are increased in number and are spread over the nucleus during epidermal keratinocyte differentiation

Centromers and associated pericentromeric repetitive sequences form constitutive heterochromatic domains in eukaryotic cells (Choo, 2001). These domains establish pericentromeric heterochromatin clusters in 3D nuclear space that can be visualized using 3D Fluorescent In Situ Hybridization (FISH) with the probes specific to the major pericentromeric repeats (Horz and Altenburger, 1981). In mammalian cells, neighbouring chromosomes tether together and form common pericentromeric heterochromatin clusters, which can also be visualized by strong DAPI staining, and both the number and positioning of these clusters change during cell differentiation (Fussner et al., 2011; Meyer-Ficca et al., 1998; Solovei et al., 2004; Solovei et al., 2009a).

To determine the dynamics of the number and nuclear positioning of pericentromeric clusters during epidermal keratinocyte differentiation, we employed 3D-FISH technology with a mouse major satellite repeat probe (Figure 3a,b). In addition, we used immunostainings to histone H3 trimethylated at lysine 9 (H3K9me3) and to Heterochromatin Protein 1α, which are markers of transcriptionally-inactive or silenced heterochromatin (Zhou et al., 2011) (Figure 3c). The results showed that the number of pericentromeric heterochromatin clusters in the keratinocyte nuclei significantly (p<10-3) increased from the basal layer to the spinous and granular layers, respectively (Figure 3 b,d).

Figure 3. Remodelling of the pericentromeric heterochromatin clusters during terminal differentiation of keratinocytes in the murine epidermis.

Figure 3

Open in a new tab

(a) Representative 200 nm single optical section images of the mouse epidermis after 3D FISH with the major satellite repeat probe. Epidermal/dermal border is indicated by dotted line. (b) Representative nuclei from the distinct epidermal layers: FISH images (left) and geometrical models of the average keratinocyte nucleus generated on the basis of 3D-reconstruction of the 70-80 confocal stacks of the whole nuclei (right). Note that in geometrical models of the average keratinocyte nuclei, orientation of the nuclei from distinct epidermal layers was purportedly changed to provide a better visual appreciation of the topological differences between them. (c) Centromeric clusters have heterochromatic character and express makers of silent chromatin like H3K9me3 which recruits protein HP1 taking part in chromatin compaction. (d) Changes of number of centromeric clusters during epidermal keratinocyte differentiation. (e) Change in radial positions of the pericentromeric clusters during terminal keratinocyte differentiation. Data in c and d represent means with 95% confidence interval; 30 nuclei in every group were measured p-values: * <0.05, **<0.01, ***<0.001. Scale bars = 10 um.

Increase in number of pericentromeric clusters in the spinous layer was also accompanied by significant increase of their radial positions compared to basal keratinocytes (p< 10-2). However, radial positions of pericentromeric clusters in the granular layer decreased compared to the spinous layer (p< 10-4) (Figure 3e). Results of the mathematical modelling (see above) for pericentromeric cluster positioning showed that, despite changes in the nuclear size and shape, their predicted radial positions within the keratinocyte nuclei in the spinous layer differed significantly from experimental data (70+0.2% versus 76+1.5%, p<10-3, Figure 3e). This suggests that more peripheral distribution of the pericentromeric clusters in the spinous versus basal epidermal keratinocytes cannot be explained by the geometrical constraints associated with changes in the nuclear shape and volume, and likely to be due to the active heterochromatin re-distribution occurring in differentiating keratinocytes.

Pericentromeric heterochromatin clusters, nucleoli and the chromosomal territory 3 show increased frequency of associations during epidermal keratinocyte differentiation

Pericentromeric heterochromatin clusters and nucleoli occupy a significant part of the nuclear volumes in mammalian cells and are often associated with each other, thus influencing the positioning of chromosomes, distinct chromatin domains and genomic loci (Alcobia et al., 2000; Carvalho et al., 2001; Guetg et al.; Haaf and Schmid, 1991; Solovei et al., 2009a). To characterize relative positioning of the pericentromeric chromatin clusters, nucleoli and chromosomal territory 3, we performed 3D-FISH analyses using probes specific for pericentromeric clusters and/or chromosomal territory 3 (whole chromosome 3 paint) combined this with immune-fluorescent visualization of nucleoli using anti-nucleophosmin/B23 antibodies (Figure 4).

Figure 4. Changes in the spatial association between pericentromeric heterochromatin clusters, nucleoli and the chromosomal territory 3 during epidermal keratinocyte differentiation.

Figure 4

Open in a new tab

(a, b). Spatial associations between the pericentromeric heterochromatin clusters and nucleoli (white arrows). (c, d). Spatial associations between the chromosome territory 3 and nucleolus (white arrows) (e, f). Spatial associations between the chromosome 3 and pericentromeric clusters located either at the peripheral (p) or internal (i) parts of the nucleus. (g) 3D reconstruction of a representative nucleus from the epidermal basal layer. (h) Summary: remodelling of keratinocyte nucleus during epidermal differentiation is likely to contribute to global changes in the transcriptional landscape in differentiating keratinocytes. Data in a, c, and e represent means with 95% confidence interval; 30 nuclei in every group were measured, p-values: * <0.05, **<0.01, ***<0.001. Scale bars = 5 um.

Chromosome territory 3 was purposely selected for these analyses, because it bears the Epidermal Differentiation Complex (EDC) locus that is crucial for both keratinocyte differentiation and epidermal barrier formation (Bazzi et al., 2007; Fessing et al., 2011; Segre, 2006), whereas it does not contain the NORs (Bartova et al., 2010). Thus, the positioning of chromosome territory 3 in the nucleus should not be directly influenced (or reflect changes) by positioning of nucleoli (see Figure 2).

3D image analyses and computational reconstructions of the nuclei revealed that the number of pericentromeric heterochromatin clusters associated with nucleoli significantly increased in the spinous and granular layers compared to basal epidermal keratinocytes (Figure 4a,b). Chromosomal territory 3 occupied a peripheral positioning in the nuclei of keratinocytes in all epidermal layers, closely associated with the nuclear membrane via its peripheral part and interacting with the nuclear interior via its internal part (Figure 4d,f). Despite the fact that positioning of the chromosomal territory 3 in the nucleus was not changed between different epidermal layers, the frequencies of close spatial associations between nucleoli and the chromosome territory 3 were significantly higher in the spinous and granular layers in comparison to the basal layer (Figure 4c,d). Furthermore, our analysis demonstrated a significant increase in the frequency of peripheral localization of the pericentrometic heterochromatin of chromosome 3 in the nuclei of the spinous and granular layers compared to the basal layer, suggesting a substantial spatial remodelling of the chromosomal territory 3 during keratinocyte differentiation (Figure 4e, f, g).

These data demonstrate that keratinocyte transition between the distinct epidermal layers is also associated with the changes in spatial relationships between pericentromeric heterochromatin clusters, nucleoli and chromosome territory 3, thus suggesting a substantial 3D re-organization of the genome in keratinocytes during terminal differentiation (Figure 4 h).

Discussion

Execution of lineage-specific cell differentiation programs requires a high degree of coordination between many levels of regulation of gene expression in the nucleus including higher-order chromatin remodelling and proper spatial arrangements of the individual chromosomes, chromatin domains and nuclear bodies (Hubner and Spector, 2010; Joffe et al., 2010; Schoenfelder et al., 2010). In differentiating cells, actively transcribed genes are spatially compartmentalized and frequently form preferential intra- and inter-chromosomal topologically associated domains or interactomes, which are regulated by lineage-specific transcription factors, whereas repressed genes also form spatially organized inactive chromatin domains (Lieberman-Aiden et al., 2009; Nora et al., 2012; Schoenfelder et al., 2010). Thus, spatial organization of the transcriptionally-active or silenced compartments in the nucleus strongly depends on its size, shape, preferential arrangements of the chromosomes, number and size of distinct nuclear bodies, etc., which are markedly changed during cell differentiation (Dundr and Misteli, 2010; Joffe et al., 2010; Schoenfelder et al., 2010).

Here, we present data of the first systematic analyses of the remodelling of nuclear architecture during terminal keratinocyte differentiation in the epidermis and demonstrate that terminally differentiated keratinocytes show marked differences in micro-anatomical organization of the nucleus compared to basal epidermal cells including: i) decrease of the nuclear volume; ii) decrease in expression of the markers of transcriptionally-active chromatin; iii) internalization and decrease in the number of nucleoli; iv) increase in the number of pericentromeric heterochromatic clusters; v) increase in the frequency of associations between nucleoli, pericentromeric clusters and chromosomal territory 3 (Figures 1-4).

Our data obtained by using a multi-color confocal microscopy of cryosections of the normal mouse foot-pad epidermis, followed by computational image reconstruction and mathematical modelling are quite consistent with the results of two-dimensional ultra-structural studies of the nucleus in differentiating epidermal keratinocytes obtained previously (Breathnach, 1971; Karasek et al., 1972; Tsuji and Cox, 1977). In particular, we show that nuclear volume is significantly decreased during epidermal keratinocyte differentiation (Figure 1), which corresponds well to the data obtained from normal human epidermis demonstrating that size of the nucleus (determined on ultrathin sections) in keratinocytes of the granular layer is markedly lower compared to cells of the basal or spinous layers (Tsuji and Cox, 1977).

Global levels of transcription and the number and size of nucleoli are tightly coupled with metabolic activity of the cell (Derenzini et al., 2009; McKeown and Shaw, 2009). Our analysis of the markers of active transcription and morphology of the nucleoli showed significant differences between keratinocytes from different layers of the epidermis: transcriptional activity was high in the basal layer and increased even more in spinous layers before decreasing significantly in the granular layer, whereas nucleoli become less numerous and show a tendency to fuse in keratinocytes of the spinous and granular epidermal layers compared to basal epidermal cells (Figure 2). These data are in agreement with studies in other cell types demonstrating that nucleoli are larger in rapidly dividing cells or in cells with very high metabolic activity (McKeown and Shaw, 2009; Sullivan et al., 2001).

In addition to assembling the ribosomal subunits, nucleoli also play an essential role in the spatial genome organization by tethering and harbouring together the NOR-bearing chromosomes (in mice – chromosomes 12, 15, 16, 17, 18 and 19) (Kalmarova et al., 2007; Solovei et al., 2004). Mathematical modelling performed in our study reveals that internalization (or centripetal repositioning) and the decrease in number of nucleoli seen in differentiated keratinocytes cannot be explained by only changes in the nuclear shape or size, but rather represent the results of active remodelling of spatial nuclear organization (Figure 2). Since nucleoli are surrounded by the perinucleolar heterochromatin, which represents the sites of silencing for many genes in the nucleus (Mao et al., 2011), changes in their positioning and number might also result in spatial re-distribution of the inactive chromatin domains as a part of the programmed cessation of a number of transcription programs associated with terminal keratinocyte differentiation.

Chromosomal centromeres are embedded into heterochromatic regions of repetitive DNA sequences (Aleixandre et al., 1987; Choo, 2001; Solovei et al., 2009a). In the interphase nucleus, centromers of different chromosomes cluster together, thus providing important spatial constrains that influence the 3D nuclear organization (Alcobia et al., 2000; Solovei et al., 2004). We show that number of the pericentromeric heterochromatin clusters is increased in the spinous and granular layers (Figure 3), suggesting that at least some pericentromeric clusters disassemble in the terminally-differentiated keratinocytes, possibly due to the changes in the positioning of selected chromosomes or to the remodelling of their chromatin structure (i.e, folding) associated with terminal keratinocyte differentiation. These data are consistent with studies of the Purkinje cells where number of pericentromeric heterochromatin clusters increases during differentiation (Solovei et al., 2004).

Interestingly, analysis of spatial associations between nucleoli and pericentromeric clusters showed that the number of associations between them also increases during keratinocyte differentiation, which is also consistent with data observed previously in terminally-differentiated Purkinje cells (Solovei et al., 2004). Such associations might occur via interactions between the nucleolar RNA-binding proteins and RNAs transcribed from the genomic regions that constitute nucleolar-associated domains including α-satellite centromeric RNAs (Nemeth et al., 2010; Wong et al., 2007). In addition, nucleoli more frequently interact with non-NOR bearing chromosomal territory 3 in terminally differentiated keratinocytes compared to basal epidermal cells (Figure 4).

We speculate that these changes might generate additional sub-compartments around chromosome territory 3, where selected genes might be relocated for cell differentiation-associated silencing. However, additional 3D-FISH and microarray data are required to correlate positioning and expression levels of genes closely located to nucleoli in granular layer keratinocytes, similarly to data on other cells published previously (Nemeth et al., 2010).

In conclusion, data presented here demonstrate that terminal keratinocyte differentiation in the epidermis and transition of the keratinocyte nucleus from a metabolically active status to an inactive condition is accompanied by marked remodelling of the 3D nuclear organization and micro-anatomy (Figure 4h). Decrease of the nuclear volume, internalization of the nucleoli and expansion of pericentromeric heterochromatin clusters forming close associations to each other in terminally differentiating cells are likely to contribute to global changes in the transcriptional landscape in differentiating keratinocytes. These data also suggest the nucleoli and pericentromeric clusters as important elements of the nuclear architecture which may control the local “transcriptional micro-environment” of distinct chromatin domains by modulating the processes of chromosome tethering and regulating their positioning, folding and/or orientation. Taken together, these data provide important background information for further analyses of the topological organization of the genome in keratinocytes during cell differentiation and reprogramming, as well as for studies of the alterations in nuclear architecture seen in pathological skin conditions including the disorders of epidermal differentiation (such as psoriasis) or epidermal benign or malignant tumours.

Material and methods

Experimental animals and tissue collection

Tissue collection was performed under protocols approved by the Bradford University and the Home Office Project License. Footpad skin of 10 day-old C57Bl/6 mice was processed for immunofluorescence or 3D immuno-FISH, as described previously (Walter et al., 2006; Fessing et al., 2011).

Immunofluorescence and 3D ImmunoFISH

Cryosections of quick frozen tissue were air dried, fixed in 4% formaldehyde for 10 minutes at room temperature and incubated with primary and secondary antibodies (list of antibodies and dilutions are provided in the Supplemental Table S1), counterstained by DAPI and embedded into Vectashield medium (Vector Labs, Burlingame, CA, USA).

3D-FISH analysis was performed with labelled DNA probes on FISH-fixed tissue sections as previously described (Solovei et al., 2009b). The probes detecting mouse major satellite repeats (MSR) and whole chromosome paints of mouse chromosome 3 were labelled with Cy3- or Biotin-dUTP by (Horz and Altenburger, 1981; Telenius et al., 1992; Henegariu et al., 2000; Solovei et al., 2004). The frozen sections were dried, heated to partially reverse crosslinking, and equilibrated in 50% formamide/2×SSC. Precipitated labelled DNA probe mixtures were applied on the tissue under glass chambers. Hybridization was performed at 37°C for 2 days, followed by washing and incubation with streptavidin-Cy5 (Rockland Immunochemicals, Gilbertsville, PA). From this stage, sections were processed for immunofluorescent labelling and counterstaining with DAPI, as described above.

Microscopy

Images were collected using a laser-scanning confocal microscope (LSM 710 or LSM510 META; Carl Zeiss, Oberkochen, Germany) equipped with UV (364 nm), Argon (488 nm), HeNe1 (543 nm) and HeNe2 (633nm) lasers. For 3D image analysis, the nuclei were scanned using a narrow band detector with a Z axial distance of 200 nm yielding separate stacks of 8-bit grayscale images for each fluorescent channel with a pixel size of 100nm. For each optical section, images were collected sequentially for all fluorophores, and axial chromatic shift was corrected for each channel according to (Solovei et al., 2009).

3D image processing and analysis, reconstruction of epidermal nuclei

Based on the detected fluorescent signals, individual nuclear surfaces were segmented by manual retrieving of points using imageJ software (http://rsbweb.nih.gov/ij/), followed by triangulation, reconstruction and editing of the reconstructed 3D image using MATLAB (Mathworks, Natick, MA). Similar reconstruction was done to visualize spatial associations between the centromeric clusters, nucleoli and chromosome territory 3 and their localization within the nucleus. We defined the association between the nucleoli and chromosomal territory 3 as a partial overlap or close contacts (touching) between the fluorescent signals depicting these structures. During the quantification process, such contacts between these structures were scored as a single association. Positioning of the peri-centromeric clusters was considered as peripheral when the fluorescent signals depicting such clusters showed close contact with the nuclear border.

Morphometric analyses of the nuclei and the number, radial positions and volumes of nucleoli and centromeric clusters

Retrieved coordinates from the surface of the nuclei were used for calculation of the nuclear volume and shape. Volume was calculated by the script written according to the recommendations of the R-Foundation for statistical computing (http://www.r-project.org/foundation/). The surface coordinates were used subsequently for fitting of the ellipsoid equation and retrieving of its axis sizes (the least square algorithm) by publically available MATLAB “minVolellipse” (Mathworks, Natick, MA) . For estimation of radial positions of centromeric clusters and nucleoli, for every nucleus coordinates of 200-400 points from the surface were retrieved in the ImageJ and along with the centroids of nucleoli and centromeric clusters obtained previously were used as input data for a script according to recommendations published elsewhere (http://www.r-project.org/foundation/). In total, 30 nuclei from every epidermal layer were analyzed.

Mathematical modelling of the nuclei and statistical analyses

Mathematical modelling of the nucleus was performed based on previous measurements, according to previously published instructions (Bolzer et al., 2005; Cremer et al., 2001; Ronneberger et al., 2008). The model nucleus had the average size for nuclei from distinct epidermal layers, average number of nucleoli and centromeric clusters which were also of average size characteristic for distinct layer. Distribution of nucleoli and clusters restricted by the nuclear volume and the volumes of other nucleoli/clusters was randomized. After generation of the nuclear models, radial positions were calculated and averaged in sets of 30. Finally, radial positions from the model were compared with radial position from experiment with the T-test. Representative models of nuclei from distinct layers were visualized in MATLAB (Mathwork, Natick, MA). Data were analyzed using the Lilliefor's normality test and then either with Student t-test (for two groups) or one-way analysis of variance followed by the Newman-Keuls procedure (for more than two groups) in Excel (Microsoft, Redmond, WA). Statistically significant differences between groups are denoted by asterisks: *P<0.05, ** P<0.01, *** P<0.001.

Supplementary Material

Suppliment

Supplemental Figure S1. Nuclei, nucleoli and centromeric clusters in murine footpad epidermis – from visualization by immunoFISH to 3D reconstruction. (a) 3D-immunoFISH was performed on 3D preserved mouse footpad epidermis with antibodies against nucleoli, FISH probes detecting mouse centromers (mouse major satellite) and chromosome territory 3. Epidermal/dermal border is indicated by dotted line. (b-d) Probes were checked for specificity on metaphase spreads. Nuclei were visualized with DAPI. (e,f) Cryosections were scanned with confocal microscope and obtained stack of images representing distinct optical sections were used for analysis and reconstruction. Scale bars: S1a = 10μm, S1b-d = 2μm.

Supplementary Figure S2. Immunostaining for Loricrin and Keratin 10 in mouse footpad epidermis. Cryo-section of mouse foot pad skin were stained with antibodies against Loricrin (a) and Keratin 10 (b). Cell nuclei were counterstained by DAPI. Epidermal/dermal border is indicated by dotted line. Scale bars = 10μm.

Supplementary Figure S3. Immnunofluorescent signal intensity of the nuclei of murine footpad epidermis stained with antibodies for different markers of transcriptionally active chromatin (see Fig. 2). Immunostaining with antibodies against RNA polymerase II large subunit phosphorylated at Ser2 of the CTD (a), H3K4me3 (b), and H3K56Ac (c). Data represent means with 95% confidence interval; signal intensity (a.u.) in 30 nuclei in every group was measured, p-values: * <0.05, **<0.01, ***<0.001.

Acknowledgments

Critical reading and comments on the manuscript of Prof. D. J. Tobin are gratefully acknowledged. This study was supported in part by the grant from the Medical Research Council UK (G0901666) to V.A.B.

Abbreviations

FISH

Fluorescent In Situ Hybridization

KC

keratinocyte

3D

three-dimensional

Footnotes

Conflict of interests

Authors declare no conflict of interests associated with this study.

References

  1. Alcobia I, Dilao R, Parreira L. Spatial associations of centromeres in the nuclei of hematopoietic cells: evidence for cell-type-specific organizational patterns. Blood. 2000;95:1608–1615. [PubMed] [Google Scholar]
  2. Aleixandre C, Miller DA, Mitchell AR, Warburton DA, Gersen SL, Disteche C, et al. p82H identifies sequences at every human centromere. Hum Genet. 1987;77:46–50. doi: 10.1007/BF00284712. [DOI] [PubMed] [Google Scholar]
  3. Bartova E, Horakova AH, Uhlirova R, Raska I, Galiova G, Orlova D, et al. Structure and epigenetics of nucleoli in comparison with non-nucleolar compartments. J Histochem Cytochem. 2010;58:391–403. doi: 10.1369/jhc.2009.955435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bazzi H, Fantauzzo KA, Richardson GD, Jahoda CA, Christiano AM. Transcriptional profiling of developing mouse epidermis reveals novel patterns of coordinated gene expression. Dev Dyn. 2007;236:961–970. doi: 10.1002/dvdy.21099. [DOI] [PubMed] [Google Scholar]
  5. Blanpain C, Fuchs E. Epidermal homeostasis: a balancing act of stem cells in the skin. Nat Rev Mol Cell Biol. 2009;10:207–217. doi: 10.1038/nrm2636. Epub 2009 Feb 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolzer A, Kreth G, Solovei I, Koehler D, Saracoglu K, Fauth C, et al. Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS Biol. 2005;3:e157. doi: 10.1371/journal.pbio.0030157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Botchkarev VA, Gdula MR, Mardaryev AN, Sharov AA, Fessing MY. Epigenetic Regulation of Gene Expression in Keratinocytes. J Invest Dermatol. 2012 doi: 10.1038/jid.2012.182. Epub ahead of print: doi: 10.1038/jid.2012.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Botchkarev VA, Metz M, Botchkareva NV, Welker P, Lommatzsch M, Renz H, et al. Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as “epitheliotrophins” in murine skin. Lab Invest. 1999;79:557–572. [PubMed] [Google Scholar]
  9. Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI. The nucleolus under stress. Mol Cell. 2010;40:216–227. doi: 10.1016/j.molcel.2010.09.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Breathnach AS. An Atlas of the Ultrastructure of Human Skin. Gloucher Place; London: 1971. [Google Scholar]
  11. Carvalho C, Pereira HM, Ferreira J, Pina C, Mendonca D, Rosa AC, et al. Chromosomal G-dark bands determine the spatial organization of centromeric heterochromatin in the nucleus. Mol Biol Cell. 2001;12:3563–3572. doi: 10.1091/mbc.12.11.3563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Choo KH. Domain organization at the centromere and neocentromere. Dev Cell. 2001;1:165–177. doi: 10.1016/s1534-5807(01)00028-4. [DOI] [PubMed] [Google Scholar]
  13. Cremer M, von Hase J, Volm T, Brero A, Kreth G, Walter J, et al. Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells. Chromosome Res. 2001;9:541–567. doi: 10.1023/a:1012495201697. [DOI] [PubMed] [Google Scholar]
  14. Deniaud E, Bickmore WA. Transcription and the nuclear periphery: edge of darkness? Curr Opin Genet Dev. 2009;19:187–191. doi: 10.1016/j.gde.2009.01.005. [DOI] [PubMed] [Google Scholar]
  15. Derenzini M, Montanaro L, Trere D. What the nucleolus says to a tumour pathologist. Histopathology. 2009;54:753–762. doi: 10.1111/j.1365-2559.2008.03168.x. [DOI] [PubMed] [Google Scholar]
  16. Dundr M, Misteli T. Biogenesis of nuclear bodies. Cold Spring Harb Perspect Biol. 2010;2:a000711. doi: 10.1101/cshperspect.a000711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eckert RL, Adhikary G, Rorke EA, Chew YC, Balasubramanian S. Polycomb group proteins are key regulators of keratinocyte function. J Invest Dermatol. 2011;131:295–301. doi: 10.1038/jid.2010.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fessing MY, Mardaryev AN, Gdula MR, Sharov AA, Sharova TY, Rapisarda V, et al. p63 regulates Satb1 to control tissue-specific chromatin remodeling during development of the epidermis. J Cell Biol. 2011;194:825–839. doi: 10.1083/jcb.201101148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fuchs E. Scratching the surface of skin development. Nature. 2007;445:834–842. doi: 10.1038/nature05659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fussner E, Djuric U, Strauss M, Hotta A, Perez-Iratxeta C, Lanner F, et al. Constitutive heterochromatin reorganization during somatic cell reprogramming. EMBO J. 2011;30:1778–1789. doi: 10.1038/emboj.2011.96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guetg C, Lienemann P, Sirri V, Grummt I, Hernandez-Verdun D, Hottiger MO, et al. The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats. EMBO J. 29:2135–2146. doi: 10.1038/emboj.2010.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Haaf T, Schmid M. Chromosome topology in mammalian interphase nuclei. Exp Cell Res. 1991;192:325–332. doi: 10.1016/0014-4827(91)90048-y. [DOI] [PubMed] [Google Scholar]
  23. Hemberger M, Dean W, Reik W. Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington's canal. Nat Rev Mol Cell Biol. 2009;10:526–537. doi: 10.1038/nrm2727. Epub 2009 Jul 2015. [DOI] [PubMed] [Google Scholar]
  24. Henegariu O, Bray-Ward P, Ward DC. Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling. Nat Biotechnol. 2000;18:345–348. doi: 10.1038/73815. [DOI] [PubMed] [Google Scholar]
  25. Hernandez-Verdun D. The nucleolus: a model for the organization of nuclear functions. Histochem Cell Biol. 2006;126:135–148. doi: 10.1007/s00418-006-0212-3. [DOI] [PubMed] [Google Scholar]
  26. Ho L, Crabtree GR. Chromatin remodelling during development. Nature. 2010;463:474–484. doi: 10.1038/nature08911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Horz W, Altenburger W. Nucleotide sequence of mouse satellite DNA. Nucleic Acids Res. 1981;9:683–696. doi: 10.1093/nar/9.3.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hubner MR, Spector DL. Chromatin dynamics. Annu Rev Biophys. 2010;39:471–489. doi: 10.1146/annurev.biophys.093008.131348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Joffe B, Leonhardt H, Solovei I. Differentiation and large scale spatial organization of the genome. Curr Opin Genet Dev. 2010 doi: 10.1016/j.gde.2010.05.009. [DOI] [PubMed] [Google Scholar]
  30. Kalmarova M, Smirnov E, Masata M, Koberna K, Ligasova A, Popov A, et al. Positioning of NORs and NOR-bearing chromosomes in relation to nucleoli. J Struct Biol. 2007;160:49–56. doi: 10.1016/j.jsb.2007.06.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Karasek J, Smetana K, Hrdlicka A, Dubinin I, Hornak O, Ohelert W. Nuclear and nucleolar ultrastructure during the late stages of normal human keratinocyte maturation. Br J Dermatol. 1972;86:601–607. doi: 10.1111/j.1365-2133.1972.tb05075.x. [DOI] [PubMed] [Google Scholar]
  32. Koster MI, Roop DR. Mechanisms regulating epithelial stratification. Annu Rev Cell Dev Biol. 2007;9:93–113. doi: 10.1146/annurev.cellbio.23.090506.123357. [DOI] [PubMed] [Google Scholar]
  33. Kunisada T, Lu SZ, Yoshida H, Nishikawa S, Mizoguchi M, Hayashi S, et al. Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor. J Exp Med. 1998;187:1565–1573. doi: 10.1084/jem.187.10.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lam YW, Trinkle-Mulcahy L, Lamond AI. The nucleolus. J Cell Sci. 2005;118:1335–1337. doi: 10.1242/jcs.01736. [DOI] [PubMed] [Google Scholar]
  35. Lanctot C, Cheutin T, Cremer M, Cavalli G, Cremer T. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat Rev Genet. 2007;8:104–115. doi: 10.1038/nrg2041. [DOI] [PubMed] [Google Scholar]
  36. Lechler T, Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin. Nature. 2005;437:275–280. doi: 10.1038/nature03922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lee CH, Kim MS, Chung BM, Leahy DJ, Coulombe PA. Structural basis for heteromeric assembly and perinuclear organization of keratin filaments. Nat Struct Mol Biol. 2012;19:707–715. doi: 10.1038/nsmb.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009;326:289–293. doi: 10.1126/science.1181369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Loomis CA. Development and morphogenesis of the skin. Adv Dermatol. 2001;17:183–210. [PubMed] [Google Scholar]
  40. Loomis CA, Harris E, Michaud J, Wurst W, Hanks M, Joyner AL. The mouse Engrailed-1 gene and ventral limb patterning. Nature. 1996;382:360–363. doi: 10.1038/382360a0. [DOI] [PubMed] [Google Scholar]
  41. Mao YS, Zhang B, Spector DL. Biogenesis and function of nuclear bodies. Trends Genet. 2011;27:295–306. doi: 10.1016/j.tig.2011.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Marella NV, Seifert B, Nagarajan P, Sinha S, Berezney R. Chromosomal rearrangements during human epidermal keratinocyte differentiation. J Cell Physiol. 2009;221:139–146. doi: 10.1002/jcp.21855. [DOI] [PubMed] [Google Scholar]
  43. McKeown PC, Shaw PJ. Chromatin: linking structure and function in the nucleolus. Chromosoma. 2009;118:11–23. doi: 10.1007/s00412-008-0184-2. [DOI] [PubMed] [Google Scholar]
  44. Meyer-Ficca M, Muller-Navia J, Scherthan H. Clustering of pericentromeres initiates in step 9 of spermiogenesis of the rat (Rattus norvegicus) and contributes to a well defined genome architecture in the sperm nucleus. J Cell Sci. 1998;111(Pt 10):1363–1370. doi: 10.1242/jcs.111.10.1363. [DOI] [PubMed] [Google Scholar]
  45. Naumova N, Dekker J. Integrating one-dimensional and three-dimensional maps of genomes. J Cell Sci. 2010;123:1979–1988. doi: 10.1242/jcs.051631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nemeth A, Conesa A, Santoyo-Lopez J, Medina I, Montaner D, Peterfia B, et al. Initial genomics of the human nucleolus. PLoS Genet. 2010;6:e1000889. doi: 10.1371/journal.pgen.1000889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, et al. Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature. 2012;485:381–385. doi: 10.1038/nature11049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Plikus M, Wang WP, Liu J, Wang X, Jiang TX, Chuong CM. Morpho-regulation of ectodermal organs: integument pathology and phenotypic variations in K14-Noggin engineered mice through modulation of bone morphogenic protein pathway. Am J Pathol. 2004;164:1099–1114. doi: 10.1016/S0002-9440(10)63197-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Poulson ND, Lechler T. Robust control of mitotic spindle orientation in the developing epidermis. J Cell Biol. 2010;191:915–922. doi: 10.1083/jcb.201008001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rippe K. Dynamic organization of the cell nucleus. Curr Opin Genet Dev. 2007;17:373–380. doi: 10.1016/j.gde.2007.08.007. [DOI] [PubMed] [Google Scholar]
  51. Ronneberger O, Baddeley D, Scheipl F, Verveer PJ, Burkhardt H, Cremer C, et al. Spatial quantitative analysis of fluorescently labeled nuclear structures: problems, methods, pitfalls. Chromosome Res. 2008;16:523–562. doi: 10.1007/s10577-008-1236-4. [DOI] [PubMed] [Google Scholar]
  52. Rowat AC, Lammerding J, Herrmann H, Aebi U. Towards an integrated understanding of the structure and mechanisms of the cell nucleus. BioEssays. 2008;30:226–236. doi: 10.1002/bies.20720. [DOI] [PubMed] [Google Scholar]
  53. Rowden G. Ultrastructural studies of keratinized epithelia of the mouse. III. Determination of the volumes of nuclei and cytoplasm of cells in murine epidermis. J Invest Dermatol. 1975;64:1–3. doi: 10.1111/1523-1747.ep12540840. [DOI] [PubMed] [Google Scholar]
  54. Scherl A, Coute Y, Deon C, Calle A, Kindbeiter K, Sanchez JC, et al. Functional proteomic analysis of human nucleolus. Mol Biol Cell. 2002;13:4100–4109. doi: 10.1091/mbc.E02-05-0271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schoenfelder S, Clay I, Fraser P. The transcriptional interactome: gene expression in 3D. Curr Opin Genet Dev. 2010;20:127–133. doi: 10.1016/j.gde.2010.02.002. [DOI] [PubMed] [Google Scholar]
  56. Segre JA. Epidermal barrier formation and recovery in skin disorders. J Clin Invest. 2006;116:1150–1158. doi: 10.1172/JCI28521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sharov AA, Weiner L, Sharova TY, Siebenhaar F, Atoyan R, McNamara CA, et al. Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation. EMBO J. 2003;22:2992–3003. doi: 10.1093/emboj/cdg291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Smirnov E, Kalmarova M, Koberna K, Zemanova Z, Malinsky J, Masata M, et al. NORs and their transcription competence during the cell cycle. Folia Biol (Praha) 2006;52:59–70. doi: 10.14712/fb2006052030059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Solovei I, Grandi N, Knoth R, Volk B, Cremer T. Positional changes of pericentromeric heterochromatin and nucleoli in postmitotic Purkinje cells during murine cerebellum development. Cytogenetic and Genome Research. 2004;105:302–310. [Google Scholar]
  60. Solovei I, Kreysing M, Lanctot C, Kosem S, Peichl L, Cremer T, et al. Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution. Cell. 2009;137:356–368. doi: 10.1016/j.cell.2009.01.052. [DOI] [PubMed] [Google Scholar]
  61. Sullivan GJ, Bridger JM, Cuthbert AP, Newbold RF, Bickmore WA, McStay B. Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli. EMBO J. 2001;20:2867–2877. doi: 10.1093/emboj/20.11.2867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Telenius H, Pelmear AH, Tunnacliffe A, Carter NP, Behmel A, Ferguson-Smith MA, et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer. 1992;4:257–263. doi: 10.1002/gcc.2870040311. [DOI] [PubMed] [Google Scholar]
  63. Tsuji T, Cox AJ. Ultrastructural studies of nuclei and nucleoli in psoriatic epidermis compared to those in normal epidermis. J Invest Dermatol. 1977;69:205–210. doi: 10.1111/1523-1747.ep12506307. [DOI] [PubMed] [Google Scholar]
  64. Walter J, Joffe B, Bolzer A, Albiez H, Benedetti PA, Müller S, et al. Towards many colors in FISH on 3D-preserved interphase nuclei. Cytogenet Genome Res. 2006;114:367–378. doi: 10.1159/000094227. [DOI] [PubMed] [Google Scholar]
  65. Wang KC, Chang HY. Molecular mechanisms of long noncoding RNAs. Mol Cell. 2011;43:904–914. doi: 10.1016/j.molcel.2011.08.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Williams RR, Broad S, Sheer D, Ragoussis J. Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei. Exp Cell Res. 2002;272:163–175. doi: 10.1006/excr.2001.5400. [DOI] [PubMed] [Google Scholar]
  67. Wong LH, Brettingham-Moore KH, Chan L, Quach JM, Anderson MA, Northrop EL, et al. Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome Res. 2007;17:1146–1160. doi: 10.1101/gr.6022807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zhang J, Bardot E, Ezhkova E. Epigenetic regulation of skin: focus on the Polycomb complex. Cell Mol Life Sci. 2012 doi: 10.1007/s00018-012-0920-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zhou VW, Goren A, Bernstein BE. Charting histone modifications and the functional organization of mammalian genomes. Nat Rev Genet. 2011;12:7–18. doi: 10.1038/nrg2905. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppliment

Supplemental Figure S1. Nuclei, nucleoli and centromeric clusters in murine footpad epidermis – from visualization by immunoFISH to 3D reconstruction. (a) 3D-immunoFISH was performed on 3D preserved mouse footpad epidermis with antibodies against nucleoli, FISH probes detecting mouse centromers (mouse major satellite) and chromosome territory 3. Epidermal/dermal border is indicated by dotted line. (b-d) Probes were checked for specificity on metaphase spreads. Nuclei were visualized with DAPI. (e,f) Cryosections were scanned with confocal microscope and obtained stack of images representing distinct optical sections were used for analysis and reconstruction. Scale bars: S1a = 10μm, S1b-d = 2μm.

Supplementary Figure S2. Immunostaining for Loricrin and Keratin 10 in mouse footpad epidermis. Cryo-section of mouse foot pad skin were stained with antibodies against Loricrin (a) and Keratin 10 (b). Cell nuclei were counterstained by DAPI. Epidermal/dermal border is indicated by dotted line. Scale bars = 10μm.

Supplementary Figure S3. Immnunofluorescent signal intensity of the nuclei of murine footpad epidermis stained with antibodies for different markers of transcriptionally active chromatin (see Fig. 2). Immunostaining with antibodies against RNA polymerase II large subunit phosphorylated at Ser2 of the CTD (a), H3K4me3 (b), and H3K56Ac (c). Data represent means with 95% confidence interval; signal intensity (a.u.) in 30 nuclei in every group was measured, p-values: * <0.05, **<0.01, ***<0.001.

NIHMS480976-supplement-Suppliment.pdf (1,014.2KB, pdf)

RESOURCES