Polycystic kidney disease in SBM transgenic mice: role of c-myc in disease induction and progression.

SBM mouse is a unique transgenic model of polycystic kidney disease (PKD) produced by dysregulation of c-myc in the kidneys. Our previous demonstration that c-myc is overexpressed in human autosomal polycystic kidney disease (ADPKD) prompted us to investigate the pathogenetic role of c-myc in the induction and progression of the cystogenic phenotype in our mouse model. In young SBM kidneys, c-myc was two- to threefold increased with persistent expression levels into adulthood, an age when c-myc is normally undetectable. In situ hybridization analysis of the c-myc transgene demonstrated intense signal specifically overlying glomerular and tubular epithelium of developing cysts in fetal and young kidneys. Increased expression of c-myc correlated with the initiation and progression of the PKD phenotype as evidenced by early tubular and glomerular cysts at E16.5. Cyst number and size increased with age, with co-development of glomerular and tubular epithelial hyperplasia. Consistently, the mean renal proliferative index was increased approximately 5- to 20-fold in noncystic and cystic tubules of newborn SBM animals compared with littermate controls. Similarly, in fetal and newborn kidneys the tubular apoptotic indices were increased approximately three- to ninefold over controls. Both proliferation and apoptotic rates in cystic tubules approached levels in developing tubules from the normal nephrogenic zone. We conclude that the pathogenesis of PKD hinges on a critical imbalance in c-myc regulation of the opposing processes of cell proliferation and apoptosis, recapitulating the cellular phenomena in developing fetal kidney.     

The role of macrophages in clearing programmed cell death in the developing kidney

The metanephric kidney develops from two tissue sources, the metanephric mesenchymal blastema and the ureteric bud epithelium. Following a complex interplay of inductive signals between these two tissues, small groups of metanephric mesenchymal cells aggregate and epithelialise to form young nephrons. As this is happening, significant numbers of cells in close proximity to the forming nephrons undergo programmed cell death or apoptosis. In this paper we investigate the clearance of developmental cell death in the mouse kidney between embryonic days 11.5 and 16.5; specifically, we address the issue of whether specialist macrophages or non-specialist neighbouring mesenchymal cells are responsible for phagocytosis and removal of dying cells. We show, using a monoclonal antibody F4/80 that specifically recognizes murine macrophages, that whenever and wherever there is cell death in the developing mesonephric or metanephric kidney there are also haemopoietically derived specialist macrophages. Moreover, in the mesonephros and from E14.5 in the metanephric kidney, we see large numbers of macrophages clearly swollen with phagocytosed apoptotic bodies. Double-labelling experiments using the DNA dye 7AAD to reveal condensed apoptotic nuclei and F4/80 to reveal macrophage plasma membranes show definitively that the majority of dying cells in the developing kidney are engulfed by macrophages.     

甘油二酯激酶γ参与大鼠胚胎脑神经上皮细胞的发育

目的探讨甘油二酯激酶γ(DGKγ)在大鼠胚胎脑神经上皮发育中的作用及可能机制。方法 18只胎龄(E)11.5、E12.5、E14.5、E16.5、E18.5大鼠胚胎用于Western blotting方法检测DGKγ在大鼠胚胎脑组织的表达,E9.5~E18.5大鼠胚胎各5只用于免疫组织化学和免疫荧光染色方法检测DGKγ在大鼠胚胎脑的分布以及DGKγ与Ki67、乙酰胆碱转移酶(Ch AT)及酪氨酸羟化酶(TH)的细胞内共定位。结果 DGKγ蛋白含量在E11.5~E14.5表达逐渐增多,E16.5表达较E14.5显著减少,E18.5表达显著增多且高于E14.5水平。E10.5~E12.5,DGKγ在脑泡壁出现表达,并随着脑泡的发育表达在端脑、除大脑脚外的中脑、后脑和末脑;E13.5~E14.5,DGKγ的强阳性表达从海马及周围的新皮质、苍白球、嗅脑和大脑脚阳性延伸到除新皮质外的脑的各区域;E15.5至出生前,DGKγ在新皮质阳性表达增强,但在大脑脚和脑桥表达减弱。免疫荧光染色显示,E14.5,与海马和苍白球相比,嗅脑的DGKγ阳性细胞中Ki67阳性率较高(87%),Ch AT阳性率较低(9%)(均P0.05),中脑顶盖的DGKγ阳性细胞中62%为TH阳性;E16.5,新皮质的DGKγ阳性细胞中Ki67阳性率为26%,较E14.5时(49%)显著降低(P0.05),Ch AT阳性率为70%,较E14.5时(45%)显著增高(P0.05);DGKγ多位于神经上皮细胞的胞质,也可见于胞膜上,或者同时位于胞质和胞核。结论 DGKγ在胚胎脑的表达模式和亚细胞分布,提示其参与了胚胎脑神经上皮的增殖、迁移和分化后神经元的发育。     

甘油二酯激酶ζ、蛋白激酶Cδ和Tis21在发育中大鼠脑的表达和定位

目的探讨甘油二酯激酶ζ(DGKζ)、蛋白激酶Cδ(PKCδ)和Tis21在大鼠胚胎脑的时空表达模式以及与脑发育的关系。方法胚龄(E)9.5~E18.5大鼠胚胎各5只,连续切片用于免疫组织化学方法检测DGKζ、p-PKCδ和Tis21在大鼠胚胎脑的表达;22只胚龄E11.5、E12.5、E14.5、E16.5和E18.5大鼠胚胎用于Western blotting,检测DGKζ、磷酸化PKCδ(p-PKCδ)、PKCδ和Tis21在大鼠胚胎脑组织的表达。结果免疫组织化学显示,于E9.5 p-PKCδ和Tis21在神经沟神经上皮呈阳性表达,DGKζ为阴性;E10.5~E11.5,DGKζ、p-PKCδ和Tis21在脑泡壁均呈阳性表达;在E12.5三者均表达于端脑、间脑、中脑腹侧和脑桥处神经上皮,而中脑背侧、峡部和小脑原基仅DGKζ呈阳性表达,p-PKCδ和Tis21呈阴性;于E13.5三者沿海马、隔、苍白球及嗅脑呈阳性分布,在新皮质呈阴性,p-PKCδ和Tis21在峡部和小脑原基出现阳性表达;于E14.5 DGKζ表达延伸到新皮质,在脑的各个区域呈现出全阳性。而p-PKCδ和Tis21表达仅延伸到中脑顶盖,在新皮质仍为阴性;于E15.5 p-PKCδ和Tis21在新皮质出现阳性表达,在脑的各个区域均呈阳性;E16.5~E18.5,三者在小脑和脑桥表达范围缩小;DGKζ和p-PKCδ可表达在神经上皮细胞的胞质,或胞质和细胞核。Western blotting显示,DGKζ和Tis21在E11.5~E14.5表达逐渐增多,E12.5较E11.5显著增多,在E16.5和E18.5逐渐下降;p-PKCδ/PKCδ显示PKCδ活性变化趋势与DGKζ表达趋势一致。结论 DGKζ、p-PKCδ和Tis21时空表达与脑发育模式一致,DGKζ和p-PKCδ在神经上皮细胞的亚细胞分布提示它们主要通过DGKζ/Tis21和PKCδ/Tis21途径参与脑的发育。     

Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation.

Inhibitors of programmed cell death (apoptosis) may regulate tissue differentiation and aberrantly promote cell survival in neoplasia. A novel apoptosis inhibitor of the IAP gene family, designated survivin, was recently found in all of the most common human cancers but not in normal, terminally differentiated adult tissues. The expression of survivin in embryonic and fetal development was investigated. Immunohistochemistry and in situ hybridization studies demonstrated strong expression of survivin in several apoptosis-regulated fetal tissues, including the stem cell layer of stratified epithelia, endocrine pancreas, and thymic medulla, with a pattern that did not overlap with that of another apoptosis inhibitor, bcl-2. A sequence-specific antibody to survivin immunoblotted a single approximately 16.5-kd survivin band in human fetal lung, liver, heart, kidney, and gastrointestinal tract. In mouse embryo, prominent and nearly ubiquitous distribution of survivin was found at embryonic day (E)11.5, whereas at E15 to -21, survivin expression was restricted to the distal bronchiolar epithelium of the lung and neural-crest-derived cells, including dorsal root ganglion neurons, hypophysis, and the choroid plexus. These data suggest that expression of survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation independently of bcl-2. Aberrations of this developmental pathway may result in prominent re-expression of survivin in neoplasia and abnormally prolonged cell viability.     

发育期小鼠肾小体细胞凋亡的研究

目的 研究小鼠肾小体发育过程中的细胞凋亡规律及形态学特点。方法 应用光镜、电镜技术和脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记法(TUNEL法)分别对不同胚龄和生后日龄小鼠肾小体内细胞凋亡进行观察。结果 胚龄14d肾小体发生时就出现细胞凋亡，胚龄18d左右达到高峰，l随后缓慢下降。肾小体内凋亡细胞以内皮细胞和足细胞多见。凋亡细胞的两种结局：1．被邻近的细胞所吞噬；2．落人肾小体的毛细血管腔或肾小囊中。结论 小鼠肾小体的整个发育过程均存在细胞凋亡现象，肾小体内细胞凋亡与肾小体发育的完善有关。     

Apoptosis, bcl2 expression, and cell cycle analyses in nickel(II)-treated normal rat kidney cells

Nickel compounds are carcinogenic to human and are potent inducers of kidney and lung tumors in experimental animals. In this study, the effects of nickel(II) acetate on apoptosis, cell cycle and bcl2 expression in normal rat kidney (NRK- 52E) cells were investigated. Nickel(II) induced apoptosis in NRK-52E cells as demonstrated by DNA laddering. Apparent DNA laddering was observed in cells treated with 480 microM for 48 hr. In the flow cytometric analysis using propidium iodide fluorescence, an increase of cell proportion in G2/M phase was shown in cells exposed to at least 320 microM of nickel(II) acetate, from 7.7% for 0 microM of nickel(II) to 16.5% for 480 microM of nickel(II) acetate. Induction of apoptotic cell death by nickel(II) was accompanied by reduction of bcl2 protein expression, while the level of p53 protein was not changed. Taken together, our data indicate that nickel(II)-induced apoptosis in NRK-52E cells is accompanied by G2/M cell cycle arrest, regardless of p53 function, and that bcl2-mediated signaling pathway may be involved in positive regulation of nickel(II)-induced apoptotic cell death in NRK-52E cells.     

Trmt112 gene expression in mouse embryonic development

Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.     

Immunohistochemical Localization of Barx2 in the Developing Fetal Mouse Submandibular Glands

The development of mouse submandibular gland (SMG) begins at embryonic day 11.5–12 (E11.5–12), during which successive rounds of epithelial clefting and branching create complex epithelial tree-like structures. Homeobox genes regulate place-dependent morphogenesis, including epithelial-mesenchymal interactions, and control the expression patterns of signaling molecules. The Barx2 containing Homeobox exerts several key roles in development. Some studies have shown that the Barx2 plays important roles in the epithelial-mesenchymal interactions of organogenesis. However, the mechanisms of Barx2 associated with the development of SMG are obscure. In this study, we demonstrated for the first time the exact spatial and temporal Barx2 expression pattern in SMG epithelial tissue during development using immunohistochemical staining and Real-Time quantitative PCR. Barx2 was expressed in the nucleus of the epithelial cells located in the proliferative and differentiative regions of the developing SMG during the early development stages (E11.5–E13.5). After the E14.5-time period, the expression gradually decreased, and at E16.5, expression mostly disappeared despite the fact that evidence of cytodifferentiation, such as the appearance of proacinar cells, distinct lumen formation, and secretory products, was beginning to be observed. Results of Real-Time PCR demonstrated that the amount of Barx2 mRNA expression in SMG was maximal on E14.5, and gradually decreased by E18.5. These results indicate that Barx2 is associated with early stage epithelial tissue development, and can be a useful epithelial marker of the SMG during early developmental stages.     

流式细胞术检测细胞凋亡比较研究

从三方面探讨检测细胞凋亡流式细胞术的方法.选用大鼠皮质神经元细胞,同时使用碘化丙锭(PI)An-nexin V/PI.JC-1法检测细胞凋亡率.结果显示三种方法的凋亡率分别是:2.60%,6.52%和18.56%,两两之间有显著性差异(P＜0.01).JC-1法最敏感,AV/PI次之,PI单染法不适合检测早期细胞凋亡.     

TET2-Mediated Spatiotemporal Changes of 5-Hydroxymethylcytosine During Organogenesis in the Late Mouse Fetus

Genomic DNA demethylation is important for mammalian embryonic development and organ function. 5-Hydroxymethylcytosine (5hmC) is considered a novel epigenetic marker. Ten-eleven translocation (TET) enzymes convert 5-methylcytosine (5mC) to 5hmC. To explore the dynamic changes of epigenetic modifications during organogenesis in the late mouse fetus, the regional distribution and histological localization of 5hmC and TET enzymes was investigated by immunohistochemical method. The liver of mouse fetus gradually matured from embryonic day (E) 12.5 to E18.5.5mC was positive in developing liver at E16.5 and E18.5. 5hmC, TET2 and TET3 were strongly positive in hepatocytes and oval cells at E18.5. The small intestinal villi were formed at E16.5. The striate border and goblet cells appeared at E18.5. 5mC was detectable from E12.5 to E18.5. 5hmC and TET2 were positive in small intestine at E12.5, E14.5, and E18.5. The alveolar was formed at E18.5. 5mC and 5hmC were detectable from E12.5 to E18.5. Only TET2 was positive in the lung of the late Kunming mouse fetus. For vertebra, mesenchymal cells formed hyaline cartilage at E15.5 and then ossify at E16.5 and E18.8. 5mC, 5hmC, and TET2 were detectable in chondrocytes and osteocytes during the late Kunming mouse fetal; TET1 expressed from E14.5 to E16.5 and TET3 expressed in bone matrix at E18.5. In summary, TET2 was strongly expressed in liver, small intestinal, lung, and vertebra in the late Kunming mouse fetus. These findings suggested that TET2 may play a more critical role than TET1 and TET3 during organogenesis in the late stage of Kunming mouse embryo. Anat Rec, 302:954–963, 2019. © 2018 Wiley Periodicals, Inc.     

Temporal and Spatial Cellular Distribution of Neural Crest Derivatives and Alpha Cells during Islet Development

Recent studies have revealed that signals from neural crest (NC) derivatives regulate the mass, proliferation, and maturation of beta cells in developing fetal pancreas. However, little is known about the cellular distribution of NC derivatives during pancreatic development or the process whereby the developing islets are enclosed. We studied the temporal and spatial distribution of NC derivatives and endocrine cells at each developmental stage. At embryonic day 10.5 (E10.5) of mouse embryo, NC derivatives that migrated to the prospective pancreatic region were distributed in close proximity to pancreatic epithelial cells. As development advanced, most NC derivatives progressively surrounded endocrine rather than exocrine cells, and were distributed in closer proximity to alpha cells rather than to beta cells. At E20, approximately 70% of the NC derivatives enclosing endocrine cells were distributed in close proximity to alpha cells. Moreover, the expression of SynCAM, a Ca(2+)-independent homophilic trans-cell adhesion molecule, was confirmed from E16.5 on and was more remarkable at the cell boundaries of alpha cells and NC derivatives. These findings suggest that NC derivatives might be distributed in close proximity to alpha cells as a result of homophilic binding of SynCAM expressed by alpha cells and NC derivatives during islet development.     

Spatiotemporal distribution of apoptosis during normal cloacal development in mice

To understand normal cloacal developmental processes, serial sagittal sections of mouse embryos were made every 6 hrs from embryonic day 11.5 (E11.5) to E13.5. During cloacal development to form the urogenital sinus and anorectal canal, fusion of the urorectal septum with the cloacal membrane was not observed, and the ventral and dorsal parts of the cloaca were continuously connected by the canal until disappearance of the cloacal membrane to open the vestibule formed by the urogenital sinus and anorectal canal to the outside at E13.5. Ventral shifting of the dorsal part of the cloaca was observed until E12.5. The dorsal part was transformed in accordance with ventral shifting. In addition, apoptosis was seen to occur around the dorsal part. However, from E12.25, apoptotic cells are observed in a linear arrangement in the urorectal septum just ventral to the peritoneal cavity. Interestingly, extension of this line reaches the area of the cloacal membrane disintegrated by apoptosis. The present findings suggest that in the early stages (until E12.0), distribution of apoptosis in mesenchyme around the dorsal part of the cloaca might be strongly related to the transformation and ventral shifting of this part. Conversely, the apoptosis pattern in urorectal septum mesenchyme in later stages (from E12.0) might be involved in transformation of the urorectal septum and disintegration of the cloacal membrane.     

Role for keratins 6 and 17 during wound closure in embryonic mouse skin.

Injury to adult skin triggers a response designed to restore its vital barrier function. A conserved aspect of this response is a rapid switch in gene expression whereby the type II keratin 6 (K6) and type I keratins 16 and 17 (K16, K17) are induced in epithelial cells at the wound edge. This induction occurs at the expense of the keratins normally expressed during terminal differentiation and correlates with the activation of epithelial cells at the wound edge, ahead of their migration into the wound site. Here, we show that the capacity to enact this switch is already acquired in E11.5 stage mouse embryos. Such early timing is well ahead of the onset of differentiation-specific gene expression (approximately E13.5) and the acquisition of barrier formation by developing epidermis (approximately E16.5). Induction of K6, K16, and K17 correlates with changes in the morphology of epithelial cells at the wound edge. The closure of embryonic wounds is significantly delayed in K17 null embryos, but not embryos null for K6. These observations significantly extend the correlation between K6, K16, and K17 expression and epithelial wound closure, and provide direct evidence that expression of these keratins, K17 in particular, is important for the timeliness of this process.     

Three‐Dimensional Visualization of Developing Neurovascular Architecture in the Craniofacial Region of Embryonic Mice

Recent studies have highlighted the mechanism of vascular and axonal guidance to ensure proper morphogenesis and organogenesis. We aimed to perform global mapping of developing neurovascular networks during craniofacial development of embryonic mice. To this end, we developed histology‐based three‐dimensional (3D) reconstructions using paraffin‐embedded serial sections obtained from mouse embryos. All serial sections were dual‐immunolabeled with Pecam1 and Pgp9.5/Gap43 cocktail antibodies. All immunolabeled serial sections were digitized with virtual microscopy to acquire high spatial resolution images. The 3D reconstructs warranted superior positional accuracy to trace the long‐range connectivity of blood vessels and individual cranial nerve axons. It was feasible to depict simultaneously the details of angiogenic sprouting and axon terminal arborization and to assess quantitatively the locoregional proximity between blood vessels and cranial nerve axons. Notably, 3D views of the craniofacial region revealed the following: Branchial arch arteries and blood capillary plexi were formed without accompanying nerves at embryonic day (E) 9.5. Cranial nerve axons began to grow into the branchial arches, developing a labyrinth of small blood vessels at E10.5. Vascular remodeling occurred, and axon terminals of the maxillary, mandibular, chorda tympani, and hypoglossal nerve axons had arborized around the lateral lingual swellings at E11.5. The diverged patterning of trigeminal nerves and the arterial branches from the carotid artery became congruent at E11.5. The overall results support the advantage of dual‐immunolabeling and 3D reconstruction technology to document the architecture and wiring of the developing neurovascular networks in mouse embryos. Anat Rec, 298:1824–1835, 2015. © 2015 Wiley Periodicals, Inc.     

Apoptosis in mouse fetuses from dams exposed to T-2 toxin at different days of gestation.

T-2 toxin (2 mg/kg b.w.) was orally inoculated to pregnant mice at gestational day (GD) 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5 and GD 16.5, respectively, and the fetuses were examined 24 hours later. The number and region of pyknotic or karyorrhectic cells varied according to inoculation date. In the GD 13.5-subgroup, a moderate to high number of pyknotic or karyorrhectic neuronal cells were observed in the central nervous system, peri-ventricular zone to subventricular zone, and pyknosis or karyorrhexis were also observed in a small number of chondroblasts and chondrocytes. In the GD 16.5-subgroup, a moderate to high number of pyknotic or karyorrhectic cells were observed in the thymus and renal subcapsular parenchyma. The nuclei of these pyknotic or karyorrhectic cells were strongly stained by the terminal deoxy nucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling method widely used for the in situ detection of apoptotic nuclei. In addition, a few fetuses from dams which were given T-2 toxin at GD 13.5 or GD 14.5 and killed at GD 17.5 showed skeletal abnormalities such as wavy ribs and short scapula. From the present findings and the well known fact that T-2 toxin readily crosses the rat placenta, it seems that T-2 toxin-induced apoptosis in the developing mouse fetuses might be a direct effect of T-2 toxin on fetuses.     

Apoptosis in the pattern formation of the ventricular wall during mouse heart organogenesis

Apoptosis is an important mechanism in organogenesis, but its role in heart development has been poorly characterized. We have here studied apoptosis in the developing ventricular wall of mouse embryonic heart. Developing mice hearts on days 11 to 16 of gestation were studied using in situ end‐labeling of degraded DNA (TUNEL), immunocytochemistry of regulatory genes Bcl‐2 and Bax, and light and electron microscopy. TUNEL end‐labeled apoptotic cells were found in the ventricular wall on days 11 to 16 of gestation. The proportions of apoptotic cells of all cells in the ventricular wall differed between the trabecular and compact regions (P = 0.003) and between the days of gestation (P = 0.0001), the calculated apoptotic index was greater in the compact region at all ages except day 14. Ultrastructural analysis showed typical apoptotic shrinkage, chromatin degradation, and apoptotic bodies in several myoblastic and myocardial endothelial cells which were also positive by DNA end‐labeling. Immunocytochemical reaction for the apoptosis checkpoint proteins in the ventricular wall showed clearly more Bcl‐2 positive cells than Bax positive cells. The numerical densities of all cells in the compact and trabecular regions remained always higher in the compact region (P = 0.04) despite the fact that apoptosis was present in both areas at the same time. In conclusion, apoptosis takes place in the developing myocardial muscle as well as the myocardial endothelium during ventricular morphogenesis on days 11 through 16 and decreases clearly on day 16. We suggest that apoptosis and its regulatory factors are closely involved in the morphogenesis of the ventricular wall of the mammalian heart. Anat Rec 256:208–217, 1999. © 1999 Wiley‐Liss, Inc.