Sonic hedgehog: restricted expression and limb dysmorphologies

Sonic hedgehog, SHH, is required for patterning the limb. The array of skeletal elements that compose the hands and feet, and the ordered arrangement of these bones to form the pattern of fingers and toes are dependent on SHH. The mechanism of action of SHH in the limb is not fully understood; however, an aspect that appears to be important is the localized, asymmetric expression of Shh. Shh is expressed in the posterior margin of the limb bud in a region defined as the zone of polarizing activity (ZPA). Analysis of mouse mutants which have polydactyly (extra toes) shows that asymmetric expression of Shh is lost due to the appearance of an ectopic domain of expression in the anterior limb margin. One such polydactylous mouse mutant, sasquatch (Ssq), maps to the corresponding chromosomal region of the human condition pre-axial polydactyly (PPD) and thus represents a model for this condition. The mutation responsible for Ssq is located 1 Mb away from the Shh gene; however, the mutation disrupts a long-range cis-acting regulator of Shh expression. By inference, human pre-axial polydactyly results from a similar disruption of Shh expression. Other human congenital abnormalities also map near the pre-axial polydactyly locus, suggesting a major chromosomal region for limb dysmorphologies. The distinct phenotypes range from loss of all bones of the hands and feet to syndactyly of the soft tissue and fusion of the digits. We discuss the role played by Shh expression in mouse mutant phenotypes and the human limb dysmorphologies.     

The notch response inhibitor DAPT enhances neuronal differentiation in embryonic stem cell-derived embryoid bodies independently of sonic hedgehog signaling.

During development of the neural tube, inhibition of the Notch response as well as the activation of the Sonic Hedgehog (Shh) response results in the formation of neuronal cell types. To determine whether Shh and Notch act independently, we tested the effects of the Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) on neuralized, embryonic stem (ES) cell-derived embryoid bodies (EBs), while varying the levels of Shh pathway activation. Shh-resistant EBs were derived from Smo null ES cells, while EBs with constitutive high level of Shh pathway activation were derived from Ptc1 null ES cells. Intermediate levels of Shh pathway activation was achieved by the addition of ShhN to the EB culture medium. It was found that DAPT-mediated inhibition of the Notch response resulted in enhanced neuronal differentiation. In the absence of Shh, more interneurons were detected, while the main effect of DAPT on EBs with an activated Shh response was the precocious loss of ventral neuronal precursor-specific markers.     

Neuroprotective effects of a Smoothened receptor agonist against postoperative cognitive dysfunction by promoting autophagy in the dentate gyrus of aged rats

ABSTRACT

Objectives: To investigate the effect of purmorphamine (PUR), a Shh co-receptor Smoothened (Smo) agonist, on postoperative cognitive dysfunction (POCD) rat models.

Methods: Eighteen-month-old male Sprague–Dawley rats were subjected to intramedullary fixation of a tibial fracture with 7% chloral hydrate anesthesia to mimic human clinical surgery. PUR was administered via an intraperitoneal injection at a dose of 15mg/kg/day for 3 consecutive days at 6 h after surgery. The aged rats were sacrificed after performing a Morris water maze test 1, 3, and 7 days postoperatively to evaluate the expression of related proteins at the appointed time.

Results: Compared to the POCD + vehicle group and sham + PUR group, the POCD + PUR group restored neurological deficit (P = 0.01). PUR administration induced upregulation of Shh expression on postoperative day 1 (P = 0.02), which continued on the third day (P = 0.008) but dropped by the 7th day (P = 0.03). Immunofluorescent analysis, similar to western blot analysis, showed a significant increase in the autophagy-marker LC3 (P = 0.006) as well as p62 degradation (P = 0.000) in the dentate gyrus of the aged rats (P = 0.000) after PUR treatment. Importantly, LC3 was mainly found in the presynaptic and postsynaptic membranes of the hippocampus.

Conclusions: These results indicate a link between Shh and autophagy in the rat model of POCD, providing new insights into Shh signaling pathway-mediated mechanisms of neuroprotection and cognitive repair after POCD. It also provides a potential entry point for the development of clinical drugs.     

Shh促进BMP9诱导的小鼠间充质干细胞C3H10T1/2的成骨分化

目的 观察Shh蛋白对骨形态发生蛋白9(BMP9)诱导的小鼠间充质干细胞(MSCs) C3H10T1/2成骨分化的影响,并初步探讨其作用机制.方法 Shh腺病毒和BMP9作用于C3H10T1/2细胞,碱性磷酸酶(ALP)检测ALP变化,茜素红S染色检测钙盐沉积,RT-PCR检测Shh、BMP9、骨桥蛋白(OPN)、骨钙素(OCN)以及成骨相关基因Id1、Id2、Id3、CTGF和Runx2的表达,Western blot检测OPN、OCN、Runx2、DLX5和p-Smad1/5/8的蛋白水平,荧光素酶报告基因检测Smad1/5/8的转录活性.结果 Shh不影响BMP9的表达,但可增强由BMP9诱导的C3H10T1/2细胞早晚期成骨分化(P＜0.05),并促进BMP9诱导的成骨相关基因的表达(P＜0.05);Shh促进了BMP9诱导的Smad荧光素酶活性(P＜0.05),但对其磷酸化并无影响.结论 Shh可促进BMP9诱导的小鼠C3H10T1/2细胞的成骨分化.     

Sonic hedgehog signaling during adrenal development

It has been speculated for a number of years that Sonic hedgehog (Shh) signaling plays an important role in adrenal development. Over the past two years several reports have described the expression and function of Shh pathway genes in the adrenal cortex, using primarily mouse models. The key findings are that Shh signals produced by a population of partially differentiated cortical cells located in the outer cortex/zona glomerulosa are received by non-cortical mesenchymal cells located predominantly in the overlying capsule. This signal is required for growth of both the capsule and the cortex, but not for cortical zonation or steroidogenic cell differentiation. Using molecular genetic tools to define the adrenocortical cell lineages that are descended from both Shh signaling and receiving cells, both capsule and cortical cells were found to have properties of adrenocortical stem and/or progenitor cells. Here we place these observations within the context of prior studies on adrenal development, postnatal adrenal maintenance and adrenocortical stem/progenitor cell lineages.     

Shh/Ihh在小鼠胚胎椎间盘形成过程中的表达

文题释义：Shh,Ihh：哺乳动物Hedgehog基因存在3种同源基因,分别为Sonic Hedgehog,India Hedgehog以及Desert Hedgehog,分别编码Shh,Ihh以及Dhh蛋白。它们在胚胎的发育、成体组织和器官干细胞的自我更新与增殖过程中发挥重要的作用。他莫昔芬：是一种选择性雌激素反应调节剂。当向CreER^T2小鼠体内注射他莫昔芬时,由他莫昔芬代谢所产生的雌激素类似物与ERT结合,从而使CreER^T2进入细胞核内发挥Cre重组酶活性。背景：研究表明,Sonic Hedgehog(Shh)在小鼠脊索细胞内有表达,India Hedgehog(Ihh)主要在小鼠胚胎椎体的软骨样细胞以及后期形成的终板中表达。但关于Shh以及Ihh在小鼠胚胎椎间盘形成过程中的表达情况并不清楚。 目的：探究Shh和 Ihh信号在小鼠胚胎椎间盘形成过程中的表达。 方法：将雄性的Shh-CreER^T2; R26-mTmG/+小鼠与雌性R26-mTmG/+小鼠进行交配,获取不同时期(E8.5,E11.5,E12.5,E14.5,E16.5,E18.5)的孕鼠,注射10 g/L的他莫昔芬10 μL/g。当小鼠胚胎处于P0时,取小鼠胚胎椎间盘组织进行基因型鉴定;同时将雄性的C57BL/6小鼠与雌性的C57BL/6小鼠进行交配,获取不同时期(E11.5,E12.5,E14.5,E16.5,E18.5)的孕鼠,取小鼠胚胎椎间盘组织进行分析。通过免疫荧光法检测Shh信号在小鼠胚胎椎间盘形成过程中的表达;免疫组织化学法检测Ihh信号在小鼠胚胎椎间盘形成过程中的表达。所有涉及动物的相关实验均获得苏州大学动物伦理委员会的批准。 结果与结论：①经PCR扩增筛选出基因型为Shh-CreERT2; R26-mTmG/+小鼠胚胎;②免疫荧光染色结果显示,在椎间盘形成过程中,髓核细胞内Shh表达水平逐渐降低;③免疫组织化学染色结果显示,在椎间盘形成过程中,髓核细胞内Ihh表达水平逐渐升高;④结果表明Shh和Ihh在椎间盘形成过程中均发挥调控作用,在一定程度上为研究椎间盘退变的分子机制奠定基础。 ORCID: 0000-0001-9429-2339(巩婷婷) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Shh和Ptch在肝细胞癌中的表达研究

目的探讨肝细胞癌（HCC）中Shh和Ptch的表达及其意义。方法应用组织芯片技术、免疫组化和原位杂交方法检测100例HCC、25例癌旁组织和5例正常肝组织中Shh和Ptch的表达情况。结果免疫组化检测Hcc中Shh和Ptch的阳性表达率分别为19．28％（16／83）、24．68％（19／77）;原位杂交检测结果分别为44．29％（31／70）、15．85％（13／82）;两种基因在HCC中的表达相关,均与非肿瘤组织的表达有显著性差异,并分别与肿瘤的大小及分化程度相关。结论Shh和Ptch的表达参与了HCC的发生,它们可能是HCC治疗的理想靶标。