Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning

gb-2007-8-9-r184 GBJ Research Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning Doss Xavier Michael m-x.doss@uni-koeln.de Chen Shuhua shuhua.chen@uni-koeln.de Winkler Johannes j.winkler@uni-koeln.de Hippler-Altenburg Rita akp61@uni-koeln.de Odenthal Margareta m.odenthal@uni-koeln.de Wickenhauser Claudia c.wickenhauser@uni-koeln.de Balaraman Sridevi sridevi1576@yahoo.com Schulz Herbert heschulz@mdc-berlin.de Hummel Oliver o.hummel@mdc-berlin.de Hübner Norbert nhuebner@mdc-berlin.de Ghosh-Choudhury Nandini choudhury@uthscsa.edu Sotiriadou Isaia sisaia1@uni-koeln.de Hescheler Jürgen J.hescheler@uni-koeln.de Sachinidis Agapios a.sachinidis@uni-koeln.de

Institute of Neurophysiology, University of Cologne, Robert-Koch Str. 39, 50931 Cologne, Germany

Institute of Pathology, University of Cologne, Joseph-Stelzmann-Str. 9, 50931 Cologne, Germany

Max-Delbrueck-Center for Molecular Medicine - MDC, Robert-Rössle Str. 10, 13092 Berlin, Germany

Department of Pathology, The University of Texas Health Science Center at San Antonio, TX 78229, USA

Genome Biology 1465-6906 2007 8 9 R184 http://genomebiology.com/2007/8/9/R184 17784959 10.1186/gb-2007-8-9-r184 11 12 2006 30 5 2007 4 9 2007 04 09 2007 2007 Doss et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Transcriptome and phenotype of BMP2⁺cells

Transcriptome analysis of BMP2⁺cells in comparison to the undifferentiated BMP2 ES cells and the control population from 7-day old embryoid bodies led to the identification of 479 specifically upregulated and 193 downregulated transcripts.

Abstract

Background

Bone morphogenetic protein (BMP)2 is a late mesodermal marker expressed during vertebrate development and plays a crucial role in early embryonic development. The nature of the BMP2-expressing cells during the early stages of embryonic development, their transcriptome and cell phenotypes developed from these cells have not yet been characterized.

Results

We generated a transgenic BMP2 embryonic stem (ES) cell lineage expressing both puromycin acetyltransferase and enhanced green fluorescent protein (EGFP) driven by the BMP2 promoter. Puromycin resistant and EGFP positive BMP2⁺cells with a purity of over 93% were isolated. Complete transcriptome analysis of BMP2⁺cells in comparison to the undifferentiated ES cells and the control population from seven-day-old embryoid bodies (EBs; intersection of genes differentially expressed between undifferentiated ES cells and BMP2⁺EBs as well as differentially expressed between seven-day-old control EBs and BMP2⁺EBs by t-test, p < 0.01, fold change >2) by microarray analysis led to identification of 479 specifically upregulated and 193 downregulated transcripts. Transcription factors, apoptosis promoting factors and other signaling molecules involved in early embryonic development are mainly upregulated in BMP2⁺cells. Long-term differentiation of the BMP2⁺cells resulted in neural crest stem cells (NCSCs), smooth muscle cells, epithelial-like cells, neuronal-like cells, osteoblasts and monocytes. Interestingly, development of cardiomyocytes from the BMP2⁺cells requires secondary EB formation.

Conclusion

This is the first study to identify the complete transcriptome of BMP2⁺cells and cell phenotypes from a mesodermal origin, thus offering an insight into the role of BMP2⁺cells during embryonic developmental processes in vivo.

Development Genome studies

Background

Bone morphogenetic protein (BMP)2 is a member of the transforming growth factor (TGF)-β superfamily and plays a crucial role in early embryonic patterning as shown by gene ablation studies 12. It is normally expressed in lateral plate mesoderm and extraembryonic mesoderm 13. BMP2⁺mesodermal cells at this stage comprise a subset of mesoderm, the lateral plate cardiogenic mesoderm 4. BMP2 expression immediately follows the transient expression of T-Brachyury in the nascent mesoderm. Interestingly, administration of soluble BMP2 to chick embryo explant cultures induces full cardiac differentiation in stage 5-7 anterior medial mesoderm, a tissue that is normally not cardiogenic 5.

Since BMP2 is a cardiogenic factor as well as expressed in the cardiogenic mesoderm, it is highly imperative to investigate the molecular nature and phenotype of the mesodermal cells expressing BMP2 during the early stages of development in the context of cardiomyogenesis. Also, it has been well documented that BMP2 is a potent apoptotic inducer and a potent neurotrophic factor, acting on target cells in a concentration gradient-dependant manner, mostly through its paracrine mode of action 678. Thus, BMP2 plays a pivotal role not only during cardiomyogenesis but also during other early embryonic patterning and lineage specification. To date, the molecular nature and phenotype of the mesodermal cells expressing BMP2 during the early stages of development have not been characterized, leaving a gap in our understanding of their molecular interactions with target cells and, thus, their role during early embryonic patterning and cell lineage commitment. This is due, in part, to the pleiotrophic effects of BMP2 and largely because of the practical difficulty in isolating pure early stage BMP2-expressing cells in sufficient quantities during early embryonic development in vivo. Extensive investigations applying the in vitro embryonic stem (ES) cell-based developmental model in the past two decades have proven its value for the elucidation of developmental processes during embryonic development, in particular, the mechanisms by which lineage commitment occurs during early embryogenesis 9.

To circumvent the practical difficulties in the isolation of BMP2-expressing cells in sufficient quantities during embryonic development in vivo, and to address the molecular nature and behaviour of the BMP2⁺mesodermal cells during their differentiation into specific somatic cell lineages, we first established an ES cell-derived transgenic BMP2 cell lineage expressing both puromycin acetyltransferase and enhanced green fluorescent protein (EGFP) under the control of the BMP2 promoter. In order to identify all possible signal transduction pathways and biological processes characteristic of the BMP2⁺cells, we performed expression studies using Affymetrix microarrays. Our study on the phenotypic identification of the ES cell-derived BMP2 lineage-specific cells shows that the early BMP2⁺population contained a heterogeneous population of predominantly NCSCs and their lineages - smooth muscle cells, epithelial like cells, astrocytes and melanocytes. When the early BMP2⁺population was further cultured under certain conditions, it contained cardiomyocytes, macrophages and osteoblasts. Interestingly, these are the cell phenotypes that need BMP2 for their phenotypic induction. Our work clearly demonstrates the presence of a multi-lineage progenitor phenotype resembling NCSCs cells in early ES cell-derived BMP2⁺cells. Moreover, identification of the key signal transduction pathways induced or repressed in BMP2⁺cells explains the observed potential of BMP2 in modulating early embryonic development, in particular the mesodermal patterning.

Results and discussion

Isolation of BMP2⁺cells from the transgenic BMP2 ES cell lineage

The transgenic BMP2 ES cell lineage was generated with the linearized pBMP2^p-puro IRES2 EGFP construct by stable transfection. Like its parental wild type CGR8, the BMP2 ES cells do not express BMP2 in the undifferentiated state (Figure 1a(i) and 1a(ii)). Expression of BMP2 during progressive differentiation induced by the hanging drop protocol (see Materials and methods) starts in the three-day-old embryoid bodies (EBs), gradually increases to a maximum in the five-day-old EBs and, thereafter, gradually decreases to a minimum in ten-day-old EBs, in the same manner as that seen in the RT-PCR results (Figure 1a). During the course of differentiation induced by the hanging drop protocol, the EGFP-expressing cells in the three- and four-day-old EBs were found to be scattered (Figure 1b). As differentiation continues, the EGFP fluorescence peaks in the five-day-old EBs and the EGFP-expressing cells are localized to a particular region in every EB, as shown in Figure 1b. The RNAs isolated from these EBs were analyzed for the expression of other candidate markers (T-bra, flk1, smooth muscle α-actin, neurofilament-H (NF-H) and also α-fetoprotein (AFP)) to demonstrate that these EBs were differentiating in the normal way as per their parental wild-type EBs (Additional data file 1). Isolation and further characterization of the BMP2⁺, puromycin-resistant cells were optimized according to the protocol described in Figure 1c. Briefly, a single cell suspension of BMP2⁺ES cells was seeded in bacteriological dishes for two days to form two-day-old EBs. These were then transferred into gelatine coated tissue culture dishes and cultured for a further two days. Thereafter, plated EBs were treated with 3 μg/ml puromycin for three days. After trypsinization of puromycin-resistant seven-day-old BMP2⁺cells, fluorescence-activated cell sorting (FACS) analysis was performed. As demonstrated in Figure 1d,e, after 3 days of puromycin treatment, EGFP fluorescing and puromycin resistant BMP2⁺cells (hereafter called BMP2⁺cells) accounted for 93% of the cells in the EBs, whereas in the control EBs without puromycin treatment (hereafter called control EBs (seven-day-old EBs)) only 11% of the cells were BMP2⁺cells. This result demonstrates a nearly 8.5-fold enrichment of BMP2⁺cells in EBs treated with puromycin. As demonstrated in Figure 1f, plating of the BMP2⁺cells in gelatine coated tissue culture dishes for another three days in the presence of puromycin results in a bright EGFP-positive BMP2⁺cell population. Furthermore, the BMP2 protein was detected by immunostaining using BMP2-specific antibodies. The undifferentiated BMP2 ES cells were included as a negative control (Figure 1h). As demonstrated, BMP2 is detected only in the cytosol, and specifically in vesicles, of the BMP2⁺cells (Figure 1g).

Figure 1

Expression pattern of BMP2 in differentiating EBs

Expression pattern of BMP2 in differentiating EBs. (a) Detection of the expression of BMP2 by qPCR on the samples from EBs derived from wild-type CGR8 ES cells (i) and RT-PCR (ii) on samples from EBs derived from BMP2 ES cells (for conditions, see Additional data file 14). The qPCR results are presented as the mean of three independent experiments ± standard deviation. (b) Expression of EGFP during differentiation of the BMP2 ES cells induced by the conventional hanging drop protocol. Scale bar represents 50 μm. (c) Protocol for isolation of puromycin resistant BMP2⁺cells after treating the plated four-day-old EBs with 3 μg/ml puromycin for three days. (d,e) FACS analysis of the trypsinized untreated control and puromycin resistant BMP2⁺cells. (f) BMP2⁺, three days after plating in gelatine-coated tissue culture dishes in the presence of 3 μg/ml puromycin. Scale bar represents 50 μm. (g,h) Detection of BMP2 in BMP2⁺cells (g) or ES cells (h) by immunohistochemistry staining. Stainings were done after the cells were trypsinized and plated on microscopic slides for 24 hours. Scale bar represents 20 μm.

Functional categorization of transcripts upregulated in BMP2⁺cells

The Affymetrix data obtained were validated by quantitative real time PCR (qPCR; Additional data file 2). To identify Gene Ontology (GO) 10 categories, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways 11 and BioCarta pathways 12 specifically enriched in BMP2⁺cells, we first analyzed genes that are upregulated in BMP2⁺cells in comparison to control EBs. Moreover, to identify BMP2⁺cell-specific genes, a three condition comparative analysis of the BMP2⁺cells to control EBs and to BMP2 ES cells was made (Tables 1 and 2).

Table 1

Functional annotations enriched among genes upregulated* in BMP2⁺cells compared to control cells in seven-day-old EBs

Category

Term

Count

p value

GOTERM_MF_5

Zinc ion binding

5.6E-5

GOTERM_CC_5

Nucleus

3.3E-4

GOTERM_BP_5

Cellular protein metabolism

3.3E-4

GOTERM_BP_5

Protein catabolism

4.9E-3

GOTERM_BP_5

Apoptosis

5.9E-3

GOTERM_BP_5

Biopolymer modification

6.0E-3

GOTERM_BP_5

Biopolymer catabolism

6.9E-3

GOTERM_BP_5

Positive regulation of programmed cell death

9.1E-3

GOTERM_MF_5

ATP binding

1.4E-2

GOTERM_BP_5

Response to unfolded protein

1.5E-2

GOTERM_BP_5

Transcription

1.5E-2

GOTERM_BP_5

Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism

2.7E-2

GOTERM_BP_5

Regulation of programmed cell death

3.8E-2

GOTERM_BP_5

Regulation of progression through cell cycle

5.1E-2

GOTERM_BP_5

Protein kinase cascade

5.3E-2

GOTERM_BP_5

Regulation of protein kinase activity

5.6E-2

GOTERM_BP_5

Small GTPase mediated signal transduction

5.6E-2

GOTERM_BP_5

Post replication repair

6.2E-2

GOTERM_MF_5

Ubiquitin-protein ligase activity

6.2E-2

GOTERM_BP_5

Regulation of apoptosis

7.4E-2

GOTERM_BP_5

Protein transport

7.5E-2

GOTERM_MF_5

Protein kinase activity

8.3E-2

KEGG_REACTION

Phytoceramide+h2o<=>fattyacid+phytosphingosine

8.7E-2

GOTERM_CC_5

Lytic vacuole

8.8E-2

KEGG_PATHWAY

MAPK signaling pathway

9.5E-2

Table 1 indicates different GO terms (level 5) and one KEGG pathway (mitogen-activated protein kinase (MAPK) signaling pathway) that are enriched in the BMP2⁺cells compared to control EBs. In the GO categories 'biological process' (GOTERM_BP), 'molecular function' (GOTERM_MF) and 'cellular component' (GOTERM_CC), several relevant categories are pooled, such as transcriptional processes (for zinc ion binding and transcription) and apoptotic processes (for apoptosis, cell death, death, programmed cell death and regulation of apoptosis) (Table 1). Genes for transcriptional activity and associated with apoptosis are also found to be specifically enriched in the BMP2⁺cells (Table 2). These results suggest that BMP2 causes direct or indirect induction of apoptotic processes. This hypothesis is supported by the observations that apoptotic effects of BMP2 promote cavitation in EBs and in mouse embryos 13. The MAPK signaling pathway involved in apoptotic processes 14 seems to be specific for the BMP2⁺cell population (Table 2). It is well established that the MAPK signaling pathway is involved in several processes, including cell cycle progression, cellular transformation apoptosis and differentiation (for a review, see 14). These results suggest that both apoptosis and transcription genes are characteristic gene expression signatures for the BMP2⁺cells.

Additional data files 3-5 list all the genes and include the change factor (CF) values belonging to the GO categories 'transcription' and 'apoptosis' and the KEGG 'MAPK pathway', respectively. Among genes highly upregulated specifically in the BMP2⁺cells, Gm397 (gene model 397) and Tbx4 are identified (Additional data file 3). Tbx4 has been shown to be expressed in the lateral mesoderm and is involved in limb outgrowth in the mouse 15 whereas the function of Gm397 is unknown.

Interestingly, among the MAPKs, several kinases belonging to both the classical and the c-Jun amino-terminal kinase (JNK) and p38 MAP kinase pathway were overexpressed in BMP2⁺cells (Additional data file 5, KEGG pathway scheme). The JNK and p38 MAPK pathway is known to be stimulated by serum and stress factors 14. The most striking gene specifically upregulated in BMP2⁺cells was Hspa1a (heat shock protein 1A; Additional data file 5), which belongs to the Hsp70 family of stress response genes. Members of this family participate in the process of folding and refolding of misfolded proteins and in the transport of proteins across membranes 16. Hsp1a is also found to be upregulated in chondrons, which includes the chondrocyte and its pericellular matrix, compared to chondrocytes 17.

Table 3 lists the developmental genes that are overexpressed in the BMP2⁺cells compared to control EBs. Among these, Dnmt3l (DNA (cytosine-5-)-methyltransferase,3-like), Fgf4 (fibroblast growth factor 4), Tdgf1 (teratocarcinoma derived growth factor), Zic1 (zinc finger protein of the cerebellum 1), Ifrd1 (interferon-related developmental regulator 1), Tbx4 (T-Box 4), and Neurod1 (neurogenic differentiation 1) were highly expressed in the BMP2⁺cells. DNA methylation of the genome is essential for mammalian development and plays crucial roles in a variety of biological processes including genomic imprinting 1819. Dnmt3l^mat-/-mice die before mid-gestation due to an imprinting defect 18. In addition, Dnmt3L is required for differentiation in the extra-embryonic tissue 18. Molecular and genetic data indicate that FGF signaling plays a major role in regulating trophoblast proliferation and differentiation 20. Fgf4 is expressed in early embryos, becoming restricted to the inner cell mass (ICM) of the blastocyst and later to the epiblast of the early post-implantation embryo 20.

Table 3

Genes of GO category 'development' upregulated at least two-fold* in BMP2⁺cells compared to control cells in seven-day-old EBs

Affymetrix ID

Gene name

Fold change

1425035_s_at

dna (cytosine-5-)-methyltransferase 3-like

14.0

1420086_x_at

fibroblast growth factor 4

9.4

1450989_at

teratocarcinoma-derived growth factor

9.0

1423477_at

zinc finger protein of the cerebellum 1

7.5

1416067_at

interferon-related developmental regulator 1

7.0

1456033_at

t-box 4

6.8

1426412_at

neurogenic differentiation 1

5.7

1418640_at

sir2 alpha

4.3

1456341_a_at

kruppel-like factor 9

4.1

1424607_a_at

xanthine dehydrogenase

3.7

1452240_at

bruno-like 4, rna binding protein (drosophila)

3.7

1452179_at

phd finger protein 17

3.6

1416455_a_at

crystallin, alpha b

3.5

1416953_at

connective tissue growth factor

3.2

1428334_at

osteopetrosis associated transmembrane protein 1

3.1

1418901_at

ccaat/enhancer binding protein (c/ebp), beta

2.8

1421151_a_at

eph receptor a2

2.8

1422556_at

guanine nucleotide binding protein, alpha 13

2.7

1434009_at

glucocorticoid receptor dna binding factor 1

2.6

1434054_at

v-maf musculoaponeurotic fibrosarcoma oncogene family, protein g (avian)

2.5

1422057_at

nodal

2.5

1436164_at

solute carrier family 30 (zinc transporter), member 1

2.5

1422033_a_at

ciliary neurotrophic factor

2.5

1449949_a_at

coxsackievirus and adenovirus receptor

2.5

1433455_at

linker of t-cell receptor pathways

2.5

1425932_a_at

cug triplet repeat, rna binding protein 1

2.4

1451383_a_at

conserved helix-loop-helix ubiquitous kinase

2.4

1455222_a_at

upstream binding protein 1

2.4

1451257_at

acyl-coa synthetase long-chain family member 6

2.4

1426858_at

inhibin beta-b

2.3

1421624_a_at

enabled homolog (drosophila)

2.3

1437540_at

mucolipin 3

2.3

1429192_at

sloan-kettering viral oncogene homolog

2.2

1452438_s_at

taf4a rna polymerase ii, tata box binding protein (tbp)-associated factor

2.2

1436907_at

neuron navigator 1

2.1

1450986_at

nucleolar protein 5

2.0

1416904_at

muscleblind-like 1 (drosophila)

2.0

*Student's t-test, p value < 0.01.

Teratocarcinoma-derived growth factor (encoded by Tdgf1, also known as Cripto-1) plays a pivotal role as a multifunctional modulator during embryogenesis and oncogenesis, and may be involved in stem cell maintenance 21. NeuroD1 is a member of the basic helix-loop-helix transcription factor family and has been shown to play a major role in development of the nervous system and formation of the endocrine system 22. The transcription factor ZIC1 plays important roles in patterning the neural plate in early vertebrate development. Zic1 expression was detected in the neural plate border, dorsal neural tube, and somites 23. Moreover, Zic1 plays an important role in early patterning of the Xenopus presumptive neurectoderm 24.

Interferon-related developmental regulator 1 (IFRD1; also known as PC4, Tis7) is a chromatin-associated protein that induces chromatin condensation and plays multiple roles in cellular processes, including transcription, DNA replication and repair 25. It is expressed early in the mouse embryo and extra-embryonic tissues during gastrulation and at mid-gestation in restricted structures (such as the central nervous system, kidney, and lung primordia), whereas it is ubiquitously expressed at late gestation 26. IFRD1 has been shown to act as a coactivator of myogenic differentiation 1 (MyoD1) and myocyte enhancer factor 2C (MEF2C) during myogenesis 27.

The three condition comparative analysis results in a set of seven BMP2⁺cell-specific genes (Table 4). Among these, the most prominently regulated genes are Zic1, Ifrd1 and Tbx4, which have been discussed previously. Ciliary neurotrophic factor (CNTF) is of particular interest. CNTF is a cytokine with neurotrophic and differentiating effects on central nervous system cells and myotrophic effects on skeletal muscle 28.

Table 4

Genes of GO category 'development' upregulated at least two-fold* in BMP2⁺cells compared to control cells in seven-day-old EBs and undifferentiated BMP2 ES cells

Affymetrix ID

Gene name

Fold change BMP2⁺versus BMP2 EBs

Fold change BMP2⁺versus BMP2 ES cells

1423477_at

zinc finger protein of the cerebellum 1

7.5

8.8

1416067_at

interferon-related developmental regulator 1

7.1

1456033_at

T-box 4

6.8

6.4

1434009_at

RIKEN cDNA 6430596G11 gene

2.6

3.4

1422033_a_at

ciliary neurotrophic factor

2.5

3.2

1425932_a_at

CUG triplet repeat, RNA binding protein 1

2.4

2.5

1416904_at

muscleblind-like 1 (Drosophila)

2.7

*Student's t-test, p value < 0.01.

GO enrichment analysis of the genes downregulated in BMP2⁺cells

To identify overrepresented GO categories or KEGG pathways specifically downregulated in BMP2⁺cells, we analyzed the data with the DAVID bioinformatics resource 29. Comparative analysis of the expression level of genes in BMP2⁺and in control EBs shows downregulated genes belong to several overrepresented GO categories, such as focal adhesion, TGF-β signaling pathway, extracellular matrix (ECM)-receptor interaction and shh signaling pathway (Table 5). Some overrepresented categories are related to the developmental processes (for example, development, organ development, embryonic development and brain development; (Table 5, entries in bold). This is not surprising, since the seven-day-old control EBs can still develop into various somatic precursor cells, as indicated in the tables (for example, vasculature development and brain development). Notably, GO categories associated with impaired developmental processes appear not to be characteristic of BMP2⁺cells when the expression levels of these genes in undifferentiated ES cells are also taken into account. These results clearly show that the BMP2⁺cells are more closely related to the undifferentiated ES cells than to the control EBs with regard to their developmental potential and plasticity.

Table 5

Functional annotations (GO, KEGG, Biocarta) enriched in transcripts downregulated* in BMP2⁺cells compared to control cells in seven-day-old EBs