Tbx1基因功能下调影响斑马鱼Tbx20和Tbx2表达

目的采用吗啡啉修饰反义寡核苷酸显微注射方法下调斑马鱼Tbx1基因表达,研究斑马鱼Tbx1基因功能下调对其他两个T盒基因Tbx20和Tbx2表达的影响。方法采用吗啡啉修饰的反义寡核苷酸显微注射方法抑制斑马鱼Tbx1基因表达,分别将2.5、5、8、10 ng吗啡啉反义寡核苷酸在斑马鱼0-4细胞期注入胚胎,并构建Tbx20,骨形成蛋白2b（Bmp2b）和Tbx2反义RNA探针,进行整体原位杂交,观察Tbx1基因下调对Tbx20、Bmp2b及Tbx2表达的影响。结果Tbx1吗啡啉寡核苷酸显微注射组胚胎表现出鳃弓、耳囊、心血管系统和胸腺的发育异常。Tbx1基因下调导致Tbx20的表达出现改变,Tbx20在心脏的表达与对照组相比明显下调,神经元的表达范围明显缩小;Tbx1基因功能下调会导致Bmp2b在心脏和咽囊的表达减低,Bmp2b在后部咽囊的表达较前部咽囊减低得更为明显;Tbx1基因功能下调胚胎,Tbx2在鳃弓的表达模式发生改变,48 hpf,Tbx2在鳃弓的表达出现从后向前逐渐减低,鳃弓的表达范围较对照组明显缩小。结论Tbx1在发育过程中,会对其他T盒基因,如Tbx20和Tbx2具有激活或抑制的调控作用。Tbx1对Tbx20的作用可能是通过影响Bmp2b的途径,继发地影响Tbx20的表达。Tbx1基因功能下调,会改变Tbx2在鳃弓的表达模式。     

Collectrin/tmem27 is expressed at high levels in all segments of the developing Xenopus pronephric nephron and in the Wolffian duct

Collectrin/tmem27 encodes a transmembrane protein that plays a critical role in amino-acid transport. Originally described as being expressed only in collecting ducts, it has subsequently also been shown to also be expressed in the S1 segment of the proximal tubule of mammalian metanephric nephrons. In this report we describe the expression of collectrin in the simple embryonic kidney of amphibians, the pronephros. Each pronephros contains a single large nephron with a proximo-distal segmentation very similar to that of mammalian metanephric nephrons. Analysis of collectrin expression in pronephroi at a variety of embryonic stages indicates that this gene is expressed at very high levels throughout the pronephric system, including proximal and distal segments and the Wolffian duct. Expression in the pronephros commences at Xenopus embryonic stage 28 which corresponds to when epithelialization begins within the pronephric mesenchyme. Like the Na+K+ATPase/atp1a1, another highly expressed pronephric marker, collectrin is also expressed in the cloaca but not in the cloacal derived posterior segment of the Wolffian duct, the rectal diverticulum. Unlike the Na+K+ATPase, which is expressed at lower levels in proximal portions of the pronephric nephron, expression of collectrin is even throughout all of the pronephric epithelia. This expression domain extends far beyond that shown to express amino-acid transporters and indicates collectrin may function in facilitating additional transport processes. Its high level of expression and broad distribution make it an excellent marker with which to examine pronephric kidney development.     

UV-mediated regulation of the anti-senescence factor Tbx2

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.     

Jagged2a-notch signaling mediates cell fate choice in the zebrafish pronephric duct

Pronephros, a developmental model for adult mammalian kidneys (metanephros) and a functional kidney in early teleosts, consists of glomerulus, tubule, and duct. These structural and functional elements are responsible for different kidney functions, e.g., blood filtration, waste extraction, salt recovery, and water balance. During pronephros organogenesis, cell differentiation is a key step in generating different cell types in specific locations to accomplish designated functions. However, it is poorly understood what molecules regulate the differentiation of different cell types in different parts of the kidney. Two types of epithelial cells, multi-cilia cells and principal cells, are found in the epithelia of the zebrafish distal pronephric duct. While the former is characterized by at least 15 apically localized cilia and expresses centrin2 and rfx2, the latter is characterized by a single primary cilium and sodium pumps. Multi-cilia cells and principal cells differentiate from 17.5 hours post-fertilization onwards in a mosaic pattern. Jagged2a-Notch1a/Notch3-Her9 is responsible for specification and patterning of these two cell types through a lateral inhibition mechanism. Furthermore, multi-cilia cell hyperplasia was observed in mind bomb mutants and Mind bomb was shown to interact with Jagged2a and facilitate its internalization. Taken together, our findings add a new paradigm of Notch signaling in kidney development, namely, that Jagged2a-Notch signaling modulates cell fate choice in a nephric segment, the distal pronephric duct.     

Role of histone modifications in defining chromatin structure and function

Chromosomes in eukaryotic cell nuclei are not uniformly organized, but rather contain distinct chromatin elements, with each state having a defined biochemical structure and biological function. These are recognizable by their distinct architectures and molecular components, which can change in response to cellular stimuli or metabolic requirements. Chromatin elements are characterized by the fundamental histone and DNA components, as well as other associated non-histone proteins and factors. Post-translational modifications of histone proteins in particular often correlate with a specific chromatin structure and function. Patterns of histone modifications are implicated as having a role in directing the level of chromatin compaction, as well as playing roles in multiple functional pathways directing the readout of distinct regions of the genome. We review the properties of various chromatin elements and the apparent links of histone modifications with chromatin organization and functional output.     

Genetic defects of pronephric cilia in zebrafish

Cilia play key roles in many aspects of embryogenesis and adult physiology in vertebrates. Past genetic screens in zebrafish identified numerous defects of ciliogenesis, including several mutations in the components of the intraflagellar transport machinery. In contrast to previous studies, here we describe a collection of mutants that affect subpopulations of cilia. Mutant embryos are characterized by a shortening and an abnormal movement of kidney cilia, and in one case also a reduction of cilia length in the Kupffer's vesicle. In contrast to that, the cilia of sensory neurons, including photoreceptor cells, hair cells, and olfactory sensory cells, appear grossly intact. Motility defects of pronephric cilia vary in mutant strains from complete paralysis to an increased frequency of movement, and are associated with left-right asymmetry defects. While ciliary ultrastructure is normal in most mutants, one of the mutant loci is essential for the formation of proper microtubule architecture in the axoneme of pronephric cilia. Mutants characterized in this study reveal intriguing genetic differences between subpopulations of embryonic cilia, and provide an opportunity to study several aspects of cilia structure and function.     

Role of adrenal hormones in regulating distal nephron structure and ion transport

Mineralocorticoid levels are an important determinant of membrane area and ion transport in the renal initial (ICT) and cortical (CCT) collecting tubules. Adrenalectomy leads to a dramatic and specific decrease in basolateral membrane area of principal (P) cells and depresses sodium reabsorption and potassium secretion. Although aldosterone replacement for 10 days restores basolateral membrane area and ATPase activity to control levels and dramatically elevates ion transport, glucocorticoids have no effect on basolateral membrane area, ion transport, or ATPase. It is suggested that the aldosterone-induced amplification of membrane area occurs as a mechanism whereby cells increase the number of ATPase pumps in the basolateral membrane. An acute (of 2-3 h) increase in aldosterone, but not dexamethasone, also stimulates potassium transport by the ICT. Future studies will have to establish whether the acute stimulation of transport by aldosterone involves a change in basolateral membrane area. It is concluded that mineralocorticoids, but not glucocorticoids, regulate sodium and potassium transport by P cells of the ICT and CCT, in part, by determining the number of ATPase pumps available for transport.     

Ultrastructural characterization of the pronephric glomerulus development in zebrafish

The zebrafish pronephros is a valuable model for studying kidney development and diseases. Ultrastructural studies have revealed that zebrafish and mammals share similarities in nephron structures such as podocytes, slit diaphragms, glomerular basement membrane, and endothelium. However, the basic ultrastructural features of the pronephric glomerulus during glomerulogenesis have not been characterized. To understand these features, it is instructive to consider the developmental process of the pronephros glomerulus. Here, we describe the ultrastructural features of pronephric glomerulus in detail from 24 h hours post‐fertilization (hpf) to 144 hpf, the period during which the pronephric glomerulus develops from initiation to its mature morphology. The pronephric glomerulus underwent progressive morphogenesis from 24 to 72 hpf, and presumptive glomerular cells were observed ventral to the aorta region at 24 hpf. The nascent glomerular basement membrane and initial lumen were formed at 36 hpf. A lumen was clearly visible in the region of the pronephros at 48 hpf. At 60 hpf, the pronephric glomerulus contained more patches of capillaries. After these transformations, the complex capillary vessel networks had formed inside the glomerulus, which was surrounded by podocyte bodies with elaborate foot processes as well as well‐formed glomerular basement membrane by 72 hpf. The number of renal glomerular cells rapidly increased, and the glomerulus presented its delicate structural features by 96 hpf. Morphogenesis was completed at 120 hpf with the final formation of the pronephric glomerulus. J. Morphol. 277:1104–1112, 2016. © 2016 Wiley Periodicals, Inc.     

Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins

Charge-charge interactions on the surface of native proteins are important for protein stability and can be computationally redesigned in a rational way to modulate protein stability. Such computational effort led to an engineered protein, CspB-TB that has the same core as the mesophilic cold shock protein CspB-Bs from Bacillus subtilis, but optimized distribution of charge-charge interactions on the surface. The CspB-TB protein shows an increase in the transition temperature by 20 degrees C relative to the unfolding temperature of CspB-Bs. The CspB-TB and CspB-Bs protein pair offers a unique opportunity to further explore the energetics of charge-charge interactions as the substitutions at the same sequence positions are done in largely similar structural but different electrostatic environments. In particular we addressed two questions. What is the contribution of charge-charge interactions in the unfolded state to the protein stability and how amino acid substitutions modulate the effect of increase in ionic strength on protein stability (i.e. protein halophilicity). To this end, we experimentally measured the stabilities of over 100 variants of CspB-TB and CspB-Bs proteins with substitutions at charged residues. We also performed computational modeling of these protein variants. Analysis of the experimental and computational data allowed us to conclude that the charge-charge interactions in the unfolded state of two model proteins CspB-Bs and CspB-TB are not very significant and computational models that are based only on the native state structure can adequately, i.e. qualitatively (stabilizing versus destabilizing) and semi-quantitatively (relative rank order), predict the effects of surface charge neutralization or reversal on protein stability. We also show that the effect of ionic strength on protein stability (protein halophilicity) appears to be mainly due to the screening of the long-range charge-charge interactions.     

In vitro induction of the pronephric duct in Xenopus explants

The earliest form of embryonic kidney, the pronephros, consists of three components: glomus, tubule and duct. Treatment of the undifferentiated animal pole ectoderm of Xenopus laevis with activin A and retinoic acid (RA) induces formation of the pronephric tubule and glomus. In this study, the rate of induction of the pronephric duct, the third component of the pronephros, was investigated in animal caps treated with activin A and RA. Immunohistochemistry using pronephric duct-specific antibody 4A6 revealed that a high proportion of the treated explants contained 4A6-positive tubular structures. Electron microscopy showed that the tubules in the explants were similar to the pronephric ducts of normal larvae, and they also expressed Gremlin and c-ret, molecular markers for pronephric ducts. These results suggest that the treatment of Xenopus ectoderm with activin A and RA induces a high rate of differentiation of pronephric ducts, in addition to the differentiation of the pronephric tubule and glomus, and that this in vitro system can serve as a simple and effective model for analysis of the mechanism of pronephros differentiation.     

Cell migration in the formation of the pronephric duct in Xenopus laevis

To determine if cell migration is involved in the formation of the pronephric duct in Xenopus, we used morphometry, ablation, and videomicroscopy of vitally stained cells to study duct formation. In St 23-24 (Nieuwkoop and Faber, 1956) embryos, a ridge of cells forms caudal to the pronephric rudiment. The ridge lengthens at approximately the same rate as the embryonic trunk from St 23 to St 31. Ablation experiments demonstrated that the ridge constitutes the pronephric duct rudiment (PDR); when the ridge was ablated at St 23-24, little or no duct formation occurred, whereas a duct formed when the pronephric rudiment was ablated and the ridge left intact. Vital dye injections showed that the PDR forms from the intermediate mesoderm ventral to myotomes IV-VIII. From St 29/30 to St 33/34, the PDR actively elongates along the ventral edge of the myotomes as far as myotome XIV, where it joins the cloaca as the pronephric duct. Videomicroscopy of vitally stained cells showed that the PDR elongates throughout its length and does not incorporate additional cells from the mesoderm over which it elongates. The results strengthen the case for a common mode of pronephric duct formation among amphibian species.     

New insights into sodium transport regulation in the distal nephron:Role of G-protein coupled receptors

The renal handling of Na~+ balance is a major determinant of the blood pressure(BP) level. The inability of the kidney to excrete the daily load of Na+ represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part(i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na~+ excretion and are the target of many different regulatory processes that modulate Na~+ retention more or less efficiently. G-protein coupled receptors(GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na~+ absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na~+ excretion, this review also highlights the complexity of these different pathways, and the connections between them.     

A unique expression pattern of Tbx10 in the hindbrain as revealed by Tbx10LacZ allele

To study the expression/function of Tbx10, a T‐box gene, Tbx10LacZ/+ mice were established by replacing the T‐box coding region with a LacZ gene. X‐gal staining showed that LacZ+ cells were localized to two‐cell populations in rhombomere 4 and rhombomere 6. No significant differences in the locations of LacZ+ cells were found between Tbx10LacZ/+ and Tbx10LacZ/LacZ mice, and the Tbx10LacZ/LacZ mice were viable and fertile. We found that the LacZ+ cells are present in both embryonic and adult mice. Histological studies suggest that the rhombomere 4‐derived LacZ+ cells are a subpopulation of the ventral interneurons in the pons.