TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves - PubMed (original) (raw)

TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves

Maurizio D'Antonio et al. J Neurosci. 2006.

Abstract

During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF) beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGFbeta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGFbeta signaling did not appear to affect other processes in which TGFbeta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death.

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Figures

Figure 1.

Figure 1.

Inactivation of the TβRII locus and assessment of the efficiency of deletion. A, Schwann cell (SC)-specific inactivation of the TβRII locus, obtained by crossing TGFβRIIf/f mice with P0 CRE mice. The length of the recombined restriction fragment in Southern blot (1.8 kb) is indicated. B, Southern blot of _Nco_I-digested DNA from tail (T) or purified Schwann cells from P0 CRE/TβRIIf/+ or P0 CRE/TβRIIf/f P5 mice (n = 5). Efficiency of deletion, normalized for cell purity (∼90%, established by S100 staining) (data not shown), is indicated. Recomb., Percentage of recombination. C, Nuclear localization of Smad2 and Smad4 was essentially absent in P0 CRE/TβRIIf/f mice even after TGFβ-1 treatment. Very occasionally, nuclear staining was detectable in the mutant mice (arrows), indicating that the recombination, although highly successful, is probably not complete. In some cases in the control wild type or P0 CRE/TβRIIf/+, nuclear localization is detectable even in the absence of TGFβ-1 (arrowheads). This is possibly attributable to the action of endogenous TGFβ. Ho, Hoechst nuclear dye; WT, wild type.

Figure 2.

Figure 2.

P0 CRE/TβRIIf/f mice myelinate normally. Transverse sections of sciatic nerves from P1, P5, P21, and adult (Ad; 4–6 months) wild-type (WT) and mutant mice were analyzed by EM. At every age, the mutant nerve appears indistinguishable from the wild type. Similarly to the wild type, in the mutant nerve at P1, Schwann cells are starting to correctly segregate the large-caliber axons in a 1:1 relationship, and a thin myelin sheath is already visible around some of them (arrows). At P5, in both wild-type and mutant nerves, large-caliber axons have reached the 1:1 relationship with Schwann cells, and most of them are surrounded by a thickening myelin sheath. At P21, myelination is almost complete. Bundles of nonmyelinated axons are segregated from each other and surrounded by nonmyelinating Schwann cells, forming Remak fibers (arrows). Similarly, the adult nerve does not show any difference from the wild type. Scale bar, 10 μm.

Figure 3.

Figure 3.

Expression of Schwann cell-specific proteins is normal in the P0 CRE/TβRIIf/f mouse. A, Transverse sections of newborn mutant and control wild-type sciatic nerves were immunolabeled with antibodies against the myelinating Schwann cell-specific protein periaxin (red). Hoechst (Ho) staining is shown in blue. No differences were observed between mutant and wild type in periaxin, indicating that precocious myelination in the mutant is unlikely. B, Western blot of protein extracts from mutant and wild-type (wt) mice at P10, P21, P30, and adult. No striking differences (as measured by densitometry, normalized with β-tubulin) are detectable in the expression of the myelin proteins P0 and periaxin and of the nonmyelinating Schwann cell-specific protein L1.

Figure 4.

Figure 4.

Schwann cell death is reduced during development and after injury in neonatal nerves of P0 CRE/TβRIIf/f mice. In vitro, Schwann cells from mutant mice are not sensitive to TGFβ killing, although they die normally after serum withdrawal. A, Percentages (mean ± SEM; n = 10) of TUNEL-positive nuclei in transverse sections of E18 and P2 sciatic nerves from wild-type and mutant mice. At both time points, the rate of death is significantly reduced in the mutant compared with the wild type (see Results). B, C, Sciatic nerves from P1 wild-type and mutant mice (n = 7) were transected, and after 24 h, TUNEL was performed on longitudinal sections. In the distal stump of the transected nerve, Schwann cell death increased significantly in both the wild type and the mutant (arrows in C). However, in the mutant, it remained 3.95-fold (**p < 0.001) lower than in the wild type (see Results). D, Death of mutant cells in vitro. When exposed to TGFβ, only 40 ± 2% of the wild-type cells survived after 24 h, whereas 73 ± 2% of the mutant cells were still alive. This is identical to survival of the untreated cells, showing that the mutant cells are completely protected from TGFβ-induced death (n = 3; **p < 0.005). In contrast, when wild-type or mutant cells were subjected to 10% serum withdrawal, death rates were similar, suggesting that the reduced cell death in the mutant was a specific response to TGFβ signaling (n = 3). P2cut, P2 nerve transfected at P1.

Figure 5.

Figure 5.

Schwann cell proliferation is reduced during development in P0 CRE/TβRIIf/f mice. The final number of cells is similar in mutant and wild-type nerves. Longitudinal sections from E18 sciatic nerves were immunolabeled with an antibody to PH3, which specifically marks the cells that are dividing. A, In the mutant nerve, the number of dividing cells (mean ± SEM; n = 10) is reduced from 1.15 ± 0.1 to 0.45 ± 0.12% (n = 8; **p < 0.001). B, Comparison between a wild-type and a mutant nerve: whereas in the wild type, several dividing nuclei are identified, in the mutant nerve, the number of PH3-positive nuclei is significantly reduced. Ho, Hoechst dye.

Figure 6.

Figure 6.

TGFβRII recombination has already taken place by E18. Schwann cells from mutant and wild-type mice were labeled with antibodies to Smad2 or Smad4 (red). Essentially no nuclear localization of Smad2 or Smad4 is detectable in the mutant cells from E18 animals, suggesting that recombination has already taken place at this time. Nuclei were visualized with Hoechst dye (Ho; blue).

Figure 7.

Figure 7.

TGFβ cooperates with neuregulin-1 in promoting Schwann cell proliferation in vitro. A, Schwann cells from wild-type (wt) and mutant mice were isolated, exposed to increasing concentrations of neuregulin-1 (NRG-1) for 10 h, and tested for BrdU incorporation. This showed that mutant cells proliferate at a reduced rate, which is similar to that seen when wild-type cells were exposed to neuregulin-1 in the presence of SB431542 (10 μ

m

), a TGFβ receptor blocker [averages of three separate experiments, each using triplicate coverslips; wild type and P0 CRE/TβRIIf/f represent littermates from three separate litters]. B, BrdU incorporation in wild-type cells exposed to increasing concentrations of neuregulin-1 in the presence or absence of 5 ng/ml TGFβ. Note that TGFβ strongly promotes the proliferation induced by low concentrations of neuregulin-1 (averages of three separate experiments, each using triplicate coverslips).

Figure 8.

Figure 8.

No differences in cell proliferation or in regulation of myelin genes are detectable between mutant and wild-type mice after adult sciatic nerve injury. A, Adult sciatic nerves from wild-type and mutant mice were cut and, 7 d after transection, the distal stumps of the cut nerves were labeled with PH3 to visualize cell proliferation. In both wild-type and mutant nerves, there was active proliferation, but there was no significant difference in the number of PH3-positive nuclei (arrows). Nuclei were visualized with Hoechst dye (Ho). B, Western blot on extracts from wild-type and mutant sciatic nerves. Adult sciatic nerves were crushed and tested for P0 and periaxin (data not shown) protein expression. Protein levels decreased in a similar manner, namely by 4.1-fold in the wild type and 3.4-fold in the mutant (normalized to GAPDH and determined by densitometric analysis). C, Similarly, the mRNA levels for P0 and periaxin were equal 7 d after nerve crush, indicating that TGFβ is not essential for these events. C, Contralateral nerve; 7d crush, 7 d after nerve crush.

Figure 9.

Figure 9.

A model of TGFβ function in developing nerves. TGFβ kills those cells that lose out in competition for axonal neuregulin-1 (NRG) but is a mitogen for cells with adequate axonal association. TGFβ is therefore a bifunctional amplifier that strengthens both the positive and negative consequences for Schwann cell (Sch) numbers that result from a competition for a limited amount of axonal survival support. Ax, Axon.

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