Xenopus Metamorphosis Research Articles

A unique role of thyroid hormone receptor β in regulating notochord resorption during Xenopus metamorphosis

Tail resorption during anuran metamorphosis is perhaps the most dramatic tissue transformation that occurs during vertebrate development. Earlier studies in highly related anuran species Xenopus laevis and Xenopus tropicalis have shown that thyroid hormone (T3) receptor (TR) plays a necessary and sufficient role to mediate the causative effect of T3 on metamorphosis. Of the two known TR genes in vertebrates, TRα is highly expressed during both premetamorphosis and metamorphosis while TRβ expression is low in premetamorphic tadpoles but highly upregulated as a direct target gene of T3 during metamorphosis, suggesting potentially different functions during metamorphosis. Indeed, gene knockout studies have shown that knocking out TRα and TRβ has different effects on tadpole development. In particularly, homozygous TRβ knockout tadpoles become tailed frogs well after sibling wild type ones complete metamorphosis. Most noticeably, in TRβ-knockout tadpoles, an apparently normal notochord is present when the notochord in wild-type and TRα-knockout tadpoles disappears. Here, we have investigated how tail notochord resorption is regulated by TR. We show that TRβ is selectively very highly expressed in the notochord compared to TRα. We have also discovered differential regulation of several matrix metalloproteinases (MMPs), which are known to be upregulated by T3 and implicated to play a role in tissue resorption by degrading the extracellular matrix (ECM). In particular, MMP9-TH and MMP13 are extremely highly expressed in the notochord compared to the rest of the tail. In situ hybridization analyses show that these MMPs are expressed in the outer sheath cells and/or the connective tissue sheath surrounding the notochord. Our findings suggest that high levels of TRβ expression in the notochord specifically upregulate these MMPs, which in turn degrades the ECM, leading to the collapse of the notochord and its subsequent resorption during metamorphosis.

Functional Studies of Transcriptional Cofactors via Microinjection-Mediated Gene Editing in Xenopus.

The anuran Xenopus laevis has been studied for decades as a model for vertebrate cell and developmental biology. More recently, the highly related species Xenopus tropicalis has offered the opportunity to carry out genetic studies due to its diploid genome as compared to the pseudo-tetraploid Xenopus laevis. Amphibians undergo a biphasic development: embryogenesis to produce a free-living tadpoles and subsequent metamorphosis to transform the tadpole to a frog. This second phase mimics the so-called postembryonic development in mammals when many organs/tissues mature into their adult form in the presence of high levels of plasma thyroid hormone (T3). The total dependence of amphibian metamorphosis on T3 offers a unique opportunity to study postembryonic development in vertebrates, especially with the recent development gene editing technologies that function in amphibians. Here, we first review the basic molecular understanding of the regulation of Xenopus metamorphosis by T3 and T3 receptors (TRs), and then describe a detailed method to use CRISPR to knock out the TR-coactivator SRC3 (steroid receptor coactivator 3), a histone acetyltransferase, in order to study its involvement in gene regulation by T3 in vivo and Xenopus development.

Functional limb muscle innervation prior to cholinergic transmitter specification during early metamorphosis in Xenopus.

In vertebrates, functional motoneurons are defined as differentiated neurons that are connected to a central premotor network and activate peripheral muscle using acetylcholine. Generally, motoneurons and muscles develop simultaneously during embryogenesis. However, during Xenopus metamorphosis, developing limb motoneurons must reach their target muscles through the already established larval cholinergic axial neuromuscular system. Here, we demonstrate that at metamorphosis onset, spinal neurons retrogradely labeled from the emerging hindlimbs initially express neither choline acetyltransferase nor vesicular acetylcholine transporter. Nevertheless, they are positive for the motoneuronal transcription factor Islet1/2 and exhibit intrinsic and axial locomotor-driven electrophysiological activity. Moreover, the early appendicular motoneurons activate developing limb muscles via nicotinic antagonist-resistant, glutamate antagonist-sensitive, neuromuscular synapses. Coincidently, the hindlimb muscles transiently express glutamate, but not nicotinic receptors. Subsequently, both pre- and postsynaptic neuromuscular partners switch definitively to typical cholinergic transmitter signaling. Thus, our results demonstrate a novel context-dependent re-specification of neurotransmitter phenotype during neuromuscular system development.

Methods for Investigating the Larval Period and Metamorphosis in Xenopus.

Anuran metamorphosis resembles postembryonic development in mammals, a period around birth when many organs/tissues mature into their adult form as circulating thyroid and stress hormone levels are high. Unlike uterus-enclosed mammalian embryos, tadpoles develop externally and undergo the dramatic changes of hormone-dependent development totally independent of maternal influence, making them a valuable model in which to study vertebrate postembryonic organ development and maturation. Various protocols have been developed and/or adapted for studying metamorphosis in Xenopus laevis and X. tropicalis, two highly related and well-studied frog species. Here, we introduce some of the methods for contemporary cell and molecular studies of gene function and regulation during metamorphosis.

Bisphenol F Disrupts Thyroid Hormone Signaling and Postembryonic Development in Xenopus laevis.

The safety of bisphenol A (BPA) alternatives has attracted much attention due to their wide use. In this study, we investigated the effects of bisphenol F (BPF), an alternative to BPA, on thyroid hormone (TH) signaling and postembryonic development in vertebrates using T3-induced and spontaneous Xenopus metamorphosis as models. We found that in the T3-induced metamorphosis assay, higher concentrations of BPF (100-10000 nM) antagonized T3-induced TH-response gene transcription and morphological changes including intestinal remodeling in a concentration-dependent manner, whereas 10 nM BPF exerted stimulatory effects on T3-induced integral metamorphosis when inhibited T3-induced TH-response gene transcription, demonstrating TH signaling disrupting effects of BPF. In the spontaneous metamorphosis assay, correspondingly, BPF inhibited development at metamorphic climax (with high endogenous TH levels), but promoted pre- and pro-metamorphic development (with low endogenous TH levels), displaying a developmental stage-dependent manner. Importantly, we observed agonistic actions of BPF on Notch signaling in intestines, showing that BPF disrupts vertebrate development possibly via multi pathways besides TH signaling. Thus, we infer the biphasic concentration-response relationship between BPF exposure and T3-induced metamorphosis could result from the interactions of TH signaling with other signaling pathways such as Notch signaling. Our study highlights the adverse influences of BPF on vertebrate development.

Organ Culture of the Xenopus Tadpole Intestine.

During Xenopus metamorphosis, most tadpole organs remodel from the larval to adult form to prepare for adaptation to terrestrial life. Organ culture serves as an important tool for studying larval-to-adult organ remodeling independent of the effects of other parts of the body. Here, I introduce a protocol for organ culture in vitro using the Xenopus laevis tadpole intestine before metamorphic climax. During culture in the absence of exogenous 3,3',5-triiodo-l-thyronine (T3), the most potent natural thyroid hormone, the intestine remains in its larval state without any metamorphic changes. In contrast, when T3 is added to the culture medium, the larval epithelium undergoes apoptosis, whereas adult stem cells appear, actively proliferate, and finally generate the differentiated adult epithelium within a week. At the same time, the surrounding nonepithelial tissues also develop. Thus, this culture model is useful for clarifying the control mechanisms of apoptosis in larval tissues, formation of adult stem cells, and cell proliferation and differentiation of adult tissues, all of which occur in harmony during natural metamorphosis. Moreover, a procedure for tissue recombination combined with organ culture provides a platform for investigating complex tissue interactions during organ remodeling. Such tissue recombination experiments will help to reveal the important role of nonepithelial tissues in larval epithelial apoptosis and/or adult stem cell development in the X. laevis intestine.

Genome-wide identification of thyroid hormone receptor targets in the remodeling intestine during Xenopus tropicalis metamorphosis

Thyroid hormone (T3) affects development and metabolism in vertebrates. We have been studying intestinal remodeling during T3-dependent Xenopus metamorphosis as a model for organ maturation and formation of adult organ-specific stem cells during vertebrate postembryonic development, a period characterized by high levels of plasma T3. T3 is believed to affect development by regulating target gene transcription through T3 receptors (TRs). While many T3 response genes have been identified in different animal species, few have been shown to be direct target genes in vivo, especially during development. Here we generated a set of genomic microarray chips covering about 8000 bp flanking the predicted transcription start sites in Xenopus tropicalis for genome wide identification of TR binding sites. By using the intestine of premetamorphic tadpoles treated with or without T3 and for chromatin immunoprecipitation assays with these chips, we determined the genome-wide binding of TR in the control and T3-treated tadpole intestine. We further validated TR binding in vivo and analyzed the regulation of selected genes. We thus identified 278 candidate direct TR target genes. We further provided evidence that these genes are regulated by T3 and likely involved in the T3-induced formation of adult intestinal stem cells during metamorphosis.

A balance of Mad and Myc expression dictates larval cell apoptosis and adult stem cell development during Xenopus intestinal metamorphosis

The Myc/Mad/Max network has long been shown to be an important factor in regulating cell proliferation, death and differentiation in diverse cell types. In general, Myc–Max heterodimers activate target gene expression to promote cell proliferation, although excess of c-Myc can also induce apoptosis. In contrast, Mad competes against Myc to form Mad–Max heterodimers that bind to the same target genes to repress their expression and promote differentiation. The role of the Myc/Mad/Max network during vertebrate development, especially, the so-called postembryonic development, a period around birth in mammals, is unclear. Using thyroid hormone (T3)-dependent Xenopus metamorphosis as a model, we show here that Mad1 is induced by T3 in the intestine during metamorphosis when larval epithelial cell death and adult epithelial stem cell development take place. More importantly, we demonstrate that Mad1 is expressed in the larval cells undergoing apoptosis, whereas c-Myc is expressed in the proliferating adult stem cells during intestinal metamorphosis, suggesting that Mad1 may have a role in cell death during development. By using transcription activator-like effector nuclease-mediated gene-editing technology, we have generated Mad1 knockout Xenopus animals. This has revealed that Mad1 is not essential for embryogenesis or metamorphosis. On the other hand, consistent with its spatiotemporal expression profile, Mad1 knockout leads to reduced larval epithelial apoptosis but surprisingly also results in increased adult stem cell proliferation. These findings not only reveal a novel role of Mad1 in regulating developmental cell death but also suggest that a balance of Mad and Myc controls cell fate determination during adult organ development.

Xenopus metamorphosis as a model to study thyroid hormone receptor function during vertebrate developmental transitions

A hormone-dependent developmental transition from aquatic to terrestrial existence occurs in all tetrapod vertebrates, such as birth, hatching, and metamorphosis. Thyroid hormones (TH) and their receptors (TRs) are key players in the tissue transformations comprising vertebrate developmental transitions. The African clawed frog, Xenopus, is a premier model for the role of TRs in developmental transitions because of the numerous and dramatic TH-dependent tissue transformations during metamorphosis and because of the endocrine, molecular, and genomic resources available. TRs are nuclear receptors that repress TH-response genes when plasma TH is minimal and that activate those same genes to induce tissue-specific gene regulation cascades when TH plasma levels increase. Tissue-specific TR expression levels help determine tissue sensitivity and responsivity to TH thereby regulating the initiation and rate of developmental change in TH-sensitive tissues which govern the tissue developmental asynchrony observed during metamorphosis. This review highlighting Xenopus presents the key experimental findings underpinning the roles TRs play in control of vertebrate developmental transitions.

Re-evaluation of thyroid hormone signaling antagonism of tetrabromobisphenol A for validating the T3-induced Xenopus metamorphosis assay

We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone (TH) signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10–500nmol/L TBBPA in the presence of 1nmol/L T3. After 96hr of exposure, TBBPA in the range of 10–500nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area (HA), mouth width (MW), unilateral brain width/brain length (ULBW/BL), and hind-limb length/snout-vent length (HLL/SVL). The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure, we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail, which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.

Optimization of the T3-induced Xenopus metamorphosis assay for detecting thyroid hormone signaling disruption of chemicals

T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone (TH) signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction. Xenopus laevis at stage 52 were exposed to series of concentrations of T3 (0.31–2.5nmol/L) for 6days. By comparing morphological changes induced by T3, we propose head area, mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration–response curves, with moderate morphological changes resulting from 1.25nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4days as exposure duration of T3, with concentrations close to 1.25nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.

Thyroid hormone regulation of adult intestinal stem cells: Implications on intestinal development and homeostasis.

Organ-specific adult stem cells are essential for organ homeostasis, tissue repair and regeneration. The formation of such stem cells often takes place during postembryonic development, a period around birth in mammals when plasma thyroid hormone concentration is high. The life-long self-renewal of the intestinal epithelium has made mammalian intestine a valuable model to study the function and regulation and adult stem cells. On the other hand, much less is known about how the adult intestinal stem cells are formed during vertebrate development. Here, we will review some recent progresses on this subject, focusing mainly on the formation of the adult intestine during Xenopus metamorphosis. We will discuss the role of thyroid hormone signaling pathway in the process and potential molecular conservations between amphibians and mammals as well as the implications in organ homeostasis and human diseases.

Molecular and cytological analyses reveal distinct transformations of intestinal epithelial cells during Xenopus metamorphosis.

BackgroundThe thyroid hormone (T3)-induced formation of adult intestine during amphibian metamorphosis resembles the maturation of the mammalian intestine during postembryonic development, the period around birth when plasma T3 level peaks. This process involves de novo formation of adult intestinal stem cells as well as the removal of the larval epithelial cells through apoptosis. Earlier studies have revealed a number of cytological and molecular markers for the epithelial cells undergoing different changes during metamorphosis. However, the lack of established double labeling has made it difficult to ascertain the identities of the metamorphosing epithelial cells.ResultsHere, we carried out different double-staining with a number of cytological and molecular markers during T3-induced and natural metamorphosis in Xenopus laevis. Our studies demonstrated conclusively that the clusters of proliferating cells in the epithelium at the climax of metamorphosis are undifferentiated epithelial cells and express the well-known adult intestinal stem cell marker gene Lgr5. We further show that the adult stem cells and apoptotic larval epithelial cells are distinct epithelial cells during metamorphosis.ConclusionsOur findings suggest that morphologically identical larval epithelial cells choose two alternative paths: programmed cell death or dedifferentiation to form adult stem cells, in response to T3 during metamorphosis with apoptosis occurring prior to the formation of the proliferating adult stem cell clusters (islets).

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis.

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis.

Epigenetic regulation of thyroid hormone-induced adult intestinal stem cell development during anuran metamorphosis.

Epigenetic modifications of histones are emerging as key factors in gene regulation by diverse transcription factors. Their roles during vertebrate development and pathogenesis are less clear. The causative effect of thyroid hormone (T3) on amphibian metamorphosis and the ability to manipulate this process for molecular and genetic studies have led to the demonstration that T3 receptor (TR) is necessary and sufficient for Xenopus metamorphosis, a process that resembles the postembryonic development (around birth) in mammals. Importantly, analyses during metamorphosis have provided some of the first in vivo evidence for the involvement of histone modifications in gene regulation by TR during vertebrate development. Furthermore, expression and functional studies suggest that various histone modifying epigenetic enzymes likely participate in multiple steps during the formation of adult intestinal stem cells during metamorphosis. The similarity between intestinal remodeling and the maturation of the mammalian intestine around birth when T3 levels are high suggests conserved roles for the epigenetic enzymes in mammalian adult intestinal stem cell development and/or proliferation.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-4-73) contains supplementary material, which is available to authorized users.

Activation of Sox3 gene by thyroid hormone in the developing adult intestinal stem cell during Xenopus metamorphosis.

The maturation of the intestine into the adult form involves the formation of adult stem cells in a thyroid hormone (T3)-dependent process in vertebrates. In mammals, this takes place during postembryonic development, a period around birth when the T3 level peaks. Due to the difficulty of manipulating late-stage, uterus-enclosed embryos, very little is known about the development of the adult intestinal stem cells. Interestingly, the remodeling of the intestine during the T3-dependent amphibian metamorphosis mimics the maturation of mammalian intestine. Our earlier microarray studies in Xenopus laevis revealed that the transcription factor SRY (sex-determining region Y)-box 3 (Sox3), well known for its involvement in neural development, was upregulated in the intestinal epithelium during metamorphosis. Here, we show that Sox3 is highly and specifically expressed in the developing adult intestinal progenitor/stem cells. We further show that its induction by T3 is independent of new protein synthesis, suggesting that Sox3 is directly activated by liganded T3 receptor. Thus, T3 activates Sox3 as one of the earliest changes in the epithelium, and Sox3 in turn may facilitate the dedifferentiation of the larval epithelial cells into adult stem cells.

Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance and the restoration of functionally-effective behavior.

Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis

BackgroundIntestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells.Methodology/Principal FindingsThe T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium.Conclusions/SignificanceOur results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.

Histone H3K79 methyltransferase Dot1L is directly activated by thyroid hormone receptor during Xenopus metamorphosis

BackgroundThyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and offers a unique opportunity to study how T3 controls postembryonic development in vertebrates. Earlier studies have demonstrated that TR mediates the metamorphic effects of T3 in Xenopus laevis. Liganded TR recruits histone modifying coactivator complexes to target genes during metamorphosis. This leads to nucleosomal removal and histone modifications, including methylation of histone H3 lysine (K) 79, in the promoter regions, and the activation of T3-inducible genes.ResultsWe show that Dot1L, the only histone methyltransferase capable of methylating H3K79, is directly regulated by TR via binding to a T3 response element in the promoter region during metamorphosis in Xenopus tropicalis, a highly related species of Xenopus laevis. We further show that Dot1L expression in both the intestine and tail correlates with the transformation of the organs.ConclusionsOur findings suggest that TR activates Dot1L, which in turn participates in metamorphosis through a positive feedback to enhance H3K79 methylation and gene activation by liganded TR.

Liganded thyroid hormone receptor induces nucleosome removal and histone modifications to activate transcription during adult stem cell development

Thyroid hormone (T3) plays important roles in regulating vertebrate development and pathogenesis. We are using T3‐ dependent Xenopus metamorphosis as a model to study how T3 regulates transcription during development. T3 exerts its metamorphic effects through T3 receptors (TRs). TRs recruit, in a T3‐dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during development is largely unknown. Here, we analyzed histone modifications at T3 target genes during intestinal metamorphosis, a process that involves de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.