Increase in liver acid lipase of thyroidectomized rats by thyroid hormones and its inhibition by actinomycin D

Acid lipase activity in the livers of thyroidectomized rats is increased by administration of L-thyroxine. The response is dose-dependent and can be demonstrated within 12 h after treatment. L-Triiodothyronine also evokes a rapid increase in acid lipase activity, and this increase can be inhibited by co-administration of actinomycin D.     

Identification of both general and region-specific embryonic CNS enhancer elements in the nestin promoter

In this report, we investigate how nestin expression is controlled in neural progenitor cells of the embryonic CNS. A 374-bp region in the second intron of the human nestin gene is sufficient, and a 120-bp sequence in this region is required, to express the lacZ reporter gene throughout the developing CNS of E9.5-10.5 transgenic mouse embryos. The 120-bp element region contains putative binding sites for nuclear hormone receptors and we show that TRs, RXR, RAR, and COUP-TF bind to these motifs. A separate enhancer, located most probably 5' to the 120-bp sequence in the second intron, controls midbrain expression at E10.5. In conclusion, our data show that the nestin enhancer in the second intron contains elements both for general and for region-specific CNS progenitor cell expression and suggest that nuclear hormone receptors play a role in the regulation of nestin expression in the early CNS.     

Thyroid hormones regulate anxiety in the male mouse

Thyroid hormone levels are implicated in mood disorders in the adult human but the mechanisms remain unclear partly because, in rodent models, more attention has been paid to the consequences of perinatal hypo and hyperthyroidism. Thyroid hormones act via the thyroid hormone receptor (TR) α and β isoforms, both of which are expressed in the limbic system. TR's modulate gene expression via both unliganded and liganded actions. Though the thyroid hormone receptor (TR) knockouts and a transgenic TRα1 knock-in mouse have provided us valuable insight into behavioral phenotypes such as anxiety and depression, it is not clear if this is because of the loss of unliganded actions or liganded actions of the receptor or due to locomotor deficits. We used a hypothyroid mouse model and supplementation with tri-iodothyronine (T3) or thyroxine (T4) to investigate the consequences of dysthyroid hormone levels on behaviors that denote anxiety. Our data from the open field and the light–dark transition tests suggest that adult onset hypothyroidism in male mice produces a mild anxiogenic effect that is possibly due to unliganded receptor actions. T3 or T4 supplementation reverses this phenotype and euthyroid animals show anxiety that is intermediate between the hypothyroid and thyroid hormone supplemented groups. In addition, T3 but not T4 supplemented animals have lower spine density in the CA1 region of the hippocampus and in the central amygdala suggesting that T3-mediated rescue of the hypothyroid state might be due to lower neuronal excitability in the limbic circuit.     

Effects of Low‐Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish

Thyroid hormones are required for vertebrate development, and disruption of the thyroid system in developing embryos can result in a large range of morphologic and physiologic changes, including in the eye and retina. In this study, our anatomic analyses following low‐dose, chronic thyroid inhibition reveal that both methimazole (MMI) exposure and rearing temperature affect eye development in a time‐ and temperature‐dependent fashion. Maximal sensitivity to MMI for external eye development occurred at 65 hr postfertilization (hpf) for zebrafish reared at 28°C, and at 69 hpf for those reared at 31°C. Changes in eye diameter corresponded to changes in thickness of two inner retinal layers: the ganglion cell layer and the inner plexiform layer, with irreversible MMI‐induced decreases in layer thickness observed in larvae treated with MMI until 66 hpf at 28°C. We infer that maximal sensitivity to MMI between 65 and 66 hpf at 28°C indicates a critical period of thyroid‐dependent eye and retinal development. Furthermore, our results support previous work that shows spontaneous escape from MMI‐induced effects potentially due to embryonic compensatory actions, as our data show that embryos treated beyond the critical period generally resemble controls     

Thyroid hormones are necessary for the metamorphosis of tarpon Megalops cyprinoides leptocephali

This study investigates the effects of thyroxine (T₄), triiodothyronine (T₃) and thiourea (TU) on the metamorphosis of tarpon Megalops cryprinoides leptocephali. TU is an anti-thyroid hormone drug that inhibits the production of T₄ and T₃ in the thyroid tissue. Fully grown tarpons leptocephali were collected at the river mouth and, in the laboratory, were immediately treated with 100 ppb T₄, 10 ppb T_3, or 300 ppm TU. The appropriate concentrations were validated in a preliminary dose response experiment. Morphological and physiological characteristics that indicate metamorphic processes were measured every 2 days. T₄ and T₃ slightly speeded up the metamorphosis of tarpons compared with the control group. The experimental treatments produced accelerated reductions in length, increases in head/body ratio, swimbladder development, and loss of body water and sodium. In contrast, TU treatment caused metamorphic stasis with complete inhibition of metamorphosis between days 6 and 8. Thyroid hormone treatment stimulated fast otolith growth while TU treatment stopped otolith growth between days 6 and 9. Leptocephali in T₄, T₃ and control groups completed metamorphosis in 10-14 days, but TU-treated tarpons remained in the metamorphic leptocephalus stage more than 22 days. In addition, the inhibition of leptocephalus metamorphosis by 300 ppm TU can be reversed in the presence of 10 ppb T₃. These results indicate that thyroid hormones are involved in regulating the metamorphosis of leptocephali.     

Cell-type-specific responses to the microbiota across all tissues of the larval zebrafish

1. Download : Download high-res image (329KB)
2. Download : Download full-size image

     

斑马鱼整体原位杂交法研究MMP15a的发育相关表达的初步观察

克隆斑马鱼基质金属蛋白酶15a（MMP15a）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP15a RNA探针,采用全胚胎原位杂交方法研究MMP15a基因在胚胎斑马鱼的表达。结果MMP15a基因在胚胎受精后一个细胞时期就开始表达,从受精后24h起,在眼睛处表达明显,从受精后48h MMP15a在胸鳍和耳囊有特异性表达至到受精后96h。MMP15a在斑马鱼胚胎发育不同时期表达明显,且在胸鳍和耳囊处有持续表达。     

Binding of thyroid hormones to human hemoglobin and localization of the binding site

Radiolabeled thyroid hormones were allowed to bind to erythrocyte cytosol and the complex was fractionated by Sephadex G-100 or by high-performance liquid chromatography (HPLC). On Sephadex G-100, four radioactive peaks (P₁P₄) were obtained, whereas HPLC gave only three radioactive peaks (P₁P₃). Chromatographic studies with human adult Hb and non-Hb cytosol protein fractions, which had been reacted with radiolabeled thyroid hormones, and immune precipitation with specific antisera for the hormones, confirmed that the first peak of Sephadex G-100 radioactivity was a mixture of Hb and non-Hb proteins, while the second peak was Hb. The third peak was free¹²⁵I and the fourth peak was unbound¹²⁵I-T₃ or¹²⁵I-T₄. The third peak of HPLC was confirmed to be a mixture of free¹²⁵I and unbound radiolabeled thyroid hormones. Scatchard analysis of the interaction between T₄ and apo-Hb, and the - and -chains of human Hb suggested the presence of the specific binding site(s) for the hormone. Interaction between T₄ and synthesized peptides, which constitute the heme pocket of the -chain of Hb (61–75, 71–85, 81–95), indicated that the T₄ binding site of Hb resides within the heme-binding cavity. It is concluded that human erythrocyte cytosol does not contain receptor for thyroid hormones and cannot be a model for studying functions of cytosol receptor for the hormones; rather, it contains binding protein with large binding capacity, including Hb and non-Hb proteins, which possibly constitute a large reservoir for the hormone in blood.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

NF‐Y is critical for the proper growth of zebrafish embryonic heart and its cardiomyocyte proliferation

The ubiquitous NF‐Y gene regulates the expression of different genes in various signaling pathways. However, the function of NF‐Y in zebrafish heart development is largely unknown. Previously we identified a same group of cell cycle related gene cluster (CCRG) was downregulated in the embryonic hearts with impeded growth due to various stresses. The promoter regions of these CCRG genes shared a most common motif for NF‐Y. Chromatin immunoprecipitation experiment demonstrated that the binding of NF‐Y to its motif was real on the CCRG candidate gene promoters. Knockdown of embryonic NF‐Y by morpholinos led to a small heart, mimicking the abnormal heart phenotype caused by other stresses. In parallel the expression of certain CCRG candidate genes was reduced in the NF‐Y A morphant hearts exposed to malignant environments. Absence of NF‐Y A also led to undermine cardiomyocyte proliferation and hence less total number of caridomyocytes per heart. Trans‐AM Elisa experiment also found that in the presence of the stresses such as TCDD and TNNT2 MO, the binding capacity of NF‐Y A subunit to its core motif was reduced. We conclude that NF‐Y sustains proper cardiomyocyte proliferation in the heart, thus it plays a positive role in promoting early zebrafish heart growth.     

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

The thyroid hormones L-thyroxine and triiodo-Lthyronine have profound effects on postenbryonic development of most vertebrates.Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action.Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process(morphogenesis,cell death,re-structuring,etc.) in free-living embryos and larvas of most anurans.This article will first describe the salient features of metamorphosis and its control by TH and other hormones.Emphasis will be laid on the key role played by TH receptor (TR),in particular the phenomenon of TR gene autoinduction,in initiating the developmental action of TH.Finally,it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.     

Dynamics of process formation during differentiation of tectal neurons in embryonic zebrafish

Neurons acquire their distinct shapes after passing through many transitional stages in early development. To reveal the dynamics and spatiotemporal sequence of process formation in situ, the growth of neurons in the optic tectum of live zebrafish embryos (54 to >100 h old) was monitored using time-lapse videorecordings. Neurons were labeled by injecting the fluorescent vital dye DiO into the cell-rich layer of the developing tectum in 50- to 70-h-old embryos. In phase 1, tectal neurons possess an apical “primary process” which reaches to the ventral aspect of the tectal neuropil. The primary process produces at its tip short transitory branches, some with growth cones, over a period of roughly 6 h. One of the growth cones then elongates rapidly and grows toward the caudal tectum via a route characteristic of efferent axons. After retraction of excess branches and growth cones, branching activity resumes at the tip of the primary process to form the dendritic tree (phase 2). The dendritic tree develops in the tectal neuropil through emission and retraction of many branches during a period of >20 h (our longest continuous time-lapse movie). The tectal territory “explored” in this way is larger than the area finally covered by the tree resulting from growth and loss of branches. The dynamics observed here directly are probably characteristic for dendrite formation in vertebrates. Moreover, consistent with the sequence of neuronal differentiation observed in vitro, the growth of the axon precedes that of the dendrites, although both emerge from a common primary process in this type of tectal neuron. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 627–639, 1997