Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage

Abstract

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.

Figure 1

Tamoxifen (TM) inducible Col2-Cre^™ transgenic mouse. (A) Schematic of the Col2-Cre^™ transgene. G.S.S., β-globin splicing sequence. ER^™, Modified form of the estrogen receptor responsive only to exogenous tamoxifen. (B–C) β-galactosidase activity assays (Lac Z staining) indicating induction of Cre by TM in Col2-Cre^™; R26R embryos. Whole embryos were stained at E12.5 after pregnant females were injected with 75 μg/g TM (75 μg TM per gram of body weight) at either E9.5 (B) or E10.5 (C). Forelimbs (FL) (B1–C1) and hindlimbs (HL) (B2–C2) were shown at a higher magnification beneath the corresponding whole embryos. a: autopod, z: zeugopod, s: stylopod, sc: scapula.

Figure 2

Control of cell-type specific Cre activity in the embryo. Lac Z staining was performed on cryostat longitudinal sections of the humerus from E17.5 Col2-Cre^™; R26R embryos. The embryos were harvested after pregnant females were injected with 25 μg/g TM (25 μg TM per gram of body weight) at E11.5 (A), E12.5 (B), E13.5 (C) or E14.5 (D). Green and red boxes in A–D identify areas that are shown below at a higher magnification. The proximal end is to the left in all panels. Red arrows: activity in chondrocytes; purple arrows: activity in primary spongiosa; black arrow: chondrocytes of deltoid tuberocity; green arrowheads: perichondrium; asterisks: activity around joint capsule.

Figure 3

Induction of Cre activity in postnatal mice. (A, B) Lac Z staining on cryostat longitudinal sections through the proximal end of the humerus from P16 animals. Mice with the genotype of either Col2-Cre^™; R26R (A) or Col2-Cre^™ (B) were injected 400 μg/g TM (400 μg TM per gram of body weight) at P12 and P14. Color-coded rectangular boxes in A and B identify areas shown at a higher magnification below (A1–A3 and B1–B3 respectively). The proximal end is to the top in all panels. 2^o: secondary ossification center.

Figure 4

Cre activity in trabecular osteoblast-lineage cells. Lac Z staining on cryostat longitudinal sections through the humerus from E17.5 embryos with the indicated genotypes. The pregnant females were injected with 25 μg/g TM (25 μg TM per gram of body weight) at either E13.5 (A, B) or 6 hours prior to harvest (C, D). The proximal half of the humerus is shown in A–D. Red boxes in A–D indicate areas shown at a higher magnification (A1–D1, respectively). Red arrows in A–C: activity in chondrocytes; purple arrows in A–C: activity in primary spongiosa; green arrowheads in A and B: perichondrium; red arrows in A1 and C1: Lac Z-positive flat cells; green arrows in A1 and C1: Lac Z-positive cuboidal cells; black arrows in A1, C1 and D1: Lac Z-negative cells; blue arrows in B1: Lac Z-positive osteocytes; green contours in A1–D1: outlines of bone surface; H: hypertrophic zone. The proximal end is to the left in all panels.

Figure 5

Expression of Colα1(II)A by trabecular osteoblast-lineage cells. (A) In situ hybridization using ³⁵S-labeled riboprobe specific to (II)A on longitudinal sections through the tibia of E18.5 wild type embryos. Signal is in red and counterstain in blue. Black arrow in A: endosteal surface of cortical bone; purple arrow in A: periosteal surface of cortical bone; asterisk in A: signal in chondrocytes; green arrows in A: primary spongiosa; blue arrow in A1: cell without signal; red arrow in A1: cell with signal. (B–C) Lac Z staining followed by in situ hybridization on frozen sections through the humerus of E17.5 Col2-Cre^™; R26R embryos induced with 25 μg/g TM for 6 hrs. Lac Z signal in blue, in situ signal in red, counterstain in magenta. Note that bone trabeculae were not stained. Also note that black arrows in B and C point to dark specks representing oversaturated in situ signal not Lac Z staining. Red arrows in B1 and C1: Lac Z-positive cells; green arrows in B1 and C1: high-expressers of Bsp or Colα1(I). The proximal end is to the left in all panels. Boxed regions are shown at a higher magnification to the right (A1–C1). “PH”: prehypertrophic chondrocytes; “H”: hypertrophic chondrocytes.

Figure 6

Osteogenic potential of Col2-Cre^™-positive cells isolated from the bone marrow. 2-month-old mice were analyzed at 12 hrs after TM delivery via oral gavage at 125 μg per gram of body weight. (A) Lac Z staining on a longitudinal section through the tibia of a Col2-Cre^™; R26R mouse. Black arrow denotes Lac Z-positive chondrocytes. (A′) The boxed area in (A) viewed at a higher magnification. Red arrows denote Lac Z-positive cells on trabecular bone surfaces. (B) An equivalent region from a Col2-Cre^™ mouse containing no Lac Z-positive cells. (C–D) Lac Z staining of sub-confluent cultures of bone marrow stromal cells isolated from Col2-Cre^™; R26R (C) versus Col2-Cre^™ (D) animals. (E–F) Lac Z staining of bone nodules from Col2-Cre^™; R26R (E) or Col2-Cre^™ (F) cultures. Purple arrow denotes a bone nodule consists predominantly of Lac Z-positive cells; purple arrowhead denotes an adjacent nodule with no Lac Z-positive cells. (E′–F′) von Kossa staining of the same bone nodules following Lac Z staining in (E–F).

Figure 7

TM dose-dependence of Cre activity in the embryo. Lac Z staining was performed on cryostat longitudinal sections of the humerus from E17.5 Col2-Cre^™; R26R embryos. The embryos were harvested after pregnant females were injected at E16.5 with either 100 (A) or 12.5 (B) μg/g TM (100 or 12.5 μg TM per gram of body weight). Green and red boxes in A and B identify areas shown below at a higher magnification (A1–A2, B1–B2, respectively). The proximal end to the left in all panels. Red arrow: a cluster of positive cells; green arrow: a single positive cell; green arrowheads: residual activity in perichondrium.

Figure 8

Direct regulation of PTHrP expression and chondrocyte morphology by Ihh. Histology (A1–A3, B1–B3, C1–C3) and in situ hybridization (A4–A7, B4–B7, C4–C7) performed on adjacent longitudinal sections of humerus from E14.5 wild type (WT) (A1–A7) and Col2-Cre^™; Smo^n/c (MT1 and MT2) (B1–B7, C1–C7 respectively) littermate embryos. The embryos were harvested after pregnant females were injected 50 μg/g TM (50 μg TM per gram of body weight) at E11.5. The proximal half of each section is shown with the articular surface to the left. The color-coded boxes in A1–C1 denote areas shown at a higher magnification in A2–A3, B2–B3 and C2–C3, respectively. Red arrows in B2 and C2 denote abnormal chondrocyte morphology; asterisks in B2 and C2 indicate irregular spacing between cells. ³⁵S-labeled probes used for in situ hybridization are as indicated directly above the panels. Signal is in red and counterstain in blue. Orange arrows in B4 and C4 denote ectopic Colα1(X) expression; color-coded contours in A6–A7, B6–B7 and C6–C7 demarcate equivalent areas in adjacent sections; asterisks in B6 and C6 indicate areas devoid of Ptch1 expression; green arrowheads in A6–C6 denote signal in perichondrium.