Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Murine HSCR-Study of RET and EDNRB pathway interaction in the enteric nervous system development

This part of the project concerned the study of mouse intestinal aganglionosis by evaluating the roles of RET and interacting molecules in the enteric nervous system development mainly through the analysis of their expression and proliferative/differentiative activities in neural crest derivatives obtained from wild type and RET C620R mutated mice.

Study of retC620R/C620R mice:
In order to address whether the intestinal aganglionosis in retC620R/C620R pups is a consequence of a growth defect of the Ret-expressing cells, neural crest cells (NCCs) destined to colonize the bowel were isolated from 8.5-9.0 d.p.c. embryos, cultured in vitro, and labeled with an anti-Ret antibody. Fluorescent immunohistochemistry and ultrastructural analysis by electronic microscopy demonstrated a significantly lower percentage of Ret-positive cells in ret C620R/C620R primary cultures compared to controls. Furthermore, in ret+/+ and ret+/C620R cells, the Ret-specific signals are often found in vesicles, near the cell membrane or in the cytoplasm. In the retC620R/C620R cells only a faint labeling was observable, restricted to the cytoplasm near neurotubules and endoplasmic reticulum. Notably, while the Ret-specific signals were observed in clusters on the cell membrane of control cells, the retC620R/C620R cells were consistently devoid of membrane labeling.
This result supports the in vitro biochemistry findings that ret C620R impairs maturation of the Ret protein and prevents it from being transported to the cell membrane, where its signal transduction function is effected (Yin et al. under revision by the Intl. J. Cancer).

Possible RET pathway interacting molecules:
During mice breeding RET C620R mouse carriers demonstrated very low fertility. Since RET has been shown to be expressed in testis, the low fertility of mutated mice could reveal a feature specific of the RET C620R mutation and linked to RET function during spermatogenesis.

Cytoskeletal components have been involved in differentiation/migration of NCC derivatives upon RET activation. TSGA10 is a new gene with putative functions in cell division during spermatogenesis and neurogenesis which has been recently proposed to have a role in neural crest development based on embryonic expression data. We demonstrated the presence of TSGA10 in mouse primary neural crest cultures at both RNA, by RT-PCR, and at the protein level, by immunocytochemistry (Behnam, Modarressi, Conti, Taylor, Puliti, Wolfe. Biochem Biophys Res Commun. 2006 344(4):1102-10).

Recent genetic and molecular data suggest the interaction between RET and EDNRB pathways and indicate that both are essential for the proliferation/differentiation of enteric NCCs. Although it is now clear that RET and EDNRB pathways control the enteric innervation development, the molecular mechanisms that mediate such control remain to be fully understood. We used a neuroblastoma cell line as model of NCCs in order to clarify the role of GDNF and ET3 on the fate of RET positive cells and on possible effects of RET and EDNRB pathways interaction on cell proliferation and differentiation. We verified expression of Ret, Gdnfra, and Ednrb in the neuroblastoma cells by RT-PCR analysis, and confirmed the presence of the RET protein by immunocytochemistry.
We used single or double treatments with GDNF and/or ET3 in synthetic medium in triplicated experiments. Cell growth was evaluated by counting cells obtained at the end of a 2/3 days treatment. Cell differentiation was evaluated by a detailed analysis of cell morphology by phase-contrast microscopy. A statistically significant increase of proliferation was observed in cell cultures after 2 days treatments with both GDNF and ET3. Cell morphology analysis indicated an increase of undifferentiated cells and of cells with only one neurite-like process (monopolar). Three days treatments of cells showed a statistically significant increase of differentiated cells after GDNF/ET3 double treatment.

Overall our data indicate a synergic effect of GDNF and ET3 on undifferentiated and monopolar cells proliferation as well as on the branching processes cells differentiation. These results are in agreement with the hypothesis of an interaction between the GDNF/RET and ET3/EDNRB pathways and confirm the use of the cell line as useful tool to further investigate on the complex relationships between RET and EDNRB molecular pathways.


Aldamaria PULITI, (Assistant Professor)
Tel.: +39-050-836234
Fax: +39-050-551290
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