Dmp1 Promoter-Driven Diphtheria Toxin Receptor Transgene Expression Directs Unforeseen Effects in Multiple Tissues

Abstract

Mice harbouring a dentin matrix protein 1 (Dmp1) promoter-driven human diphtheria toxin (DT) receptor (HDTR) transgene (Tg) have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT) treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT) littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2), was also found in many non-skeletal tissues in Tg mice; indicative of direct "off-target" DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.

Keywords: bone; diphtheria toxin receptor; osteocyte.

Figure 1

Diphtheria toxin (DT) injection leads to changes in bone formation despite inefficient ablation of osteocytes. Calcein double labelling (five-day interval) reveals robust levels of bone formation in vehicle-treated transgenic (Tg) (A) and WT mice (C) and diminished levels in only Tg (B), but not WT mice (D) five days after DT treatment; these changes were seen in the endosteal, but not the periosteal surfaces (E,F). White arrows indicate the inner and outer of two labels. Statistical comparisons: ** p < 0.05 vehicle and DT treated. TRAP staining for osteoclast activity for single DT-treated Tg (G; n = 3) and WT littermates (H; n = 3) (arrows indicate TRAP-positive osteoclasts) showed no significate differences between the two groups (I). Tg (J–L n = 4), but not WT (M–O; n = 4) mouse bones show marrow pathology, with marked congestion and distention of marrow sinusoidal blood vessels (*) at seven days after single DT (K) and more severe changes after five consecutive days of DT treatment (L); no comparable DT-induced changes in marrow composition were seen in WT mice (N,O). Significant osteocyte ablation (<30% empty lacunae; shown by arrowhead (), viable osteocyte; shown by arrow (), empty lacuna) was only observed in Tg mice treated with DT for five consecutive days (M) and only low levels (<10%) in WT and Tg mice after single DT treatment (P). Statistical comparisons: * p < 0.05 WT and Tg. Assessment of DT-induced apoptosis by TUNEL staining after single DT treatment in cortical bone of Tg (Q) and WT (R) revealed a very low number of apoptosis-positive osteocytes (arrow); arrowheads indicate negative cells. White-dotted boxes show a magnification of similar regions to better visualise the presence or lack of apoptotic cells. Negative (S) and positive (T) controls demonstrate a lack of staining in negative and many stained cells in positive control. Scale bar, 200 µm in the full overview images and 50 µm in the insets. Graphs represent the means ± SEM; ns = not significant.

Figure 1

Diphtheria toxin (DT) injection leads to changes in bone formation despite inefficient ablation of osteocytes. Calcein double labelling (five-day interval) reveals robust levels of bone formation in vehicle-treated transgenic (Tg) (A) and WT mice (C) and diminished levels in only Tg (B), but not WT mice (D) five days after DT treatment; these changes were seen in the endosteal, but not the periosteal surfaces (E,F). White arrows indicate the inner and outer of two labels. Statistical comparisons: ** p < 0.05 vehicle and DT treated. TRAP staining for osteoclast activity for single DT-treated Tg (G; n = 3) and WT littermates (H; n = 3) (arrows indicate TRAP-positive osteoclasts) showed no significate differences between the two groups (I). Tg (J–L n = 4), but not WT (M–O; n = 4) mouse bones show marrow pathology, with marked congestion and distention of marrow sinusoidal blood vessels (*) at seven days after single DT (K) and more severe changes after five consecutive days of DT treatment (L); no comparable DT-induced changes in marrow composition were seen in WT mice (N,O). Significant osteocyte ablation (<30% empty lacunae; shown by arrowhead (), viable osteocyte; shown by arrow (), empty lacuna) was only observed in Tg mice treated with DT for five consecutive days (M) and only low levels (<10%) in WT and Tg mice after single DT treatment (P). Statistical comparisons: * p < 0.05 WT and Tg. Assessment of DT-induced apoptosis by TUNEL staining after single DT treatment in cortical bone of Tg (Q) and WT (R) revealed a very low number of apoptosis-positive osteocytes (arrow); arrowheads indicate negative cells. White-dotted boxes show a magnification of similar regions to better visualise the presence or lack of apoptotic cells. Negative (S) and positive (T) controls demonstrate a lack of staining in negative and many stained cells in positive control. Scale bar, 200 µm in the full overview images and 50 µm in the insets. Graphs represent the means ± SEM; ns = not significant.

Figure 2

Transgenic mice exhibit generalized adverse health status with DT treatment. WT, but more markedly Tg mice showed substantial weight loss. This was apparent within two days after a single DT injection in Tg mice (A), which worsens to reach over 20% of starting body weight by Day 7. Tg mice also developed signs of distress and pain during the six-day period of DT treatment. These signs were manifested by a hunched back as a sign of distress (B). The change in gait, which might be due to pain (C), reduced activity (D), loud vocalisation (E), increased respiratory rate (F), deteriorated body condition (G) and deterioration of coat condition (H), which suggests lack of grooming and personal care. These signs suggest that DT impacts severely on the welfare particularly of treated Tg mice. Graphs represent the means ± SEM. Statistical comparisons: * denotes p < 0.05 between DT-treated WT and Tg mice; ns = not significant

Figure 3

Multiple tissues show the misexpression of Dmp1-driven HDTR mRNA and broad expression of Dmp1. (A) A 9.6-kb transgenic cassette (50-flanking region, exon 1, intron 1 and part of exon 2 of the mouse Dmp1 gene) was fused to human DTR cDNA using a toxin receptor-mediated cell knockout system and injected into fertilized egg pronucleus. Blue arrow indicates direction of transcription [18,28]. Semi-quantitative mRNA analysis reveals Dmp1 mRNA expression in only bone and brain and a lack of selective HDTR mRNA expression, with the distribution in almost all tissues examined from Tg, but not in WT mice (B), respectively. Quantitative PCR of HDTR mRNA transgene expression analysis revealed that HDTR has a broad expression in multiple tissues (C). Samples were normalised to GAPDH. Immunohistochemical staining against HDTR protein in selected tissues revealed the expression of HDTR protein in osteocytes (D, arrow; ) as wells as osteoblasts (D, arrowhead; ), bone marrow cells (E, arrowhead) and around vessels (E, arrow), kidney tubules (F, arrow), and lung alveolar wall (G, arrow). Respective tissues show negative staining in WT (H–K). (L–O) are PBS negative controls. Scale bar, 200 µm.

Figure 3

Multiple tissues show the misexpression of Dmp1-driven HDTR mRNA and broad expression of Dmp1. (A) A 9.6-kb transgenic cassette (50-flanking region, exon 1, intron 1 and part of exon 2 of the mouse Dmp1 gene) was fused to human DTR cDNA using a toxin receptor-mediated cell knockout system and injected into fertilized egg pronucleus. Blue arrow indicates direction of transcription [18,28]. Semi-quantitative mRNA analysis reveals Dmp1 mRNA expression in only bone and brain and a lack of selective HDTR mRNA expression, with the distribution in almost all tissues examined from Tg, but not in WT mice (B), respectively. Quantitative PCR of HDTR mRNA transgene expression analysis revealed that HDTR has a broad expression in multiple tissues (C). Samples were normalised to GAPDH. Immunohistochemical staining against HDTR protein in selected tissues revealed the expression of HDTR protein in osteocytes (D, arrow; ) as wells as osteoblasts (D, arrowhead; ), bone marrow cells (E, arrowhead) and around vessels (E, arrow), kidney tubules (F, arrow), and lung alveolar wall (G, arrow). Respective tissues show negative staining in WT (H–K). (L–O) are PBS negative controls. Scale bar, 200 µm.

Figure 4

DT induces severe atrophy in primary lymphoid organs. H & E staining of spleens from Tg (A,B) and WT (C,D) mice shows atrophy and diminished white pulp (*) seven days after single DT treatment in Tg (B), but less so in WT (D) mice; scale bar 250 µm. (E,F) Tg exhibit more severe thymic atrophy than WT (G,H) mice seven days post-DT. Scale bar 200 µm.

Figure 5

DT induces severe kidney damage. Tg (A–C) and WT (D–F) mice both show overt tubular necrosis (arrows; ) as early as three days after DT treatment (B,E), before any overt osteocyte death (Figure 1J–P). Kidney shows an apparent recovery in these pathological changes seven days post DT (C,F) with increased serum calcium and phosphorous levels in treated Tg mice seven days post DT (G,H). Scale bar 200 µm. Graphs represent means ± SEM. Statistical comparisons: * p < 0.05 and ** p < 0.01 vehicle and DT treated within each genotype; (a) denotes significant differences between vehicle-treated WT and Tg mice (p < 0.05).

Figure 6

Dephosphorylation of EF-2 reveals direct DT toxicity in multiple tissues in Tg mice. Bones of DT-treated Tg and WT mice show equivalent pEF-2 levels to control mice seven days post DT treatment (A: blots; B: quantification), which suggest minimal effects in bone. In kidney, spleen, lung and liver, there is a significantly lower pEF-2 level indicative of direct targeting of these tissues by DT. In addition, muscle and heart also show non-significant changes in pEF-2 levels compared to control mice seven days post treatment. (A) Representative image of one out of four; (B) quantification of all groups, n = 4. * p < 0.05 and ** p < 0.01 compared with vehicle treated Tg mice.