Daniel R. Buchholz

Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis)

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.

Developmental plasticity mirrors differences among taxa in spadefoot toads linking plasticity and diversity.

Developmental plasticity is found in most organisms, but its role in evolution remains controversial. Environmentally induced phenotypic differences may be translated into adaptive divergence among lineages experiencing different environmental conditions through genetic accommodation. To examine this evolutionary mechanism, we studied the relationship between plasticity in larval development, postmetamorphic morphology, and morphological diversity in spadefoot toads, a group of closely related species that are highly divergent in the larval period and body shape and are distributed throughout temperate areas of both the New and the Old World. Previous studies showed that accelerated metamorphosis is adaptive for desert-dwelling spadefoot toads. We show that even under common garden conditions, spadefoot toad species show divergent reaction norms for the larval period. In addition, experimentally induced changes in the larval period caused correlated morphological changes in postmetamorphic individuals such that long larval periods resulted in relatively longer hindlimbs and snouts. A comparative analysis of morphological variation across spadefoot toad species also revealed a positive correlation between the larval period and limb and snout lengths, mirroring the effects of within-species plasticity at a higher taxonomic level. Indeed, after ≈110 Ma of independent evolution, differences in the larval period explain 57% of the variance in relative limb length and 33% of snout length across species. Thus, morphological diversity across these species appears to have evolved as a correlated response to selection for a reduced larval period in desert-dwelling species, possibly diverging from ancestral plasticity through genetic accommodation.

Transgenic Analysis Reveals that Thyroid Hormone Receptor Is Sufficient To Mediate the Thyroid Hormone Signal in Frog Metamorphosis

ABSTRACT Thyroid hormone (T3) has long been known to be important for vertebrate development and adult organ function. Whereas thyroid hormone receptor (TR) knockout and transgenic studies of mice have implicated TR involvement in mammalian development, the underlying molecular bases for the resulting phenotypes remain to be determined in vivo, especially considering that T3 is known to have both genomic, i.e., through TRs, and nongenomic effects on cells. Amphibian metamorphosis is an excellent model for studying the role of TR in vertebrate development because of its total dependence on T3. Here we investigated the role of TR in metamorphosis by developing a dominant positive mutant thyroid hormone receptor (dpTR). In the frog oocyte transcription system, dpTR bound a T3-responsive promoter and activated the promoter independently of T3. Transgenic expression of dpTR under the control of a heat shock-inducible promoter in premetamorphic tadpoles led to precocious metamorphic transformations. Molecular analyses showed that dpTR induced metamorphosis by specifically binding to known T3 target genes, leading to increased local histone acetylation and gene activation, similar to T3-bound TR during natural metamorphosis. Our experiments indicated that the metamorphic role of T3 is through genomic action of the hormone, at least on the developmental parameters tested. They further provide the first example where TR is shown to mediate directly and sufficiently these developmental effects of T3 in individual organs by regulating target gene expression in these organs.

A Dominant-Negative Thyroid Hormone Receptor Blocks Amphibian Metamorphosis by Retaining Corepressors at Target Genes

ABSTRACT The total dependence of amphibian metamorphosis on thyroid hormone (T3) provides a unique vertebrate model for studying the molecular mechanism of T3 receptor (TR) function in vivo. In vitro transcription and developmental expression studies have led to a dual function model for TR in amphibian development, i.e., TRs act as transcriptional repressors in premetamorphic tadpoles and as activators during metamorphosis. We examined molecular mechanisms of TR action in T3-induced metamorphosis by using dominant-negative receptors (dnTR) ubiquitously expressed in transgenic Xenopus laevis. We showed that T3-induced activation of T3 target genes and morphological changes are blocked in dnTR transgenic animals. By using chromatin immunoprecipitation, we show that dnTR bound to target promoters, which led to retention of corepressors and continued histone deacetylation in the presence of T3. These results thus provide direct in vivo evidence for the first time for a molecular mechanism of altering gene expression by a dnTR. The correlation between dnTR-mediated gene repression and inhibition of metamorphosis also supports a key aspect of the dual function model for TR in development: during T3-induced metamorphosis, TR functions as an activator via release of corepressors and promotion of histone acetylation and gene activation.

Recruitment of N-CoR/SMRT-TBLR1 corepressor complex by unliganded thyroid hormone receptor for gene repression during frog development.

ABSTRACT The corepressors N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) interact with unliganded nuclear hormone receptors, including thyroid hormone (T3) receptor (TR). Several N-CoR/SMRT complexes containing histone deacetylases have been purified. The best studied among them are N-CoR/SMRT complexes containing TBL1 (transducin beta-like protein 1) or TBLR1 (TBL1-related protein). Despite extensive studies of these complexes, there has been no direct in vivo evidence for the interaction of TBL1 or TBLR1 with TR or the possible involvement of such complexes in gene repression by any nuclear receptors in any animals. Here, we used the frog oocyte system to demonstrate that unliganded TR interacts with TBLR1 and recruits TBLR1 to its chromatinized target promoter in vivo, accompanied by histone deacetylation and gene repression. We further provide evidence to show that the recruitment of TBLR1 or related proteins is important for repression by unliganded TR. To investigate the potential role for TBLR1 complexes during vertebrate development, we made use of T3-dependent amphibian metamorphosis as a model. We found that TBLR1, SMRT, and N-CoR are recruited to T3-inducible promoters in premetamorphic tadpoles and are released upon T3 treatment, which induces metamorphosis. More importantly, we demonstrate that the dissociation of N-CoR/SMRT-TBLR1 complexes from endogenous TR target promoters is correlated with the activation of these genes during spontaneous metamorphosis. Taken together, our studies provide in vivo evidence for targeted recruitment of N-CoR/SMRT-TBLR1 complexes by unliganded TR in transcriptional repression during vertebrate development.

Phylogenetic relationships of Pelobatoidea re-examined using mtDNA.

Pelobatoidea is a clade of ancient anurans with obscure relationships to the remaining clades of frogs. We used partial sequences of two mitochondrial genes (cytochrome b and 16S RNA) from all Pelobatoidea subclades, including all species of Pelobatidae and Pelodytidae and four outgroup taxa (Xenopus, Ascaphus, Discoglossus, and Rana), to propose a phylogenetic hypothesis for relationships within Pelobatoidea. Maximum likelihood and Bayesian analyses support the monophyly of Pelobatoidea, but our hypothesis of internal relationships differs substantially from all previous hypotheses. Megophryidae is sister to Pelobates, and this clade is sister to Pelodytes. The most basal clade within Pelobatoidea is formed by Scaphiopus and Spea. The family Pelobatidae, as previously defined is not monophyletic, and it is split into Eurasian spadefoot toads Pelobates which retain the name Pelobatidae and North American spadefoot toads Scaphiopus and Spea which comprise the revived taxon Scaphiopodidae. Our analysis uncovers the existence of morphologically cryptic taxa within previously recognized species of the genus Spea and reveals marked genetic differentiation within Iberian Pelodytes. We discuss biogeographic implications and the evolution of fossoriality in the light of the new phylogenetic hypothesis.

The Xenoestrogen Bisphenol A Inhibits Postembryonic Vertebrate Development by Antagonizing Gene Regulation by Thyroid Hormone

Bisphenol A (BPA), a chemical widely used to manufacture plastics, is estrogenic and capable of disrupting sex differentiation. However, recent in vitro studies have shown that BPA can also antagonize T(3) activation of the T(3) receptor. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. This study proposed to identify critical T(3) pathways that may be disrupted by BPA based on molecular analysis in vivo. Because amphibian metamorphosis requires T(3) and encompasses the postembryonic period in mammals when T(3) action is most critical, we used this unique model for studying the effect of BPA on T(3)-dependent vertebrate development at both the morphological and molecular levels. After 4 d of exposure, BPA inhibited T(3)-induced intestinal remodeling in premetamorphic Xenopus laevis tadpoles. Importantly, microarray analysis revealed that BPA antagonized the regulation of most T(3)-response genes, thereby explaining the inhibitory effect of BPA on metamorphosis. Surprisingly, most of the genes affected by BPA in the presence of T(3) were T(3)-response genes, suggesting that BPA predominantly affected T(3)-signaling pathways during metamorphosis. Our finding that this endocrine disruptor, well known for its estrogenic activity in vitro, functions to inhibit T(3) pathways to affect vertebrate development in vivo and thus not only provides a mechanism for the likely deleterious effects of BPA on human development but also demonstrates the importance of studying endocrine disruption in a developmental context in vivo.

Identification of direct thyroid hormone response genes reveals the earliest gene regulation programs during frog metamorphosis

Thyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis.

Origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis

In the amphibian intestine during metamorphosis, de novo stem cells generate the adult epithelium analogous to the mammalian counterpart. Interestingly, to date the exact origin of these stem cells remains to be determined, making intestinal metamorphosis a unique model to study development of adult organ‐specific stem cells. Here, to determine their origin, we made use of transgenic Xenopus tadpoles expressing green fluorescent protein (GFP) for recombinant organ cultures. The larval epithelium separated from the wild‐type (Wt) or GFP transgenic (Tg) intestine before metamorphic climax was recombined with homologous and heterologous nonepithelial tissues and was cultivated in the presence of thyroid hormone, the causative agent of metamorphosis. In all kinds of recombinant intestine, adult progenitor cells expressing markers for intestinal stem cells such as sonic hedgehog became detectable and then differentiated into the adult epithelium expressing intestinal fatty acid binding‐protein, a marker for absorptive cells. Notably, whenever the epithelium was derived from Tg intestine, both the adult progenitor/stem cells and their differentiated cells expressed GFP, whereas neither of them expressed GFP in the Wt‐derived epithelium. Our results provide direct evidence that stem cells that generate the adult intestinal epithelium originate from the larval epithelium, through thyroid hormone‐induced dedifferentiation.— Ishizuya‐Oka, A.,Hasebe, T., Buchholz, D. R., Kajita, M., Fu, L., Shi, Y.‐B. The origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis. FASEB J. 23, 2568–2575 (2009)

A Causative Role of Stromelysin-3 in Extracellular Matrix Remodeling and Epithelial Apoptosis during Intestinal Metamorphosis in Xenopus laevis

The matrix metalloproteinases are a family of proteases capable of degrading various components of the extracellular matrix. Expression studies have implicated the involvement of the matrix metalloproteinase stromelysin-3 (ST3) in tissue remodeling and pathogenesis. However, the in vivo role of ST3 has been difficult to study because of a lack of good animal models. Here we used intestinal remodeling during thyroid hormone-dependent metamorphosis of Xenopus laevis as a model to investigate in vivo the role of ST3 during postembryonic organ development in vertebrates. We generated transgenic tadpoles expressing ST3 under control of a heat shock-inducible promoter. We showed for the first time in vivo that wild type ST3 but not a catalytically inactive mutant was sufficient to induce larval epithelial cell death and fibroblast activation, events that normally occur only in the presence of thyroid hormone. We further demonstrated that these changes in cell fate are associated with altered gene expression in the intestine and remodeling of the intestinal basal lamina. These results thus suggest that ST3 regulates cell fate and tissue morphogenesis through direct or indirect ECM remodeling.