The interplay between epigenetic changes and the p53 protein in stem cells.

Epigenetic programs regulate the development and maintenance of organisms over a lifetime. These programs are carried out through chemical modifications of DNA and proteins such as histones and transcription factors. These epigenetic modifications are less stable than genetic alterations and even reversible under a variety of circumstances, such as developmental changes, regeneration of tissues, cell divisions, aging, and pathological conditions observed in many cancers. The p53 protein not only enforces the stability of the genome by the prevention of genetic alterations in cells but also plays a role in regulating the epigenetic changes that can occur in cells. The full-length p53 protein is largely inactive in stem cells but, when activated, helps to commit these cells to developmental lineages through a series of epigenetic changes. Just as p53 impacts epigenetic change, the enzyme activities that carry out epigenetic protein modifications act on the p53 protein and its splice variants in stem and progenitor cells to silence or activate its transcriptional activities. Thus, there is a great deal of cross-talk between the p53 protein and epigenetic programs. This review collects the diverse experimental evidence that leads to these conclusions. This in turn permits new ideas and directions for the treatment of cancers, reactivating developmental pathways for tissue regeneration and responses to the impact of aging.

Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.

Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor.

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.

Frizzled gene expression and negative regulation of canonical WNT-ß-catenin signaling in mouse F9 teratocarcinoma cells.

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-ß-catenin pathway. The upregulation of Wnt6 and activation of ß-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-ß-catenin signaling, thereby allowing PE cells to differentiate.

Parp-1 deficiency in ES cells promotes invasive and metastatic lesions accompanying induction of trophoblast giant cells during tumorigenesis in uterine environment.

Embryonic stem (ES) cells deficient in poly(ADP-ribose) polymerase-1 (Parp-1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp-1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild-type (Parp-1(+/+) ) and Parp-1(-/-) ES cells. The weights of the tumors derived from Parp-1(-/-) ES cells were lower than those of the tumors derived from Parp-1(+/+) ES cells (P < 0.05). The Parp-1(-/-) tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp-1(-/-) tumors, but not for the Parp-1(+/+) tumors (P < 0.05). Invasions were more frequently observed with the Parp-1(-/-) tumors compared with the Parp-1(+/+) tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp-1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.

A mutation in the Ter gene causing increased susceptibility to testicular teratomas maps to mouse chromosome 18.

Little is known about inherited susceptibility to spontaneous germ cells tumours in humans or other species. The Ter mutation in laboratory mice is novel in that it acts codominantly to reduce germ cell numbers on many inbred strain backgrounds and to enhance dramatically inherited predisposition to spontaneous testicular teratocarcinomas in strain 129 inbred mice. We have adopted a PCR-based, DNA pooling method for mice with 'extreme' phenotypes (small testes versus normal-sized testes) to identify a candidate linkage to the Ter locus. Two independent mapping approaches confirmed this evidence for Ter linkage near D18Mit62 on mouse chromosome 18, and suggest a possible human homologue on chromosome 5q.

Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors.

BACKGROUND: Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-ß signaling and activation of the ß-catenin pathway can induce EMT in intratumoral cancer cells. METHODS: We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC). RESULTS: Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating ß-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-ß was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-ß levels and tumor grade. CONCLUSIONS: Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.

The conserved translocase Tim17 prevents mitochondrial DNA loss.

Maintenance of an intact mitochondrial genome is essential for oxidative phosphorylation in all eukaryotes. Depletion of mitochondrial genome copy number can have severe pathological consequences due to loss of respiratory capacity. In Saccharomyces cerevisiae, several bifunctional metabolic enzymes have been shown to be required for mitochondrial DNA (mtDNA) maintenance. For example, Ilv5 is required for branched chain amino acid biosynthesis and mtDNA stability. We have identified OXA1 and TIM17 as novel multicopy suppressors of mtDNA instability in ilv5 cells. In addition, overexpression of TIM17, but not OXA1, prevents the complete loss of mtDNA in cells lacking the TFAM homologue Abf2. Introduction of the disease-associated A3243G mutant mtDNA into human NT2 teratocarcinoma cells frequently causes mtDNA loss. Yet when human TIM17A is overexpressed in NT2 cybrids carrying A3243G mtDNA, the proportion of cybrid clones maintaining mtDNA increases significantly. TIM17A overexpression results in long-term mtDNA stabilization, since NT2 cybrids overexpressing TIM17A maintain mtDNA at levels similar to controls for several months. Tim17 is a conserved suppressor of mtDNA instability and is the first factor to be identified that can prevent mtDNA loss in a human cellular model of mitochondrial disease.

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes.

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.

Targeted Vezf1-null mutation impairs vascular structure formation during embryonic stem cell differentiation.

OBJECTIVE: Vezf1 encodes an early zinc finger transcription factor that is essential for normal vascular development and functions in a dose-dependent manner. Here, we investigated the role of Vezf1 during processes of endothelial cell differentiation and maturation by studying mutant Vezf1 embryonic stem (ES) cells using the in vitro embryoid body differentiation model and the in vivo teratocarcinoma model. METHODS AND RESULTS: Vezf1-/- ES cell-derived embryoid bodies failed to form a well-organized vascular network and showed dramatic vascular sprouting defects. Our results indicate that the retinol pathway is an important mediator of Vezf1 function and that loss of Vezf1 results in reduced retinol/vitamin A signaling and aberrant extracellular matrix (ECM) formation. Unexpectedly, we also uncovered defects during in vitro differentiation of Vezf1-/- ES cells along hematopoietic cell lineages. Vezf1-/- ES cell-derived teratocarcinomas were able to spontaneously differentiate into cell types of all 3 germ layers. However, histological and immunohistochemical examination of these tumors showed decreased cell proliferation, delayed differentiation, and large foci of cells with extensive deposition of ECM. Embryoid bodies and teratocarcinomas derived from heterozygous ES cells displayed an intermediate phenotype. CONCLUSIONS: Together, these results suggest that Vezf1 is involved in early differentiation processes of the vasculature by regulating cell differentiation, proliferation, and ECM distribution and deposition.

[Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency].

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E_ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency. [corrected].

LINC00324 facilitates cell proliferation through competing for miR­214­5p in immature ovarian teratocarcinoma.

Immature ovarian teratocarcinoma (IOT) is a rare and malignant type of ovarian teratoma, and the molecular mechanisms underlying the pathogenesis and malignant phenotype of IOT remain uncharacterized. The present study examined a long non­coding RNA (lncRNA), long­chain intergenic non­coding RNA324 (LINC00324), which may serve a crucial role in pathogenesis of IOT. According to the results, LINC00324 was upregulated in IOT tissues and cells, as determined by reverse transcription­quantitative PCR, and its depletion impaired cell proliferation ability and improved cell apoptosis ability in IOT. Furthermore, LINC00324 acted as a miR­214­5p sponge to derepress cyclin dependent kinase 6 (CDK6), cyclin D1 (CCND1), murine double minute homolog 2 (MDM2), and mouse double minute 4 (MDM4) expression, thus increasing IOT cell proliferation and repressing apoptosis. Taken together, these results demonstrated that LINC00324 could serve as a competing endogenous RNA to facilitate IOT cell proliferation by regulation of miR­214­5p­CDK6/CCND1/MDM2/MDM4 network, which possibly provide a novel therapeutic target for IOT.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Transcriptional regulation of Rex1 (zfp42) in normal prostate epithelial cells and prostate cancer cells.

Rex1 (zfp42) was identified by our laboratory because of its reduced expression in F9 teratocarcinoma stem cells after retinoic acid (RA) treatment. The Rex1 (Zfp42) gene is currently widely used as a marker of embryonic stem cells. We compared the transcriptional regulation of the human Rex1 gene in NTera-2 (NT-2) human teratocarcinoma, normal human prostate epithelial cells (PrEC), and prostate cancer cells (PC-3) by promoter/luciferase analyses. Oct4, Sox2, Nanog, and Dax1 transcripts are expressed at higher levels in NT-2 and PrEC cells than in PC-3 cells. Co-transfection analyses showed that YY1 and Rex1 are positive regulators of hRex1 transcription in NT-2 and PrEC cells, whereas Nanog is not. Serial deletion constructs of the hRex1 promoter were created and analyzed, by which we identified a potential negative regulatory site that is located between -1 and -0.4 kb of the hRex1 promoter. We also delineated regions of the hRex1 promoter between -0.4 kb and the TSS that, when mutated, reduced transcriptional activation; these are putative Rex1 binding sites. Mutation of a putative Rex1 binding site in electrophoretic mobility shift assays (EMSA) resulted in reduced protein binding. Taken together, our results indicate that hRex1 binds to the hRex1 promoter region at -298 bp and positively regulates hRex1 transcription, but that this regulation is lost in PC-3 human prostate cancer cells. This lack of positive transcriptional regulation by the hRex1 protein may be responsible for the lack of Rex1 expression in PC-3 prostate cancer cells.

Normal establishment of epithelial tight junctions in mice and cultured cells lacking expression of ZO-3, a tight-junction MAGUK protein.

ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3(-/-) mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3(-/-) cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca(2+) switch was indistinguishable between wild-type and ZO-3(-/-) cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.

MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells.

MicroRNAs (or miRNAs) are small non-coding RNAs (21-25 nucleotides) that are involved in a wide range of activities related to the development and differentiation of cells. Comparison of the miRNA expression profiles of mouse P19 embryonic carcinoma cells with those of differentiated neural stem cells showed that the expression level of 65 miRNAs changed (2-fold) after differentiation. MiR-124a was dramatically upregulated (more than 20-fold) while miRNAs of the miR-302 family and those in the miR-290-295 cluster were strongly down-regulated. Further analysis revealed that some important factors such as Oct4 and Sox2 appeared to be involved in the regulation of these miRNAs. These results may contribute to a better understanding of miRNA-regulated neural differentiation in early mouse embryos.

Analysis of Human Stem Cell Transcription Factors.

Transcription factors NANOG, OCT4, SOX2, and NESTIN are expressed in both human embryonic stem cells (hESCs) and cancer stem cells and they play a crucial role in maintaining characteristics of stemness such as self-renewal and pluripotency. This article evaluates the expression of variants of the main stem cell-specific transcription factors NANOG and OCT4 critically and accurately with specific primers designed for identifying the most important variants that maintain stemness. We have examined four variants of NANOG along with a processed pseudogene and seven variants of OCT4 in human teratocarcinoma cell lines (NTERA2D1, SuSa, GCT-27, and 833KE), hESCs, and ovarian cancer cells by reverse transcriptase-polymerase chain reaction. In addition, we have examined their expression in NTERA2D1 cells on differentiation with all-trans-retinoic-acid. We show that NANOG1 is expressed in all teratocarcinoma cells and can be distinguished from NANOGP8, which is an expressed pseudogene. NANOG2 was not expressed in any of the cell lines, including ESCs. OCT4A was expressed in all cells, whereas the variant OCT4B-variant 3 was expressed only in NTERA2D1 cells. On differentiation of NTERA2D1 with retinoic acid, only NANOGP8 and OCT4A were expressed. In ovarian cancer cells, only 3/6 expressed NANOG1 and OCT4A. All malignant cells from patients with ovarian cancer (N = 6) expressed NANOG1 and OCT4A. These results demonstrate the necessity to precisely evaluate the expression of stem cell transcription factors when defining stemness.

Teratocarcinosarcoma of the nasal cavity and paranasal sinuses: report of 3 cases with assessment for chromosome 12p status.

Sinonasal teratocarcinosarcoma (SNTCS) is a rare malignant neoplasm with 63 reported cases to date. Patients are exclusively adults, with a mean age of 60 years and marked male predominance. Histologically, these tumors are characterized by the presence of admixed epithelial and mesenchymal components. The histogenesis of SNTCS remains uncertain and genetic studies have not been reported to date. Two SNTCSs from the archives of Memorial Sloan-Kettering Cancer Center and one submitted from St Luke's-Roosevelt Hospital Center were evaluated by fluorescent in situ hybridization for amplification of chromosome12p, an event usually associated with the genesis of bona fide germ cell neoplasms (including mediastinal and testicular teratomas). Microscopic examination revealed admixed epithelial and mesenchymal elements in all 3 cases; benign squamous and glandular epithelium and neuroepithelial tissue were identified, the squamous epithelium demonstrating "fetal-like" cytoplasmic clearing. Mesenchymal proliferations were recognized ranging from well-differentiated smooth muscle to high-grade sarcoma. A malignant germ cell component was not identified in any of the cases. Fluorescent in situ hybridization evaluation demonstrated only 2 copies of chromosome 12 per case. Although the histogenesis of SNTCS remains uncertain, we have found an absence of 12p amplification in 3 cases. Our findings suggest that 12p amplification, if it occurs at all in this setting, is exceptional and that SNTCS is a somatic-type neoplasm exhibiting divergent differentiation rather than a germ cell tumor.

Retinoic acid and homeobox gene regulation.

Hox genes have been shown to be important regulators of pattern formation in vertebrates. Retinoic acid has been shown to affect the expression of Hox genes in vitro and in vivo, and some of its effects on development correspond to changes in Hox gene expression. The idea that retinoic acid is not simply a powerful pharmocological agent, but rather that it plays an important role in creating the normal expression patterns of Hox genes, is provided by the recent identification of retinoic acid responsive enhancers near Hox genes.