Gene Expression In Early Development Research Articles

Multiomics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and noncanonical Wnt signaling dysregulation.

ASXL1 (additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo protein-truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS; OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, distinctive facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies, including heart defects and severe skeletal defects giving rise to a typical BOS posture. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia. We used primary cells from individuals with BOS (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multiomics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data show that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, which encodes a planar cell polarity pathway protein that acts through noncanonical Wnt signaling to direct tissue patterning and cell migration. This multiomics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.

Differential nuclear import sets the timing of protein access to the embryonic genome

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins’ access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

Shaken, rattled and rolled: The effects of hatchery-rearing techniques on endogenous cortisol production, stress-related gene expression, growth and survival in larval Lake Sturgeon, Acipenser fulvescens

Sturgeon, one of the most endangered families of vertebrates, are of high conservation concern with population restoration efforts worldwide. Sturgeon species are often raised in conservation hatcheries to supplement wild populations with little understanding of effects of hatchery-related stressors on early development. Lake Sturgeon hatcheries deviate from natural conditions by coating adhesive embryos in clay to prevent clumping, embryos are then added to tumbling jars, colloquially referred to as McDonald Jars; a cone or cylinder-shaped container with constant flow, causing the embryos to roll and move throughout the water column. In the wild, Lake Sturgeon embryos adhere to substrate in fast-flowing rivers, where orientation is maintained throughout embryogenesis. In this study, we examined the effects of hatchery early-rearing environment, i.e. the use of tumbling jars, on gene expression, endogenous cortisol production, growth, and survival in larval Lake Sturgeon. We compared pre- and post-hatch tumbled larvae to non-tumbled larvae that were allowed to adhere to substrate throughout embryogenesis. We hypothesized that deviating from natural rearing conditions, with the use of tumbling jars, would result in changes to cortisol production, growth and mortality in post-hatch, tumbled larvae. Endogenous cortisol production was delayed one week for fish in the tumbled treatment (two-factor ANOVA; P < .05) when compared to non-tumbled fish. Tumbled fish were also smaller throughout development, with lasting effects on growth 62 days after hatching (P < .05). The tumbled fish had 23% mortality post-hatch compared to 6% in the non-tumbled group (Cox Proportional-Hazards Model; P < .0001). There were differences in gene expression in early development at specific time points, between the two treatment groups, in StAR and hsp70 (P < .05). The observed developmental effects on endogenous cortisol production and growth in the tumbled embryos could be counterproductive to the goal of producing a hatchery-reared fish that can survive in the wild. As conservation hatcheries aim to release individuals that will supplement and reproduce with wild populations, changes to developmental timing, growth and survival should be avoided. The findings of this study suggest that tumbling jars may not be appropriate in conservation hatcheries for Lake Sturgeon as they may have long-term negative consequences on individual fitness.

A two-pore channel protein required for regulating mTORC1 activity on starvation

BackgroundTwo-pore channels (TPCs) release Ca2+ from acidic intracellular stores and are implicated in a number of diseases, but their role in development is unclear. The social amoeba Dictyostelium discoideum proliferates as single cells that aggregate to form a multicellular organism on starvation. Starvation is sensed by the mTORC1 complex which, like TPC proteins, is found on acidic vesicles. Here, we address the role of TPCs in development and under starvation.ResultsWe report that disruption of the gene encoding the single Dictyostelium TPC protein, TPC2, leads to a delay in early development and prolonged growth in culture with delayed expression of early developmental genes, although a rapid starvation-induced increase in autophagy is still apparent. Ca2+ signals induced by extracellular cAMP are delayed in developing tpc2− cells, and aggregation shows increased sensitivity to weak bases, consistent with reduced acidity of the vesicles. In mammalian cells, the mTORC1 protein kinase has been proposed to suppress TPC channel opening. Here, we show a reciprocal effect as tpc2− cells show an increased level of phosphorylation of an mTORC1 substrate, 4E-BP1. mTORC1 inhibition reverses the prolonged growth and increases the efficiency of aggregation of tpc2− cells.ConclusionTPC2 is required for efficient growth development transition in Dictyostelium and acts through modulation of mTORC1 activity revealing a novel mode of regulation.

Developmental transcriptomic analyses for mechanistic insights into critical pathways involved in embryogenesis of pelagic mahi-mahi (Coryphaena hippurus).

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac β-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

Shared Enhancer Activity in the Limbs and Phallus and Functional Divergence of a Limb-Genital cis-Regulatory Element in Snakes

The amniote phallus and limbs differ dramatically in their morphologies but share patterns of signaling and gene expression in early development. Thus far, the extent to which genital and limb transcriptional networks also share cis-regulatory elements has remained unexplored. We show that many limb enhancers are retained in snake genomes, suggesting that these elements may function in non-limb tissues. Consistent with this, our analysis of cis-regulatory activity in mice and Anolis lizards reveals that patterns of enhancer activity in embryonic limbs and genitalia overlap heavily. In mice, deletion of HLEB, an enhancer of Tbx4, produces defects in hindlimbs and genitalia, establishing the importance of this limb-genital enhancer for development of these different appendages. Further analyses demonstrate that the HLEB of snakes has lost hindlimb enhancer function while retaining genital activity. Our findings identify roles for Tbx4 in genital development and highlight deep similarities in cis-regulatory activity between limbs and genitalia.

Non-genetic cancer cell plasticity and therapy-induced stemness in tumour relapse: 'What does not kill me strengthens me'.

Therapy resistance and tumour relapse after drug therapy are commonly explained by Darwinian selection of pre-existing drug-resistant, often stem-like cancer cells resulting from random mutations. However, the ubiquitous non-genetic heterogeneity and plasticity of tumour cell phenotype raises the question: are mutations really necessary and sufficient to promote cell phenotype changes during tumour progression? Cancer therapy inevitably spares some cancer cells, even in the absence of resistant mutants. Accumulating observations suggest that the non-killed, residual tumour cells actively acquire a new phenotype simply by exploiting their developmental potential. These surviving cells are stressed by the cytotoxic treatment, and owing to phenotype plasticity, exhibit a variety of responses. Some are pushed into nearby, latent attractor states of the gene regulatory network which resemble evolutionary ancient or early developmental gene expression programs that confer stemness and resilience. By entering such stem-like, stress-response states, the surviving cells strengthen their capacity to cope with future noxious agents. Considering non-genetic cell state dynamics and the relative ease with which surviving but stressed cells can be tipped into latent attractors provides a foundation for exploring new therapeutic approaches that seek not only to kill cancer cells but also to avoid promoting resistance and relapse that are inherently linked to the attempts to kill them.

Gene expression in early development

Gene expression in early development

Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA¯) encoding γ-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA¯ cells, the expression of a growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA¯ cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA¯ cells (YakAOE/gcsA¯) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway.

Phosphorylation of Actin-related Protein 2 (Arp2) Is Required for Normal Development and cAMP Chemotaxis in Dictyostelium

Phosphorylation of the actin-related protein 2 (Arp2) subunit of the Arp2/3 complex on evolutionarily conserved threonine and tyrosine residues was recently identified and shown to be necessary for nucleating activity of the Arp2/3 complex and membrane protrusion of Drosophila cells. Here we use the Dictyostelium diploid system to replace the essential Arp2 protein with mutants that cannot be phosphorylated at Thr-235/6 and Tyr-200. We found that aggregation of the resulting mutant cells after starvation was substantially slowed with delayed early developmental gene expression and that chemotaxis toward a cAMP gradient was defective with loss of polarity and attenuated F-actin assembly. Chemotaxis toward cAMP was also diminished with reduced cell speed and directionality and shorter pseudopod lifetime when Arp2 phosphorylation mutant cells were allowed to develop longer to a responsive state similar to that of wild-type cells. However, clathrin-mediated endocytosis and chemotaxis under agar to folate in vegetative cells were only subtly affected in Arp2 phosphorylation mutants. Thus, phosphorylation of threonine and tyrosine is important for a subset of the functions of the Arp2/3 complex, in particular an unexpected major role in regulating development.

Differential gene expression profiles during embryonic heart development in diabetic mice pregnancy

Congenital heart defects (CHD) are one of the most common defects in offspring of diabetic mothers. There is a clear association between maternal diabetes and CHD; however the underlying molecular mechanism remains unknown. We hypothesized that maternal diabetes affects with the expression of early developmental genes that regulate the essential developmental processes of the heart, thereby resulting in the pathogenesis of CHD. We analyzed genome-wide expression profiling in the developing heart of embryos from diabetic and control mice by using the oligonucleotide microarray. Microarray analysis revealed that a total of 878 genes exhibited more than 1.5 fold changes in expression level in the hearts of experimental embryos in either E13.5 or E15.5 compared with their respective controls. Expression pattern of genes that is differentially expressed in the developing heart was further examined by the real-time reverse transcriptase-polymerase chain reaction. Several genes involved in a number of molecular signaling pathways such as apoptosis, proliferation, migration and differentiation in the developing heart were differentially expressed in embryos of diabetic pregnancy. It is concluded that altered expression of several genes involved in heart development may contribute to CHD in offspring of diabetic mothers.

Progestins and Antiprogestins Affect Gene Expression in Early Development in Zebrafish (Danio rerio) at Environmental Concentrations

Progesterone (P4) and synthetic progestins (gestagens) from contraceptives and hormone therapy occur in treated wastewater and surface water, and they may have adverse effects on aquatic organisms. Little is known about the molecular and reproductive effects of P4 and synthetic progestins in fish, and effects of the antiprogestin mifepristone (RU486, an abortive) are unknown. We aimed at elucidating effects on the hormone system by quantitative determination of transcriptional changes of target genes induced by 2, 20, and 200 ng/L P4, RU486, norethindrone (NET), and levonorgestrel (LNG). We exposed zebrafish embryos for 144 h post fertilization (hpf) to these compounds and analyzed expressional changes of ar, esr1, vtg1, hsd17ß3, and progesterone (pgr), mineralo- (mr), and glucocorticoid (gr) receptors, each at 48, 96, and 144 hpf. Concentrations of NET and LNG were constant during exposure, while P4 and RU486 decreased. P4 and RU486 were the most potent steroids. Significant up to 4-fold induction of pgr, ar, mr, and hsd17b3 occurred at 2 ng/L P4 and higher, while RU484 inhibited pgr expression. NET and LNG modulated some transcripts mainly above 2 ng/L. The expressional chances occurring at environmental levels may translate to negative interference with differentiation of brain and gonads, and consequently reproduction.

Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.

Rap1 Activation in Response to cAMP Occurs Downstream of Ras Activation during Dictyostelium Aggregation

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC(-)/rasG(-)/[act15]:carA and rasG(-) cells but only slightly reduced in rasC(-) cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC(-)/rasG(-)/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation.

Genetic and epigenetic defects at the 6q24 imprinted locus in a cohort of 13 patients with transient neonatal diabetes: new hypothesis raised by the finding of a unique case with hemizygotic deletion in the critical region

Background: Transient neonatal diabetes (TND) is a rare form of diabetes usually present in the first few days after birth that resolves within 1 year but that has a tendency...

Understanding Hypoxia-Induced Gene Expression in Early Development: In Vitro and In Vivo Analysis of Hypoxia-Inducible Factor 1-Regulated Zebra Fish Insulin-Like Growth Factor Binding Protein 1 Gene Expression

Insulin-like growth factor binding protein 1 (IGFBP-1) is a hypoxia-inducible gene that plays an important role in regulating embryonic growth and development under hypoxic stress. The molecular mechanisms underlying hypoxia-induced IGFBP-1 gene expression in the embryonic tissues are not well understood. Here we report that the hypoxia-inducible factor 1 (HIF-1) pathway is established in early embryogenesis and mediates hypoxia-induced IGFBP-1 expression. Hypoxia increased the HIF-1 activity, and HIF-1alpha overexpression or CoCl2 treatment resulted in elevated IGFBP-1 expression in zebra fish embryos. Although the zebra fish IGFBP-1 promoter contains 13 consensus hypoxia response elements (HREs), deletion and mutational analysis revealed that only the HRE positioned at -1090/-1086 is required for the hypoxia and HIF-1 induction. Further experiments revealed that there is an HIF-1 ancillary sequence (HAS) adjacent only to the functional HRE. Mutation of this HAS greatly reduced the responsiveness of the IGFBP-1 promoter to hypoxia and HIF-1. The HAS does not directly bind to HIF-1 or affect the binding of the HRE to HIF-1. The HAS is bound to a nuclear protein(s), and this HAS binding activity is reduced by hypoxia. These results suggest that HIF-1 mediates hypoxia-induced IGFBP-1 gene expression in early development by selectively interacting with the -1090/-1086 HRE and its adjacent HAS.

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.

PldB, a Putative Phospholipase D Homologue in Dictyostelium discoideum Mediates Quorum Sensing during Development

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB- cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB- cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB- overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB- cells with wild-type cells does not cause the wild-type cells to behave like pldB- cells.

235 THE INVESTIGATION OF mRNA EXPRESSION OF SEVERAL CHROMATIN REMODELLING GENES DURING BOVINE PREIMPLANTATION DEVELOPMENT

The efficiency of obtaining live calves from somatic cell nuclear transfer remains quite low. One factor implicated in this failure is inadequate chromatin remodelling of the donor nucleus. Five Polycomb group (PcG) genes were investigated as potential remodelling factors in bovine oocytes and preimplantation embryos. These genes (Cbx6, Eed, Edr1, Yy1, and Zfp144) are involved in transcriptional activation and cell cycle regulation. We hypothesize that inadequate expression may cause the developmental abnormalities seen following cloning. This study is aimed at characterizing normal expression, prior to comparative studies with cloned embryos. Three single abattoir-derived in vitro-matured (IVM) oocytes or in vitro-produced (IVP) embryos from each of the following stages: 2-cell, 4-cell, 8-cell, 16–32 cell, morula, Day 7 blastocyst, and Day 8 hatched blastocyst, were studied. Messenger RNA was isolated from individual samples with Dynabeads (Dynal, Inc., Lake Success, NY, USA) and then cDNA was created and amplified with a SMART cDNA synthesis kit (BD Biosciences Clontech, Palo Alto, CA, USA). Products were diluted 1:10 and used to amplify target genes by PCR. PCR products were sequenced for confirmation of identity. All amplified embryo samples expressed the housekeeping genes Poly(A) polymerase and actin. Similarly, all embryos up to the 8-cell stage expressed GDF9, an oocyte-specific gene, while IFN-tau, involved in maternal recognition of pregnancy, was expressed in one morula and all blastocyst samples. Cbx6 was not expressed at any stage. The other four genes were all expressed fairly consistently throughout the pre-implantation period. Eed transcript was detected in all samples, with the exception of one oocyte, one 4-cell embryo, and one 8-cell embryo. Edr1 transcript was detected in all samples except for two oocytes, one 16–32-cell embryo, and one Day 7 blastocyst. Yy1 was expressed in all but one oocyte, one 2-cell embryo, two 4-cell embryos, and one Day 7 blastocyst. Finally, Zfp144 transcript was detected in one oocyte and in all embryos until the 16–32 stage, and then was not detected until seen in one Day 7 and all Day 8 blastocysts. Cbx6, yet to be fully characterized in any species, is also known as the neuronal pentraxin receptor, and is involved in transport and clearance of synaptic debris. It is known to have the characteristic chromobox domain of the Cbx family, of which several family members play a role during pre-implantation development. Eed, Edr1, Yy1, and Zfp144 are expressed throughout the pre-implantation period, although levels of Zfp144 mRNA appear to drop during the embryonic genome activation and then reappear by the late blastocyst stage. This consistent expression may suggest a role for the proteins in chromatin remodelling or modulating patterns of gene expression in early development. Further studies are required to determine if these factors are expressed incorrectly in cloned embryos, potentially providing a link to the abnormalities observed post nuclear transfer.

Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRalpha P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.