Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues.
BackgroundDirected differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation.Methodology/Principal FindingsWe analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons.Conclusions/SignificanceOur results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.
SummaryPostnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocytespecific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER T2 ) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER T2 mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER T2 transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach. Keywordschondrocyte; Cre-mediated recombination; conditional knockout; tamoxifen; X-Gal staining Chondrocyte maturation and cartilage formation involves multiple steps that are regulated by numerous growth factors, their downstream signaling molecules and transcription factors. Conventional and tissue-specific gene deletions provide powerful tools to investigate roles of specific genes in chondrocyte maturation. However, embryonic lethality is often encountered because of the essential role of various genes in early embryonic development (Akiyama et al., 2004;Karaplis et al., 1994;Lanske et al., 1996Lanske et al., , 1999Razzaque et al., 2005;Sakamoto et al., 2005;St-Jacques et al., 1999). Furthermore, some gene deletions result in such severe skeletal malformation that interpretation of the phenotype is challenging even if the animal survive birth. An inducible conditional gene deletion approach is an exquisite method to determine the function of a gene that is either embryonic lethal or associated with marked abnormalities of morphology and tissue architectures. In this report, we present a chondrocytespecific and tamoxifen-inducible Cre transgenic mouse model. Type II collagen is a chondrocyte-specific protein and its expression is detected in growth plate and articular chondrocytes in long bones and other cartilage tissues in the body. Type II collagen promoter (col2a1) has been used in a variety of animal models to achieve tissuespecific gene expression in chondrocytes (Schipani et al., 1997;Stricker et al., 2002;Takeda et al., 2001;Ueta et al., 2001;Weir et al., 1996). In the present studies, we generated transgenic mouse lines in which the Cre recombinase was fused to a mutated ligand-binding domain of human estrogen receptor (ER) driven by the col2a1 promoter (Col2a1-CreER T2 ; Metzger et al., 2005). The fusion protein has been reported to be translocated into the nuclei in response to estrogen antagonist tamoxifen or 4-hydroxy tamoxifen (4-OH tamoxifen), an active metabolite of tamoxifen in vivo ...
Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial–temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell–cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.
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