Chen Xuesong, Macica Carolyn M, Dreyer Barbara E, Hammond Vicki E, Hens Julie R, Philbrick William M, Broadus Arthur E
Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8020, USA.
J Bone Miner Res. 2006 Jan;21(1):113-23. doi: 10.1359/JBMR.051005. Epub 2005 Oct 10.
The PTHrP gene generates low-abundance mRNA and protein products that are not easily localized by in situ hybridization histochemistry or immunohistochemistry. We report here a PTHrP-lacZ knockin mouse in which beta-gal activity seems to provide a simple and sensitive read-out of PTHrP gene expression.
PTH-related protein (PTHrP) is widely expressed in fetal and adult tissues, typically as low-abundance mRNA and protein products that maybe difficult to localize by conventional methods. We created a PTHrP-lacZ knockin mouse as a means of surveying PTHrP gene expression in general and of identifying previously unrecognized sites of PTHrP expression.
We created a lacZ reporter construct under the control of endogenous PTHrP gene regulatory sequences. The AU-rich instability sequences in the PTHrP 3' untranslated region (UTR) were replaced with SV40 sequences, generating products with lacZ/beta gal kinetics rather than those of PTHrP. A nuclear localization sequence was not present in the construct.
We characterized beta-galactosidase (beta-gal) activity in embryonic whole mounts and in the skeleton in young and adult animals. In embryos, we confirmed widespread PTHrP expression in many known sites and in several novel epidermal appendages (nail beds and footpads). In costal cartilage, beta-gal activity localized to the perichondrium but not the underlying chondrocytes. In the cartilaginous molds of forming long bones, beta-gal activity was first evident at the proximal and distal ends. Shortly after birth, the developing secondary ossification center formed in the center of this PTHrP-rich chondrocyte population. As the secondary ossification center developed, it segregated this population into two distinct PTHrP beta-gal+ subpopulations: a subarticular subpopulation immediately subjacent to articular chondrocytes and a proliferative chondrocyte subpopulation proximal to the chondrocyte columns in the growth plate. These discrete populations remained into adulthood. beta-gal activity was not identified in osteoblasts but was present in many periosteal sites. These included simple periosteum as well as fibrous tendon insertion sites of the so-called bony and periosteal types; the beta-gal-expressing cells in these sites were in the outer fibrous layer of the periosteum or its apparent equivalents at tendon insertion sites. Homozygous PTHrP-lacZ knockin mice had the expected chondrodysplastic phenotype and a much expanded region of proximal beta-gal activity in long bones, which appeared to reflect in large part the effects of feedback signaling by Indian hedgehog on proximal cell proliferation and PTHrP gene expression.
The PTHrP-lacZ mouse seems to provide a sensitive reporter system that may prove useful as a means of studying PTHrP gene expression.
甲状旁腺激素相关蛋白（PTHrP）基因产生的mRNA和蛋白质产物丰度较低，通过原位杂交组织化学或免疫组织化学方法不易定位。我们在此报告一种PTHrP - lacZ基因敲入小鼠，其中β - 半乳糖苷酶（β - gal）活性似乎为PTHrP基因表达提供了一种简单而灵敏的检测方法。
甲状旁腺激素相关蛋白（PTHrP）在胎儿和成人组织中广泛表达，通常以低丰度的mRNA和蛋白质产物形式存在，可能难以通过传统方法定位。我们创建了一种PTHrP - lacZ基因敲入小鼠，作为一种全面检测PTHrP基因表达以及识别以前未被认识的PTHrP表达位点的手段。
我们构建了一个受内源性PTHrP基因调控序列控制的lacZ报告基因构建体。PTHrP 3'非翻译区（UTR）中富含AU的不稳定序列被SV40序列取代，产生具有lacZ /β - gal动力学而非PTHrP动力学的产物。构建体中不存在核定位序列。
我们在胚胎整体以及幼年和成年动物的骨骼中对β - 半乳糖苷酶（β - gal）活性进行了表征。在胚胎中，我们证实PTHrP在许多已知位点以及几个新的表皮附属器（甲床和脚垫）中广泛表达。在肋软骨中，β - gal活性定位于软骨膜而非下方的软骨细胞。在形成长骨的软骨模型中，β - gal活性首先在近端和远端明显。出生后不久，在这个富含PTHrP的软骨细胞群体中心形成了发育中的次级骨化中心。随着次级骨化中心的发育，它将这个群体分成两个不同的PTHrPβ - gal +亚群：紧邻关节软骨细胞的关节下亚群和生长板中软骨细胞柱近端的增殖软骨细胞亚群。这些离散的群体一直持续到成年。在成骨细胞中未发现β - gal活性，但在许多骨膜部位存在。这些部位包括简单的骨膜以及所谓的骨型和骨膜型纤维肌腱插入部位；这些部位中表达β - gal的细胞位于骨膜的外层纤维层或其在肌腱插入部位的明显对应层。纯合PTHrP - lacZ基因敲入小鼠具有预期的软骨发育不良表型，并且长骨近端β - gal活性区域大大扩大，这似乎在很大程度上反映了印度刺猬信号对近端细胞增殖和PTHrP基因表达的反馈信号作用。
PTHrP - lacZ小鼠似乎提供了一个灵敏的报告系统，可能被证明是研究PTHrP基因表达的一种有用手段。
