![]() ![]() ![]() First strand cDNA (+) was generated from each RNA extract (including No RT controls, –) and used as a template for PCR to analyze expression of the genes F48E3.8, C54G7.3, the eggshell chitin synthase chs-1 and the pharyngeal chitin synthase chs-2. Synchronous non-permissive (germline-ablated) worms of this strain were grown on solid media and used to collect RNA from various time-points following L1 arrest at hatching and continuing into adulthood. elegans and Deacetylate Chitin In Vitro Figure 3ĭevelopmental time course of F48E3.8 and C54G7.3 gene expression in germline-ablated C. All sections were counterstained with hematoxylin and eosin bright-field ( c, e, g) and dark-field ( d, f, h, i) views are shown.Pharyngeal Polysaccharide Deacetylases Affect Development in the Nematode C. ( i) In situ hybridization with the 35S-labeled PPR cRNA in a section of decalcified proximal tibia of a newborn wild-type mouse. In the high-magnification images from 2-week-old CL2 mutant ( e and f), the arrow indicates the periosteal surface. ( c– h) In situ hybridization with the 35S-labeled DT7 cRNA in serial section of decalcified proximal tibia of newborn CL2 mouse ( c and d),and a wild-type littermate ( g and h). ( b) Southern blot analysis of the RT-PCR products with probe S: RT-PCR products from line not expressing the transgene (lanes 1 and 6), CL1 (lanes 2, 3, 7, and 8), and CL2 (lanes 4 and 9), with negative control in lane 5 and –RT controls in lanes 6–9. Oligonucleotide S was used as probe for Southern blot analysis of RT-PCR products, oligonucleotides S1 and A were used for RT-PCR of total RNA, and oligonucleotides S2 and A1 were used to generate the DT7 probe. Restriction sites for the enzymes HindIII, PvuII, and ScaI are also indicated. Locations of the translation initiation codon (ATG), the receptor mutation (H223R), and the stop codon (Stop) in the cDNA encoding the human PPR mutant, HKrk-H223R, are seen. ( a) Shown is the scheme of the transgene construct with the location of probes A and B for Southern blot analysis of genomic DNA. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. ![]() In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. ![]() In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen’s metaphyseal chondrodysplasia. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. ![]()
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