Progressive down-regulation of Hmgn1 expression during mouse embryogenesis.
Whole-mount in situ hybridization and immunostaining analyses revealed that the expression of Hmgn1
is selectively down-regulated during mouse embryogenesis. In 7.5-day-old mouse embryos (E7.5), the expression of Hmgn1
was high in the epiblast, weak in the extra-embryonic ectoderm, and absent from the ectoplacental cone (Fig. , frame a). At E8.5 and E9.5, high Hmgn1
expression was observed throughout the entire embryo, except in the heart (Fig. , frames b and c), an organ known to begin differentiating early (14
). At E10.5, fairly strong expression of Hmgn1
was widespread through the embryo and was prominent in the forelimb and hind limb buds, branchial arches, and tail bud (Fig. , frames d to f) but was totally absent from the heart. At subsequent developmental stages, the expression level of Hmgn1
selectively declines, coincident with the onset of differentiation in many organs, as more easily seen in tissue sections (see below).
FIG.1. Expression patterns of Hmgn1 during mouse embryogenesis. (A) Whole-mount in situ hybridization with digoxigenen-labeled Hmgn1 antisense probes. At E7.5 strong expression is observed in the embryonic region (arrowhead). No signal is observed in the ectoplacental (more ...)
Quantitative RNA dot blot analysis fully supports the conclusion of the in situ analyses (Fig. ). In these blots, the amount of RNA in each spot is normalized to the transcription levels of eight housekeeping genes; therefore, the intensity of the spot indicates the relative mRNA abundance in a tissue. The relative levels of Hmgn1 RNA were highest in 7-day-old embryos and then gradually decreased; the RNA levels in 17-day-old embryo were only 50% of those of the 7-day-old embryos. In differentiated tissues such as the adult heart and spleen, the levels of Hmgn1 RNA are only about 10% of the level present in young embryos (Fig. ).
To discern the developmental pattern of Hmgn1 expression in the internal organs of the embryos, we analyzed the presence of HMGN1 protein in multiple serial sections by immunohistochemistry using affinity-purified anti-mouse HMGN1 antibody. Representative results from several tissues (Fig. ) (images are best viewed at large magnifications; see Fig. S1 to S3 in the supplemental material) are described below.
FIG. 2. HMGN1 expression correlates with cellular differentiation rather than cell proliferation. The localization of HMGN1 and PCNA in developing mouse tissues was detected by immunofluorescence. Images at higher magnifications are presented in Fig. S1 to S3 (more ...) Neural tube.
In E10.5 transverse sections, HMGN1 protein is visible throughout the embryo, including the surface ectoderm, neural tube, dorsal root ganglia, and the mesenchymal cells (Fig. ). The neural tube is already differentiated into an outer marginal layer, an intermediate mantle layer, and inner ependymal layer; HMGN1 is strongly expressed in all the three layers. By E14.5 HMGN1 expression is localized primarily to the dorsal half of the mantle layer (Fig. , frames c), which expresses higher HMGN1 levels than the ventral half of the mantle layer (Fig. , frames d), an indication that HMGN1 expression correlates with neural tube differentiation (19
At E12.5, HMGN1 was detected in both the epithelial and mesenchymal layers of the developing stomach (Fig. ), most prominently in the outermost mesothelium. At E16.5, when the stomach underwent additional differentiation, HMGN1 expression in the epithelium was most prominent in the relatively undifferentiated basal layer (Fig. , bl in frames c).
At E12.5 HMGN1 was detected throughout the developing lung, both in the epithelial and in the dense mesenchymal cells (Fig. , me) surrounding the bronchioles (Fig. ). However at E16.5, when the bronchioles are fully differentiated but the distal epithelium Fig. , de still forms alveoli, expression of HMGN1 is localized to the developing distal epithelium and is greatly decreased in the bronchiols (Fig. , br in frames d).
To examine whether the HMGN1 expression levels are related to cellular proliferation, we compared the expression patterns of HMGN1 with that of PCNA. Comparison of the PCNA-stained panels with the HMGN1-stained panels reveals that the expression of HMGN1 and PCNA do not always overlap, which is an indication that in the developing embryo the levels of HMGN1 are not linked to cellular proliferation per se, a finding consistent with previous observations in other experimental systems (16
Our analyses revealed that in every tissue examined, the expression of HMGN1 was down-regulated as organ differentiation proceeded during development. However, the levels of the protein remained high in progenitor cells, such as the basal skin layer cells that are committed and poised for further differentiation and renewal. Thus, the dot blot RNA analysis, the in situ hybridization, and the immunofluorescence analyses indicate a differentiation-related down-regulation of HMGN1 expression.
Reciprocal expression patterns of Hmgn1 and Sox9 in the developing limb bud.
To gain additional insights into the role of HMGN1 in developmental processes, we focused on its possible involvement in the differentiation of the limb bud, where Hmgn1 expression correlated inversely with mesenchymal chondrogenic differentiation. Whole-mount in situ hybridization demonstrates clearly that the expression of Hmgn1 is down-regulated during development and is complementary to that of Sox9, a major transactivator involved in the initiation and propagation of chondrogenesis (Fig. ). Thus, in the E10.5 forelimb bud (and hind bud; data not shown), Hmgn1 was strongly expressed in a broad anterior and distal region and down-regulated in the proximal region (Fig. ), where mesenchymal cells condense and differentiate into chondrocytes, as evidenced by the onset of Sox9 expression (Fig. ). As chondrogenesis initiated in the more distal digit region over time, expression of Hmgn1 was localized to the future interdigit region and down-regulated in the future digit region (Fig. ), coinciding with activation of Sox9 expression (Fig. ). In the E12.5 forelimb bud, Hmgn1 expression is most prominent in the interdigit mesenchyme (Fig. ), while Sox9 transcripts are most abundant in future digit regions (Fig. ). In the E13.5 and E14.5 forelimb buds, Hmgn1 expression is most prominent in the interdigital mesenchyme (Fig. ), while Sox9 transcripts are most abundant in the cartilage of phalanges (Fig. ). A similar pattern of reciprocal Hmgn1 and Sox9 expression was observed during hind limb bud development (data not shown). Thus, during the development of both limbs, the decline of Hmgn1 expression preceded that of Sox9, the latter being expressed in regions with significantly diminished levels of HMGN1 (Fig. ).
FIG. 3. Reciprocal expression patterns of Hmgn1 and Sox9 during limb bud development. (A to J) Whole-mount in situ hybridization analysis of Hmgn1 and Sox9 expression in E10.5 to E14.5 forelimbs. At E10.5 Hmgn1 is strongly expressed in the distal region of limb (more ...)
More detailed immunohistochemical analyses of sections from the developing limb bud region verified the reciprocal expression of Hmgn1 and Sox9. The expression of Hmgn1 decreased while that of Sox9 increased as the prechondrogenic mesenchyme condensed in the cartilage primordia. Thus, in the E10.5 and E12.5 forelimb buds, Sox9 protein was observed in the proximal region (Fig. ) and digit cartilages (Fig. ), while HMGN1 protein was expressed in the surface ectoderm and throughout the distal mesenchyme region but absent from the prechondrogenic mesenchyme (Fig. ). In the more fully differentiated E14.5 forelimb, HMGN1 protein was abundant in the perichondrium of the phalanges but depleted from the cartilage, where Sox9 protein was expressed (Fig. ). At higher magnification the difference in the expression patterns of HMGN1 and Sox9 in the developing phalanges was very clear: the expression of Sox9 was confined to the cartilage, while HMGN1 was expressed in the surrounding perichondrium but not in cartilage (Fig. and ; see Fig. S4 in the supplemental material).
Reciprocal expression patterns of Hmgn1 and Sox9 in micromass cultures.
To gain additional insights into the role of HMGN1 in chondrocyte differentiation, we analyzed the pattern of Hmgn1 and Sox9 expression in a micromass culture system (Fig. ), which has been commonly used as a model to study in vitro chondrogenesis. When plated at high density, mouse limb bud mesenchymal cells differentiate into chondrocytes and form cartilage nodules (Fig. ). The progress of differentiation can be assessed by Alcian blue staining, which is most prominent in differentiated nodules (Fig. ). Under our growth conditions, nodules of differentiated chondrocytes can be seen within 3 days of plating (Fig. , +) and distinct, strongly staining nodules containing differentiated chondrocytes are clearly visible after 5 days of plating. In situ hybridization with digoxigenin-labeled RNA probes indicates that the levels of Hmgn1 transcripts decrease while those of Sox9 increase during differentiation. Thus, 2 days after plating, Hmgn1 was expressed throughout most of the monolayer cells. At day 3, Hmgn1 expression was down-regulated in the regions in which the cells condensed into the evolving nodules (Fig. , +), and by day 5, Hmgn1 transcripts were highly depleted and practically absent in the differentiated cartilage nodule (Fig. , top panels). In contrast, Sox9 expression was first observed in the small cell aggregates detectable after 2 days of culturing. In the evolving nodules, the levels of Sox9 RNA gradually increases and is prominent in the fully differentiated, 5-day-old nodules (Fig. , bottom panels).
FIG. 4. Reciprocal expression patterns of Hmgn1 and Sox9 during chondrogenesis in vitro in micromass cultures. (A) Schematic diagram of micromass culture method. Mesenchymal limb bud cells obtained from E10.5 embryos are dissociated by trypsin-collagenase treatment, (more ...)
Confocal immunofluorescence analysis of the fully formed 5-day-old nodules revealed reciprocity in HMGN1 and Sox9 protein levels at the single-cell resolution. The fully differentiated nodules present after 5 days of growth in micromass culture were clearly depleted of HMGN1 protein, while the undifferentiated cells surrounding the nodules expressed high levels of HMGN1 protein (Fig. , frame a). In contrast, Sox9 levels were high in the nodules but undetectable in the undifferentiated cells surrounding the nodules. Although higher magnifications revealed some residual HMGN1 protein in the nodule, a merge of the DNA and HMGN1 confocal images clearly demonstrates a reduced level of HMGN1 protein in the cells at the center of the nodules. In contrast, Sox9 levels are highest in the cells at the center of the nodule and gradually decrease toward the periphery of the nodule; Sox9 is absent from the cells surrounding the nodules. In the DNA-Sox9 image merge, the center of the nodule is green (high protein levels) and the surrounding cells are red (high DNA, low protein), while in the DNA-HMGN1 merge the opposite is visible: the center is red due to low HMGN1 (light green), while the surrounding cells are green due to relatively high levels of HMGN1 (Fig. , frame b).
Thus, the expression levels of Hmgn1 and Sox9 in the micromass cultures faithfully reproduce the Hmgn1 and Sox9 expression patterns observed in mouse embryos. Chondrocyte differentiation is associated with decreased levels of HMGN1 protein and increased levels of Sox9. Taken together, the data demonstrate reciprocity in the expression of Hmgn1 and Sox9.
HMGN1 modulates chondrocyte differentiation.
The reciprocal expression of HMGN1 and Sox9 raises the possibility that HMGN1 modulates the rate of chondrocyte differentiation. To test this possibility, we first compared the rate of nodule formation in micromass cultures prepared from cells derived from the limb buds of wild-type and Hmgn1−/− E10.5 embryos. On the basis of Alcian blue staining, Hmgn1−/− cultures were found to be differentiated faster than Hmgn1+/+ cultures at all time points examined (Fig. ). Quantification of Alcian blue staining confirmed that the rate of differentiation of the micromass prepared from Hmgn1−/− limb bud cells was significantly faster than that prepared from Hmgn1+/+ limb buds. After 4 days in culture, the amount of Alcian blue stain recovered from the Hmgn1−/− cultures was almost twice that obtained from Hmgn1+/+ cultures, and after 5 days it was still more than 1.5 times higher (Fig. ). Thus, genetic inactivation of Hmgn1 accelerated differentiation.
FIG.5. Loss of HMGN1 enhances the rate of differentiation. (A) Alcian blue staining of micromass cultures derived from E10.5 limb bud mesenchymal cells of Hmgn1−/− or Hmgn1+/+ embryos after different lengths of time in culture. (more ...)
To further test this possibility, we examined the effect of HMGN1 on cartilage nodule formation by reexpressing HMGN1 protein in Hmgn1−/− cultures. To this end, E10.5 limb bud mesenchymal cells prepared from Hmgn1−/− embryo were transiently transfected with vectors expressing either YFP (control) or the HMGN1-YFP fusion protein. Fluorescent analysis of the cells verified that the expression level of the control plasmid transcribing YFP was similar to that of the plasmid transcribing the HMGN1-YFP fusion protein (Fig. , frames a and b). The number and staining intensity of the Alcian blue-positive nodule in the cultures expressing the HMGN1-YFP fusion protein were significantly lower than those in the cultures expressing the control YFP protein (compare Fig. , frames d and e, and B). Likewise, the levels of Sox9 in cells expressing HMGN1-YFP were lower than in those expressing only YFP (Fig. , frames g and h). Thus, overexpression of HMGN1 inhibited the formation of differentiated nodules and the expression of Sox9, suggesting that down-regulation of HMGN1 is required for chondrocyte differentiation.
FIG. 6. HMGN1 reduces the rate of chondrocyte differentiation. (A) E10.5 limb bud mesenchyme cells derived from Hmgn1−/− embryos were transfected with plasmids expressing either YFP (Control), HMGN1-YFP (HMGN1) fusion protein or HMGN1(S20,24E)-YFP (more ...)
To test whether the HMGN1-induced inhibition of nodule formation is related to the ability of the protein to bind to chromatin, we expressed the double point mutant HMGN1(S20,24E)-YFP fusion protein, rather than the wild-type fusion protein, in the Hmgn1−/−
E10.5 limb bud cells. The HMGN1(S20,24E) mutant enters the nucleus but does not bind nucleosomes (26
). In contrast to the wild-type HMGN1, this mutant did not affect Sox9
expression (Fig. , frames f and i), and the rate of nodule formation was comparable to that observed in cells transfected with the control plasmid expressing YFP (Fig. , compare frames d and g to f and i, and B). Quantification of the effect by measuring the Alcian blue staining in the cultures (Fig. ) revealed that the amount of stain in the cultures expressing wild-type HMGN1 was 60% of that in control cells transfected with plasmids expressing either YFP or the HMGN1(S20,24E) mutant (Fig. ). Thus, reexpression of wild-type but not mutant HMGN1 in Hmgn1−/−
limb bud cells reduced the rate of Sox9
expression and chondrocyte differentiation. We therefore conclude that the interaction of HMGN1 with chromatin affects the rate of chondrocyte differentiation by modulating the expression levels of chondrogenic factors such as Sox9
Specific binding of HMGN1 to the Sox9 gene.
The interrelationship between HMGN1 and Sox9 expression raised the possibility that HMGN1 protein is directly involved in the regulation of Sox9 gene expression. We therefore tested whether HMGN1 protein was directly associated with the Sox9 gene by using a ChIP assay with affinity-purified antibodies against mouse HMGN1. The ChIP analyses were performed with chromatin isolated either from nonchondrogenic MEFs or from E10.5 limb bud cells, which contain both prechondrogenic cells and chondrocytes (Fig. ). The relative amounts of Hmgn1 transcripts and protein in the MEFs and limb bud cells are similar, while those of Sox9 are almost 10 times higher in the limb bud cells (Fig. ). However, although Sox9 expression is detected in the E10.5 limb bud, most of the cells are only poised to express the gene; high-level Sox9 expression occurs at later developmental stages, when HMGN1 is significantly down-regulated (Fig. ).
The DNA purified from the immunoprecipitated chromatin was amplified with 21 primer sets spanning an approximately 10-kb long genomic region encompassing the Sox9
gene and its 4.4-kb 5′ and 5.7-kb 3′ flanking regions. In limb bud cells, but not in MEFs, these analyses identified three regions in the Sox9
chromatin as enriched in HMGN1: a region 2 kb upstream of the promoter, exon 2, and exon 3 (Fig. ). The DNA sequences of the three regions do not contain any common motifs (data not shown), supporting previous findings that the interaction of HMGN1 with chromatin is not regulated by the sequence of the nucleosomal DNA (28
To test whether the presence of HMGN1 is associated with changes in chromatin structure, we first focused on the region spanned by primer set 3 (Fig. ), since its location was in the 5′ region of the gene and may be involved in the regulation of Sox9 gene expression. We digested nuclei isolated from E10.5 limb bud cells or MEFs with different amounts of DNase I and used PCR to determine the amount of undigested DNA in a 748-bp long region amplified by forward primer 3 and reverse primer 4. The yield of the resulting amplified fragment is a measure of the relative rate of DNA digestion in chromatin. A DNase I-hypersensitive site between primer sets 3 and 4 in the Sox9 promoter in nuclei prepared from E10.5 limb bud cells was digested faster than in MEF nuclei (Fig. ), suggesting that the chromatin structure of Sox9 promoter in limb bud cells, which are poised to begin Sox9 expression, is less compact than the corresponding region in MEFs. Similar results were obtained by analysis of other regions of the Sox9 gene, suggesting that the HMGN1-containing gene is more susceptible to DNase I digestion. Similar analysis of the DNase I sensitivity of the beta-globin gene, which is not expressed in either MEFs or limb bud cells, showed only minor differences between the two cell types (Fig. ). Our finding both in vivo and in vitro that the expression of Hmgn1 precedes that of Sox9 and that HMGN1 is associated with Sox9 chromatin in limb bud but not in MEFs argues for a role for HMGN1 in Sox9 gene regulation during chondrocyte development.
Enhanced binding of HMGN2 on the Sox9 gene in Hmgn1−/− mice.
The lack of phenotype in Hmgn1−/− mice raises the possibility that homeostatic mechanisms, perhaps involving HMGN2, compensate for loss of HMGN1 protein. The levels of HMGN2 RNA and protein in Hmgn1−/− mice is the same as in their Hmgn1+/+ littermates (not shown). ChIP analysis with affinity pure antibodies to HMGN2 revealed the levels of this protein in the 10.5-day limb bud Sox9 chromatin are higher than in the Sox9 chromatin of MEFs (Fig. ). Interestingly, while HMGN1 was enriched in distinct regions of the gene (Fig. ), HMGN2 protein was enriched along the entire length of the gene (Fig. ). Significantly, in the Sox9 chromatin derived from the limb bud of Hmgn1−/− mice, the level of HMGN2 was enriched in several regions, especially those spanning primer sets 6 to 10 and 17 to 19 (Fig. ). The increased level of HMGN2 in the Sox9 chromatin of the Hmgn1−/− limb bud suggests functional redundancy among HMGN proteins.
FIG. 8. Enhanced binding of HMGN2 on the Sox9 gene in Hmgn1−/− mice. (A) ChIP assay for HMGN2 on Sox9 in MEF and limb bud cells. Enrichment of each DNA sequence in the HMGN2 immunoprecipitate relative to the input DNA was normalized to the expression (more ...)