Published papers

The mouse hair coat comprises protective "primary" and thermo-regulatory "secondary" hairs. Primary hair formation is ectodysplasin (Eda) dependent, but it has been puzzling that Tabby (Eda-/y) mice still make secondary hair. We report that Dickkopf 4 (Dkk4), a Wnt antagonist, affects an auxiliary pathway for Eda-independent development of secondary hair. A Dkk4 transgene in wild-type mice had no effect on primary hair, but secondary hairs were severely malformed. Dkk4 action on secondary hair was further demonstrated when the transgene was introduced into Tabby mice: the usual secondary follicle induction was completely blocked. The Dkk4-regulated secondary hair pathway, like the Eda-dependent primary hair pathway, is further mediated by selective activation of Shh. The results thus reveal two complex molecular pathways that distinctly regulate subtype-based morphogenesis of hair follicles, and provide a resolution for the longstanding puzzle of hair formation in Tabby mice lacking Eda. © 2010 Greenbaum et al.

Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell's identity by showing similarities or differences among two or multiple cell types; (5) to find regulatory pathways and/or networks affected by gene manipulations, such as overexpression or repression of gene expression; (6) to find downstream target genes of transcription factors; (7) to find downstream target genes of cell signaling; (8) to examine the effects of environmental manipulation of cells on gene expression patterns; and (9) to find the effects of genetic manipulation in embryos and adults. Here, we describe strategies for executing these experiments and monitoring changes of cell state with gene expression microarrays in application to mouse embryology. Both statistical assessment and interpretation of data are discussed. We also present a protocol for performing microarray analysis on a small amount of embryonic materials. © 2010 Elsevier Inc.

Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.

ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically ''undifferentiated'' cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly when introduced back into morulae or blastocysts, the V+S+ population is not effective at contributing to the epiblast and can contribute to the extra-embryonic visceral and parietal endoderm, while the V2S+ population generates high contribution chimeras. Taken together our data support a model in which ES cell culture has trapped a set of interconvertible cell states reminiscent of the early stages in blastocyst differentiation that may exist only transiently in the early embryo.

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells. © 2010 Macmillan Publishers Limited. All rights reserved.

During the preimplantation phase of pregnancy the endometrial stroma differentiates into decidua, a process that implies numerous morphological changes and is an example of physiological transdifferentiation. Here we show that UIII rat endometrial stromal cells cultured in the presence of calf serum acquired morphological features of decidual cells and expressed decidual markers. To identify genes involved in decidualization we compared gene expression patterns of control and decidualized UIII cells using cDNA microarray. We found 322 annotated genes exhibiting significant differences in expression (>3-fold, fold discovery rate (FDR) >0.005), of which 312 have not been previously related to decidualization. Analysis of overrepresented functions revealed that protein synthesis, gene expression, and chromatin architecture and remodeling are the most relevant modified functions during decidualization. Relevant genes are also found in the functional terms differentiation, cell proliferation, signal transduction, and matrix/structural proteins. Several of these new genes involved in decidualization (Csdc2, Trim27, Eef1a1, Bmp1, Wt1, Aes, Gna12, and Men1) are shown to be also regulated in uterine decidua during normal pregnancy. Thus, the UIII cell culture model will allow future mechanistic studies to define the transcriptional network regulating reprogramming of stromal cells into decidual cells. © 2009 Wiley-Liss, Inc.

Sweat glands play a fundamental role in thermal regulation in man, but the molecular mechanism of their development remains unknown. To initiate analyses, we compared the model of Eda mutant Tabby mice, in which sweat glands were not formed, with wild-type (WT) mice. We inferred developmental stages and critical genes based on observations at seven time points spanning embryonic, postnatal and adult life. In WT footpads, sweat gland germs were detected at E17.5. The coiling of secretory portions started at postnatal day 1 (P1), and sweat gland formation was essentially completed by P5. Consistent with a controlled morphological progression, expression profiling revealed stage-specific gene expression changes. Similar to the development of hair follicles - the other major skin appendage controlled by EDA - sweat gland induction and initial progression were accompanied by Eda-dependent up-regulation of the Shh pathway. During the further development of sweat gland secretory portions, Foxa1 and Foxi1, not at all expressed in hair follicles, were progressively up-regulated in WT but not in Tabby footpads. Upon completion of WT development, Shh declined to Tabby levels, but Fox family genes remained at elevated levels in mature sweat glands. The results provide a framework for the further analysis of phased down-stream regulation of gene action, possibly by a signaling cascade, in response to Eda.

We describe the identification and characterization of Trim43a, Trim43b, and Trim43c genes, whose expression are restricted to preimplantation stages and peak at the 8-cell to morula stage. We identified a 5 kb DNA fragment that covers upstream region of Trim43a as a putative promoter, which can drive the expression of mStrawberry fluorescent protein in a manner similar to endogenous Trim43 genes. Trim43 genes will be useful stage-specific markers for the study of preimplantation embryos.

We present CisFinder software, which generates a comprehensive list of motifs enriched in a set of DNA sequences and describes them with position frequency matrices (PFMs). A new algorithm was designed to estimate PFMs directly from counts of n-mer words with and without gaps; then PFMs are extended over gaps and flanking regions and clustered to generate non-redundant sets of motifs. The algorithm successfully identified binding motifs for 12 transcription factors (TFs) in embryonic stem cells based on published chromatin immunoprecipitation sequencing data. Furthermore, CisFinder successfully identified alternative binding motifs of TFs (e.g. POU5F1, ESRRB, and CTCF) and motifs for known and unknown co-factors of genes associated with the pluripotent state of ES cells. CisFinder also showed robust performance in the identification of motifs that were only slightly enriched in a set of DNA sequences.

To examine transcription factor (TF) network(s), we created mouse ESC lines, in each of which 1 of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable tetracycline-repressible locus. Of the 50 TFs, Cdx2 provoked the most extensive transcriptome perturbation in ESCs, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. ChIP-Seq revealed that CDX2 binds to promoters of upregulated target genes. By contrast, genes downregulated by CDX2 did not show CDX2 binding but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also downregulated by the induction of at least 15 other TFs, suggesting a common initial step for ESC differentiation mediated by interference with the binding of core TFs to their target genes. These ESC lines provide a fundamental resource to study biological networks in ESCs and mice. © 2009 Elsevier Inc. All rights reserved.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Transcription factor Oct3/4 is an indispensable factor in the self-renewal of ES cells. In this study, we searched for a protein that would interact with Oct3/4 in ES cells and identified an orphan nuclear hormone receptor, Dax1. The association of Dax1 with Oct3/4 was mediated through the POU-specific domain of Oct3/4. Ectopic expression of Dax1 inhibited Oct3/4-mediated activation of an artificial Oct3/4-responsive promoter. Expression of Dax1 in ES cells also reduced the activities of Nanog and Rex1 promoters, while knockdown of Dax1 increased these activities. Pulldown and gel shift assays revealed that the interaction of Dax1 with Oct3/4 abolished the DNA binding activity of Oct3/4. Chromatin immunoprecipitation assay results showed that Dax1 inhibited Oct3/4 binding to the promoter/enhancer regions of Oct3/4 and Nanog. Furthermore, overexpression of Dax1 resulted in ES cell differentiation. Taken together, these data suggest that Dax1, a novel molecule interacting with Oct3/4, functions as a negative regulator of Oct3/4 in ES cells. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three 'cell lineage trajectories', which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation.

Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 3′-untranslated region (UTR) and CpG di-nucleotides in the 5′-UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells.

Background. Rex1/Zfp42 has been extensively used as a marker for the undifferentiated state of pluripotent stem cells. However, its function in pluripotent stem cells including embryonic stem (ES) cells remained unclear although its involvement in visceral endoderm differentiation in F9 embryonal carcinoma (EC) cells was reported. Results. We showed the function of Rex1 in mouse ES cells as well as in embryos using the conventional gene targeting strategy. Our results clearly indicated that Rex1 function is dispensable for both the maintenance of pluripotency in ES cells and the development of embryos. However, Rex1-/- ES cells showed the defect to induce a subset of the marker genes of visceral endoderm, when differentiated as embryoid body, as found in EC cells. Conclusion. Rex1 should be regarded just as a marker of pluripotency without functional significance like the activity of alkaline phosphatase. © 2008 Masui et al; licensee BioMed Central Ltd.

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

Background: Identification of protein-protein interactions is an important first step to understand living systems. High-throughput experimental approaches have accumulated large amount of information on protein-protein interactions in human and other model organisms. Such interaction information has been successfully transferred to other species, in which the experimental data are limited. However, the annotation transfer method could yield false positive interologs due to the lack of conservation of interactions when applied to phylogenetically distant organisms. Results: To address this issue, we used phylogenetic profile method to filter false positives in interologs based on the notion that evolutionary conserved interactions show similar patterns of occurrence along the genomes. The approach was applied to Mus musculus, in which the experimentally identified interactions are limited. We first inferred the protein-protein interactions in Mus musculus by using two approaches: i) identifying mouse orthologs of interacting proteins (interologs) based on the experimental protein-protein interaction data from other organisms; and ii) analyzing frequency of mouse ortholog co-occurrence in predicted operons of bacteria. We then filtered possible false-positives in the predicted interactions using the phylogenetic profiles. We found that this filtering method significantly increased the frequency of interacting protein-pairs coexpressed in the same cells/tissues in gene expression omnibus (GEO) database as well as the frequency of interacting protein-pairs shared the similar Gene Ontology (GO) terms for biological processes and cellular localizations. The data supports the notion that phylogenetic profile helps to reduce the number of false positives in interologs. Conclusion: We have developed protein-protein interaction database in mouse, which contains 41109 interologs. We have also developed a web interface to facilitate the use of database http://lgsun.grc.nia.nih.gov/ mppi/. © 2008 Yellaboina et al; licensee BioMed Central Ltd.

The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. © 2008 Wiley-Liss, Inc.

Background: The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR). Results: We found that gene expression changes occurred in aging gonads, but were generally different from those in somatic organs during aging. For example, only two functional categories of genes previously associated with aging in muscle, kidney, and brain were confirmed in ovary: genes associated with complement activation were upregulated, and genes associated with mitochondrial electron transport were downregulated. The bulk of the changes in gonads were mostly related to gonad-specific functions. Ovaries showed extensive gene expression changes with age, especially in the period when ovulation ceases (from 6 to 16 months), whereas testes showed only limited age-related changes. The same trend was seen for the effects of CR: CR-mediated reversal of age-associated gene expression changes, reported in somatic organs previously, was limited to a small number of genes in gonads. Instead, in both ovary and testis, CR caused small and mostly gonad-specific effects: suppression of ovulation in ovary and activation of testis-specific genes in testis. Conclusion: Overall, the results are consistent with unique modes of aging and its modification by CR in testis and ovary. © 2008 Sharov et al; licensee BioMed Central Ltd.

Background: Target genes of a transcription factor (TF) Pou5f1 (Oct3/4 or Oct4), which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES) cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP)-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results: To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR) criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after Pou5f1 suppression) and published ChIP data, we identified 420 tentative target genes (TTGs) for Pou5f1. The majority of TTGs (372) were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion: We have identified the most reliable sets of direct target genes for key pluripotency genes - Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly. © 2008 Sharov et al; licensee BioMed Central Ltd.