Studies of chimeric mice support the importance of IHHPTHrP interactions

Despite this suggestion from the phenotype of PTHrP~l~ mice that the negative feedback loop actually functions to fine-tune the pace of differentiation of chondrocytes, the postulated importance of this loop thus far is largely based on experiments in which large amounts of IHH or PTHrP are introduced into bones cultured in vitro. Further evidence involving more modest experimental perturbations in vivo are needed to establish the role of the feedback loop. The model also leaves unclear which chondrocytes respond directly to PTHrP. The PTHrP~l~ and PTH/PTHrP receptor^l~ mice have foreshortened proliferative columns, but the cells with the greatest expression of PTH/PTHrP receptor mRNA are prehypertrophic chondrocytes, which appear unchanged in the mutant mice. One possible explanation of these findings might be that PTHrP acts on the prehypertrophic chondrocytes, which then, in turn, stimulate proliferation of adjacent proliferating chondrocytes by secreting a factor such as IGF1. To explore more fully the cell—cell interactions in intact growth plates, we generated embryonic stem (ES) cells missing both copies of the PTH/PTHrP receptor gene. Through appropriate matings, these cells were genetically marked with a gene expressing ^-galactosidase in all cells descended from the marked ES cell. The mutant ES cells were then introduced into blastocysts, the blastocysts were inserted into the uteri of pseudo-pregnant foster mothers, and the bones of resultant chimeric embryos were examined at various times (Chung et al 1998).

When control, wild-type ES cells marked with ^-galactosidase were injected into blastocysts, the resultant bones were normal, with cohorts of blue cells (after staining for ^-galactosidase) forming the expected columns of proliferating chondrocytes. In contrast, the mutant PTH|PTHrP receptor chondrocytes differentiated prematurely and became hypertrophic, even though they were surrounded by the much more numerous normal chondrocytes in proliferative columns. This result demonstrates the cell autonomy of the PTH|PTHrP receptor chondrocyte phenotype and eliminates the model in which PTHrP acts only on prehypertrophic cells to stimulate proliferation of chondrocytes through the actions of another ligand.

Strikingly, the columns of genetically normal chondrocytes did not appear normal at all. Instead, the normal chondrocyte columns were much longer than expected. The extra length of columns was roughly in proportion to the contribution of PTH/PTHrP receptorchondrocytes in the chimeric growth plates. More mutant cells were associated with longer columns of normal cells. One possible model that could explain these results is shown in Fig. 2. The mutant prehypertrophic cells are expected to synthesize IHH much closer to the ends of bones than normal, because of the premature differentiation of the

FIG. 2. Abnormalities in chimeric growth plates. Dark cells represent cells derived from PTHjPTHrP receptor^1 ~ ES cells. These cells leave the proliferative pool early and synthesize IHH much closer to the end of the bone than normal cells would. This ectopically produced IHH then increases the synthesis of PTHrP at the end of the bone; this acts on the normal chondrocytes to delay their differentiation.

FIG. 2. Abnormalities in chimeric growth plates. Dark cells represent cells derived from PTHjPTHrP receptor^1 ~ ES cells. These cells leave the proliferative pool early and synthesize IHH much closer to the end of the bone than normal cells would. This ectopically produced IHH then increases the synthesis of PTHrP at the end of the bone; this acts on the normal chondrocytes to delay their differentiation.

PTH/P THrP receptor^1 ~ cells. This IHH might then initiate a cascade that leads to an increase in PTHrP synthesis at the ends of the bone. This increase in PTHrP expression would be expected to delay the differentiation of the normal chondrocytes but not affect the differentiation of the PTH/PTHrP receptor chondrocytes.

To test this hypothesis, we analysed Ihh mRNA by in situ hybridization. As expected, Ihh was synthesized both in its normal location in the normal prehypertrophic layer of cells and in ectopic, mutant prehypertrophic cells near the top of the growth plate. Patched (Pic) is a gene that encodes the IHH receptor and is itself stimulated by IHH action. The expression of Ptc is a good index of IHH action. In situ hybridization for Ptc mRNA demonstrated expression in its normal location adjacent to the normal prehypertrophic cells and in chondrocytes adjacent to the mutant cells ectopically expressing Ihh. Thus, the abnormally located Ihh stimulates gene expression as expected. Further, in situ hybridization for PTHrP mRNA showed an increase in expression by cells at the top of the growth plate, just as was found in the growth plates of PTH/PTHrP receptor^/ ~ mice. The abnormal location of Ihh and Ptc expression and the increase in PTHrP expression are all predicted by the model in Fig. 2 and support the idea that minor changes in the expression of Ihh and PTHrP can be sensed by growth plate chondrocytes in ways demanded by the feedback loop hypothesis.

To further substantiate this hypothesis, ES cells missing both the PTH/PTHrP receptor and the Ihh genes were established and used to generate chimeric mice. In preliminary experiments, the mutant chondrocytes differentiated prematurely, just as the PTH/PTHrP receptor~\~ chondrocytes had differentiated. This result is expected, because normal cells surrounding the [Ihh / ; PTH/PTHrP receptor~\~\ chondrocytes are expected to secrete Ihh and make up for the loss of Ihh from the relatively low number of mutant chondrocytes. Thus, the double knockout chondrocytes resemble the PTH/PTHrP receptor^/ ~ cells in chimeric growth plates. Importantly, however, the normal chondrocyte columns are not lengthened in the chimeric limbs containing [Ihh / ; PTH/PTHrP receptor~/~\ chondrocytes. This is precisely the result predicted from the hypothesis of Fig. 2, namely that IHH made by the mutant cells triggers the cascade leading to lengthening of the normal columns. Without IHH, the mutant cells cannot trigger the synthesis of PTHrP and, thereby, stimulate the proliferation of adjacent cells. Thus, the negative feedback loop between IHH and PTHrP can synchronize and determine the pace of chondrocyte differentiation in the growth plate.

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