TGF‐beta Signalling and Development of Mouse Midbrain Dopaminergic Neurons and Hindbrain Serotonergic Neurons

Abstract

Tyrosine hydroxylase (TH) positive, midbrain dopaminergic neurons (mDA) and hindbrain serotonergic neurons (5‐HT) represent clinically important ventral neuronal subpopulations. Dysregulation of mDA has been linked to the development of depression and drug addiction, while their degeneration leads to the characteristic symptoms of Parkinson's disease. Dysfunction of hindbrain 5HT neurons has been associated with several neurological and psychiatric diseases. In previous studies we have shown that members of the transforming growth factor beta family are regulators of development and maintenance of mDA neurons. In this work we aim to understand the role that TGF‐beta signalling plays on the development of mDA and hindbrain 5HT neurons. For this purpose, we have generated a conditional knockout mouse model in which a region of the midbrain and hindbrain, including the mDA and rostral 5HT neurons, lacks the TGF‐beta Receptor II (TGFBR2). This model was generated using the Cre‐lox system by crossing the En1‐Cre line, in which the expression of the recombinase Cre is controlled by the Engrailed 1 promoter, and the TGFBR2‐flox line, in which the TGFBR2 gene is floxed. Through the use of immunohistochemistry on brain cryosections, and counts of TH immunopositive cells we found significant reduced numbers in both substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) of the mutants at E18 and even at E16. In contrast, at E14 comparable numbers of TH immunopositive mDA neurons in cKO and WT were obtained. Using in situ hybridization we have calculated the volume of Cck and Ahd2 expression at E14 and our results show dramatic loss of Cck and Ahd2 expression. Development of rostral and caudal 5‐HT subpopulations was also affected by loss of TGF‐beta signaling. The number of dorsal raphe, median raphe, and paramedian rahe 5‐HT neurons was significantly reduced in the conditional knock out, compared to the wildtype, accompanied by decreased expression of Pet and Gata2.In order to understand the molecular mechanism by which TGF‐beta acts in this process, we performed a cDNA microarray to analyse the transcriptome of the ventral midbrain of conditional knockout mice and compared it with that of wild type mice. Among the genes differentially regulated by the loss of the TGFBR2, we found genes that fall into the category of receptors, ion transporters, phosphatases and molecular chaperones, among others. Some of the differentially regulated genes have been shown to be implicated in processes like inflammation and endosomal sorting. Future analysis will be focused on understanding the function these genes have during the generation of the midbrain DA neurons, in order to shed some light onto the mechanism by which TGF‐beta acts on these process.Support or Funding InformationDFG