Nodal Gene Research Articles

Novel Mutations of NODAL Gene in Chinese Patients with Congenital Heart Disease

Congenital heart disease (CHD) is one of most common birth defects threatening newborns' health. Over the past few decades, a variety of CHD-causing gene mutations have been identified, but the pathogenic mechanism of congenital heart disease is yet not very clear. The aim of this study was to identify potential pathologic mutations in the NODAL gene and to gain insight into the etiology of CHD. By using amplification with polymerase chain reaction and sequence analysis of NODAL in 800 patients with nonsyndromic CHD and 250 healthy controls, we identified 3 nonsynonymous variants. One of them was first identified in the present study. These variants were not observed in 250 controls. To our knowledge, this is the first study to suggest that NODAL may be involved in the etiology of nonsyndromic CHD in a Chinese population.

Nodal activates smad and extracellular signal-regulated kinases 1/2 pathways promoting renal cell carcinoma proliferation.

Expression of the nodal gene is high in a number of tumor cell types and may promote tumor growth. The expression of lefty, an inhibitor of nodal is often reduced in tumor cells. To the best of our knowledge, few studies have investigated the expression of nodal and lefty in renal cell carcinoma (RCC) cells. In the present study, quantitative polymerase chain reaction assays demonstrated that the level of nodal expression in RCC cells was high compared with that of adjacent non-tumor tissue cells, while the opposite pattern was observed for the level of lefty expression. Furthermore, lefty overexpression in RCC cells inhibited the expression of nodal. Nodal overexpression promoted RCC cell proliferation and invasion, and inhibited RCC cell apoptosis. Nodal downregulation and lefty overexpression led to similar observations: The inhibition of RCC cell proliferation and invasion, and the promotion of RCC cell apoptosis. The results of the present study suggested that the expression of nodal promoted RCC growth by activating the smad and extracellular signal-regulated kinases 1/2 pathways. The expression of lefty in RCC cells was lower than that in adjacent non-tumor cells, which may result in the overexpression of nodal, thereby promoting the growth of RCC. The results of the present study may therefore be useful for the development of novel biomarkers for RCC tumor diagnosis, and suggest a potential target gene for the treatment of RCC.

Molecular and functional characterization of grass carp squint/nodal-related 1: a potential regulator of activin signaling in teleost pituitary cells

Nodal, a member of the transforming growth factor-β superfamily, plays important roles in embryogenesis in vertebrates, including fish. However, the functional characterization of the fish nodal-related gene in nonembryonic cells is still unclear. In teleost, three nodal-related genes, nodal-related (ndr)1/squint, ndr2/cyclops, and ndr3/southpaw have been reported. In this study, a full-length cDNA for grass carp squint (gcSqt) was cloned, and its transcript was detected in the selected organs, including pituitary, brain, heart, head kidney, kidney, spleen, and gonad. To further define its functional role, recombinant grass carp squint (rgcSQT) was produced in Escherichia coli in a homodimer form. Furthermore, we examined the effects of rgcSQT on activin and its receptor gene expression with the use of grass carp pituitary cell as a model. Results showed that rgcSQT stimulated the mRNA expression of activin βA and βB subunit, as well as activin receptor ActRIB and ActRIIB. These findings not only contribute to the understanding of nonembryonic functions of nodal gene in fish, but they also provide new insight into the regulation of activin signaling in vertebrates.

Left-right symmetry breaking in mice by left-right dynein may occur via a biased chromatid segregation mechanism, without directly involving the Nodal gene

Ever since cloning the classic iv (inversed viscerum) mutation identified the “left-right dynein” (lrd) gene in mice, most research on body laterality determination has focused on its function in motile cilia at the node embryonic organizer. This model is attractive, as it links chirality of cilia architecture to asymmetry development. However, lrd is also expressed in blastocysts and embryonic stem cells, where it was shown to bias the segregation of recombined sister chromatids away from each other in mitosis. These data suggested that lrd is part of a cellular mechanism that recognizes and selectively segregates sister chromatids based on their replication history: old “Watson” versus old “Crick” strands. We previously proposed that the mouse left-right axis is established via an asymmetric cell division prior to/or during gastrulation. In this model, left-right dynein selectively segregates epigenetically differentiated sister chromatids harboring a hypothetical “left-right axis development 1” (“lra1”) gene during the left-right axis establishing cell division. Here, asymmetry development would be ultimately governed by the chirality of the cytoskeleton and the DNA molecule. Our model predicts that randomization of chromatid segregation in lrd mutants should produce embryos with 25% situs solitus, 25% situs inversus, and 50% embryonic death due to heterotaxia and isomerism. Here we confirmed this prediction by using two distinct lrd mutant alleles. Other than lrd, thus far Nodal gene is the most upstream function implicated in visceral organs laterality determination. We next tested whether the Nodal gene constitutes the lra1 gene hypothesized in the model by testing mutant’s effect on 50% embryonic lethality observed in lrd mutants. Since Nodal mutation did not suppress lethality, we conclude that Nodal is not equivalent to the lra1 gene. In summary, we describe the origin of 50% lethality in lrd mutant mice not yet explained by any other laterality-generating hypothesis.

Regulation of growth factors‐associated cell migration by C‐phycocyanin scaffold in dermal wound healing

1. The present study examined the role of C-phycocyanin (C-pc) in relation to growth factors and cell migration during wound healing. 2. Histological and biochemical studies showed that C-pc scaffold significantly (P < 0.01) increased hydroxyl proline, total hexamine and protein content, and decreased uronic acid content in the wound tissues during a time course study in newly formed skin. 3. Reverse transcription polymerase chain reaction array of mouse growth factors in wound tissue showed overexpression (up to 10-fold) of growth factors, such as Cxcl12, Fgf18, Lefty 1, Lefty 2, Rabep 1 and Zip91, and downregulation (up to -10-fold) of Amh, Bmp 7 and Nodal genes in a 6-day period in C-pc treated groups. Also, Csf 3, Fgf 22, Mdk, Igf 2, transforming growth factor (TGF)-α 1 and interleukin (IL)-1β showed an upregulation of more than 30-fold than the control groups. TGF-β subfamily cytokine growth factors, such as Bmp 2, 4 and 8b, and other growth factors, such as Cxcl 1, showed the highest activity on day 3, showing a transient type of regulation. Western blot analysis showed a positive correlation between gene activity and protein expressions of Bmp 8b, Bmp4, Bmp2 and Cxcl 1. Day 6 in the C-pc group showed the highest csf3 and IL-1β expression. 4. C-pc had no direct effect on keratinocyte migration. However, keratinocytes that were co-cultured with fibroblasts showed a significantly higher rate of migration in the presence of C-pc, showing an indirect effect of C-pc on keratinocyte migration. 5. In conclusion, biodegradable C-pc scaffold might help to serve as an alternate scaffold material for wound healing.

Low oxygen levels induce the expression of the embryonic morphogen Nodal

Low oxygen (O(2)) levels characterize the microenvironment of both stem cells and rapidly growing tumors. Moreover, hypoxia is associated with the maintenance of stem cell-like phenotypes and increased invasion, angiogenesis and metastasis in cancer patients. Metastatic cancers, such as breast cancer and melanoma, aberrantly express the embryonic morphogen Nodal, and the presence of this protein is correlated with metastatic disease. In this paper, we demonstrate that hypoxia induces Nodal expression in melanoma and breast cancer cells concomitant with increased cellular invasion and angiogenic phenotypes. Of note, Nodal expression remains up-regulated up to 48 h following reoxygenation. The oxygen-mediated regulation of Nodal expression occurs via a combinatorial mechanism. Within the first 24 h of exposure to low O(2), there is an increase in protein stability. This increase in stability is accompanied by an induction of transcription, mediated by the HIF-1α-dependent activation of Notch-responsive elements in the node-specific enhancer of the Nodal gene locus. Finally, Nodal expression is maintained upon reoxygenation by a canonical SMAD-dependent feed-forward mechanism. This work provides insight into the O(2)-mediated regulation of Nodal, a key stem cell-associated factor, and reveals that Nodal may be a target for the treatment and prevention of hypoxia-induced tumor progression.

Is Pitx2 Growing Up?

In this issue of Circulation: Cardiovascular Genetics , Kirchhof et al1 present an interesting study on the expression of the developmental transcription factor Pitx2c in adult heart and its potential role in atrial fibrillation (AF). AF is the most common sustained cardiac arrhythmia, with a prevalence that increases with age and affects 1% to 2% of the general population.2 Despite recent advances in AF research, underlying pathophysiologic mechanisms are not fully understood. In general, initiation and perpetuation of AF needs a trigger for its onset and a vulnerable substrate for its maintenance.3 Focal ectopic activity can act as a trigger to initiate AF on the basis of a single- or multiple-circuit reentry.4 Especially, cardiomyocyte sleeves around the pulmonary veins have been shown as an origin of focal activity.5 Underlying mechanisms are early and delayed after depolarizations as a result of disturbances in depolarizing inward (Na+ or Ca2+) or repolarizing outward (K+) currents.6 Article see p 123 Once initiated, AF itself induces structural and electric remodeling, which stabilizes reentry and AF maintenance.6 Structural remodeling due to signaling through angiotensin, profibrotic cytokines like transforming growth factor-β, or microRNAs leads to upregulation of extracellular matrix proteins (eg, collagen) and downregulation of matrix-degrading enzymes (eg, matrix metalloproteinase), resulting in progressive atrial fibrosis. Electric remodeling is caused by expressional changes in ion channel subunits. AF induces calcium overload and increases activity of protein phosphatases, leading to downregulation of depolarizing L-type calcium channels and upregulation of repolarizing potassium channels. Measurable substrate of these changes in atrial electric properties is the shortening of action potential duration (APD), leading to shortening of refractory period and enabling reentry mechanisms. In most cases, therefore, AF is self-terminating (paroxysmal) in the beginning, developing to more sustained forms (persistent and permanent) over …

Abrogation of p53 function leads to metastatic transcriptome networks that typify tumor progression in human breast cancer xenografts

Development of chromosomal instability (CIN) and consequent phenotypic heterogeneity represent common events during breast cancer progression. Breast carcinomas harboring extensive chromosomal aberrations display a more aggressive behavior characterized by chemoresistance and the propensity to give rise to distant metastases. The tumor suppressor p53 plays a key role in the maintenance of chromosomal stability and tissue homeostasis through activation of cell cycle checkpoints following DNA damage and control of centrosome duplication that ensures equal chromosome segregation during cell division. Furthermore, p53 suppresses CD44 expression and the acquisition of stem cell-like properties responsible for epithelial to mesenchymal transition (EMT) and metastasis. In this study we employed MCF-7 breast cancer cells with endogenous wild-type p53, an engineered MCF-7 variant (vMCF-7(DNP53)) overexpressing a dominant negative p53val135 mutant, and cells re-cultured from vMCF-7(DNP53) tumor xenografts. We carried out an integrative transcriptome and cytogenetic analysis to characterize the mechanistic linkage between loss of p53 function, EMT and consequent establishment of invasive gene signatures during breast cancer progression. We demonstrate that abrogation of p53 function drives the early transcriptome changes responsible for cell proliferation, EMT and survival, while further transcriptome changes that occur during in vivo tumor progression are mechanistically linked to the development of CIN leading to a more invasive and metastatic breast cancer phenotype. Here we identified distinct novel non-canonical transcriptome networks involved in cell proliferation, EMT, chemoresistance and invasion that arise following abrogation of p53 function in vitro and development of CIN in vivo. These studies also have important translational implications since some of the nodal genes identified here are 'druggable' making them appropriate molecular targets for the treatment of breast carcinomas displaying mutant p53, EMT, CIN and high metastatic potential.

A conserved mechanism for vertebrate mesoderm specification in urodele amphibians and mammals

Understanding how mesoderm is specified during development is a fundamental issue in biology, and it has been studied intensively in embryos from Xenopus. The gene regulatory network (GRN) for Xenopus is surprisingly complex and is not conserved in vertebrates, including mammals, which have single copies of the key genes Nodal and Mix. Why the Xenopus GRN should express multiple copies of Nodal and Mix genes is not known. To understand how these expanded gene families evolved, we investigated mesoderm specification in embryos from axolotls, representing urodele amphibians, since urodele embryology is basal to amphibians and was conserved during the evolution of amniotes, including mammals. We show that single copies of Nodal and Mix are required for mesoderm specification in axolotl embryos, suggesting the ancestral vertebrate state. Furthermore, we uncovered a novel genetic interaction in which Mix induces Brachyury expression, standing in contrast to the relationship of these molecules in Xenopus. However, we demonstrate that this functional relationship is conserved in mammals by showing that it is involved in the production of mesoderm from mouse embryonic stem cells. From our results, we produced an ancestral mesoderm (m)GRN, which we suggest is conserved in vertebrates. The results are discussed within the context of a theory in which the evolution of mechanisms governing early somatic development is constrained by the ancestral germ line–soma relationship, in which germ cells are produced by epigenesis.

The embryonic morphogen, Nodal, is associated with channel‐like structures in human malignant melanoma xenografts

Formation of channel-like structures, also termed vasculogenic mimicry (VM), describes the ability of aggressive melanoma cells to form PAS-positive anastomosing structures that correlate with tumor virulence. This phenomenon may indicate differentiation plasticity, a feature melanoma cells may share with stem cells in the developing embryo. Recent studies have indicated that VM and tumorigenicity of human malignant melanoma may depend on the signaling pathways of an embryonic morphogen, Nodal. However, given the secretory nature of Nodal protein and melanoma cell heterogeneity, it remains unclear whether the Nodal-expressing cells participate directly or indirectly in VM that is potentially related to tumorigenic growth. We have developed a humanized murine xenograft model in which developing human melanomas may be sequentially studied during early stages of tumorigenic growth within a physiological human dermal microenvironment. Nodal protein localized diffusely to melanoma cell membranes, with occasional foci of accentuated reactivity in patterns suggestive of channel formation. Similar findings were detected in a limited number of patient-derived tumors. In situ hybridization confirmed Nodal mRNA to be restricted to tumor cells within xenografts that formed arborizing networks in patterns consistent with VM. These data indicate that Nodal gene expression is associated with formation of VM-like structures in a physiologically relevant model of human melanoma tumorigenesis, and further support a key role for Nodal expression in the formation of channel-like structures. The humanized xenograft model should be useful in future studies to define the mechanistic pathways responsible for VM and melanoma progression.

04-P007 A simplified gene regulatory network for mesendoderm formation is revealed by knockdown of single Nodal and Mix genes in axolotl embryos

04-P007 A simplified gene regulatory network for mesendoderm formation is revealed by knockdown of single Nodal and Mix genes in axolotl embryos

Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly

The cyclopic and laterality phenotypes in model organisms linked to disturbances in the generation or propagation of Nodal-like signals are potential examples of similar impairments resulting in birth defects in humans. However, the types of gene mutation(s) and their pathogenetic combinations in humans are poorly understood. Here we describe a mutational analysis of the human NODAL gene in a large panel of patients with phenotypes compatible with diminished NODAL ligand function. Significant reductions in the biological activity of NODAL alleles are detected among patients with congenital heart defects (CHD), laterality anomalies ( e.g. left–right mis-specification phenotypes), and only rarely holoprosencephaly (HPE). While many of these NODAL variants are typical for family-specific mutations, we also report the presence of alleles with significantly reduced activity among common population variants. We propose that some of these common variants act as modifiers and contribute to the ultimate phenotypic outcome in these patients; furthermore, we draw parallels with strain-specific modifiers in model organisms to bolster this interpretation.

Respecification of ectoderm and altered Nodal expression in sea urchin embryos after cobalt and nickel treatment

In the sea urchin embryo, Nodal is the earliest known signal to play a role in the specification of the oral ectodermal territory. Nodal, a TGF-beta ligand, is first expressed in the presumptive oral ectoderm at ∼7 H of development. Nodal overexpression produces a distinctive bell-shaped phenotype with expanded oral ectoderm, which resembles the oralized phenotype obtained as a result of nickel (Ni) treatment. To date, a detailed analysis of gene expression in Ni-treated embryos has not been undertaken. Because treatment with cobalt (Co) produces similar results to those seen with Ni treatment in other systems, we were interested in determining how Co influences sea urchin embryonic development. Here we report that Co also induces oralization of the ectoderm, and the effects of Ni and Co depend on functional Nodal signaling. Although both metals upregulate nodal gene expression, they do not initiate nodal transcription precociously. Analysis of the perturbation of Nodal receptor function suggests that Ni and Co contribute to nodal upregulation in the absence of nodal autoregulation, but cannot fully oralize the ectoderm in the absence of Nodal signaling.

Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

BackgroundThe present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling.MethodsThe gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG_U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR.ResultsHierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4 mRNA expression failed to show correlation with any of the relevant parameters. ConclusionThe characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma.

Molecular insights into evolution of the vertebrate gut: focus on stomach and parietal cells in the marsupial, Macropus eugenii

Gastrulation in vertebrate embryos results in the formation of the primary germ layers: ectoderm, mesoderm and endoderm, which contain the progenitors of the tissues of the entire fetal body. Extensive studies undertaken in Xenopus, zebrafish and mouse have revealed a high degree of conservation in the genes and cellular mechanisms regulating endoderm formation. Nodal, Mix and Sox gene factor families have been implicated in the specification of the endoderm across taxa. Considerably less is known about endoderm development in marsupials. In this study we review what is known about the molecular aspects of endoderm development, focusing on evolution and development of the stomach and parietal cells and highlight recent studies on parietal cells in the stomach of Tammar Wallaby, Macropus eugenii. Although the regulation of parietal cells has been extensively studied, very little is known about the regulation of parietal cell differentiation. Intriguingly, during late-stage forestomach maturation in M. eugenii, there is a sudden and rapid loss of parietal cells, compared with the sharp increase in parietal cell numbers in the hindstomach region. This has provided a unique opportunity to study the development and regulation of parietal cell differentiation. A PCR-based subtractive hybridization strategy was used to identify candidate genes involved in this phenomenon. This will allow us to dissect the molecular mechanisms that underpin regulation of parietal cell development and differentiation, which have been a difficult process to study and provide markers that can be used to study the evolutionary origin of these cells in vertebrates.

Left–right asymmetric expression of Pitx is regulated by the asymmetric Nodal signaling through an intronic enhancer in Ciona intestinalis

Left-right asymmetric expression of Pitx as well as of Nodal has been observed in some ascidian species, but a mechanism that regulates the asymmetric expression of Pitx remains largely unclear. We addressed the transcription regulatory mechanism of the left-right asymmetric expression of Pitx gene in the ascidian, Ciona intestinalis. We first identified an intronic enhancer that drives Ci-Pitx left-sided expression in the epidermis and found that a single FoxH1 binding site present in this enhancer is essential for its activity. Also, we have shown that the enhancer requires Nodal signaling to drive the Ci-Pitx expression in the left epidermis. In C. intestinalis, left-sided expression of the Nodal gene has not been reported so far. We have confirmed that Ci-Nodal is expressed in the left epidermis of embryos at the tailbud stage only when they are allowed to develop within the chorion. Then, to test the importance of the FoxH1 binding site, we carried out knock down experiments using morpholino antisense oligonucleotides against Ci-FoxHa, the only FoxH gene that is expressed in the same stage as the left-sided expression of Ci-Pitx is observed. Knocking down of the function of Ci-FoxHa led to the down regulation of the expression of Ci-Pitx in the left epidermis. The present results suggest that the regulatory mechanism controlling the left-right asymmetric expression of the Pitx genes is well conserved between C. intestinalis and vertebrates.

Cis‐regulatory control of the nodal gene, initiator of the sea urchin oral ectoderm gene network

Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. The cis‐regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. A 5′ cis‐regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression.

A conserved role for the nodal signaling pathway in the establishment of dorso‐ventral and left–right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.

Transgenic mouse lines expressing Cre recombinase specifically in posterior notochord and notochord

During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward.

The Expression Signature of M3 AML Suggests Direct and Indirect Effects of PML-RARA on Target Genes.

In order to better understand the pathogenesis of acute promyelocytic leukemia (APL, FAB M3), we sought to determine its gene expression signature by comparing the expression profiles of 14 APL samples to that of other AML subtypes (M0, M1, M2, M4, n=62) and to fractionated normal whole bone marrow cells (CD34 cells, promyelocytes, PMNs, n=5 each). We used ANOVA and SAM (Significance Analysis of Microarrays) to select genes that were highly expressed in APL cells and that displayed low to no expression in other AML subtypes. The APL signature was then further refined by filtering genes whose expression in APL was not significantly different from that of normal promyelocytes, yielding 1121 annotated genes that reliably distinguish APL from the other FAB subtypes using unsupervised hierarchical clustering, both in training and validation datasets. Fold change differences in expression between M3 and other AML FAB classes were striking, for example: GABRE 35.4, HGF 21.3, ANXA8 21.3, PTPRG 16.9, PTGDS 12.1, PPARG 11.1, STAB1 9.8. A large proportion of the APL versus other FAB dysregulome was recapitulated when we compared APL expression to that of the normal pattern of myeloid development. We identified 733 annotated genes with significantly different expression in APL versus normal myeloid cell fractions. These dysregulated genes were assigned to 4 classes:1.persistently expressed CD34 cell-specific genes,2.repressed promyelocyte-specific genes,3.prematurely expressed neutrophil-specific genes and4.genes with high expression in APL and low/no expression in normal myeloid cell fractions.Expression differences in several of the most dysregulated genes were validated by qRT-PCR. We then examined the expression of the APL signature genes in myeloid cell lines and tumors from a murine APL model. The bona fide M3 signature was not apparent in resting NB4 cells (which contain t(15;17), and which express PML-RARA), nor in PR-9 cells following Zn induction of PML-RARA expression, suggesting that neither cell line accurately models the gene expression signature of primary APL cells. Most of the nodal genes of the mCG-PML-RARA murine APL dysregulome (Yuan, et al, 2007) are similarly dysregulated in human M3 cells; however, the human and mouse dysregulomes do not completely coincide. Finally, we have begun investigating which APL signature genes are direct transcriptional targets of PML-RARA. The promoters of the APL signature genes were analyzed for the presence of known PML-RARA binding sites using multiple computational methods. The analyses demonstrated that several transcription factors (EBF3, TWIST1, SIX3, PPARG) have putative retinoic acid response elements (RAREs) in their upstream regulatory regions. Additionally, we examined the promoters of some of the most upregulated genes (HGF, PTGDS, STAB1) for known consensus sites of these transcription factors, and found that all have putative binding sites for at least one. These results suggest that PML-RARA may initiate a transcriptional cascade that relies not only on its own activity, but also on the actions of downstream transcription factors. In summary, our studies indicate that primary APL cells have a gene expression signature that is consistent and highly reproducible, but different from commonly used human APL cell lines and a mouse model of APL. The molecular mechanisms that govern this unique signature are currently under investigation.