Functions of TGF-β2 and GDNF in the development of the mouse nervous system: evidence from double mutant mice

Belal Rahhal's picture
Belal Mahmoud Mustafa Rahhal
Current Affiliation: 
Faculty of Medicine & Health Sciences, Department of Biomedical Sciences, An-Najah National University, Nablus, Palestine
Preferred Abstract (Original): 

1. Abstract: A major area of investigations in neuroscience is directed at understanding factors that participate in neural survival versus death. A number of neurotrophic growth factors have been identified that promote neuronal survival and differentiation. The transforming growth factors-beta (TGF-ß) constitute a family of multifunctional cytokines. Their functions include control of cell proliferation, differentiation and regulation of cell survival and death. Glial cell line-derived neurotrophic factor (GDNF) itself is distantly related to TGF-ß. It maintains survival of various neuronal populations such as midbrain dopaminergic neurons and motoneurons. Many recent advancements have revealed that growth factors acting in synergy can regulate neuronal survival much more potently than any individual factor alone. Much evidence suggests that GDNF may require cofactors to act as a neurotrophic factor. The present work aims at elucidation of TGF-ß2 and GDNF synergism in vivo through generation of TGF-ß2/GDNF double mutant mice. As mutant mice lacking TGF-ß2 or GDNF die during birth, double mutant mice have to be generated by first breeding heterozygous mice to generate double heterozygous mice and finally double mutant mice. The expected ratio was 1:16 embryos. A total of 270 embryos was btained of which 11 instead of the expected 17 were double mutant mice. The embryos were obtained from embryonic days 12 to 18. To test the hypothesis of whether TGF- ß2/GDNF synergistically promote neuron survival, a wide spectrum of neuron populations was analysed at E18, the latest stage accessible before birth and consequently death of the mutant mice. At E18.5 there was a significantly decreased number of neurons detectable in the motoneuron population, the sympathetic ganglionic neurons as well as some parasympathetic neuron populations. The loss of parasympathetic neurons was comparable to the phenotype of GDNF mutant mice, whereas lumbar motoneurons and neurons of the SCG resulted in a clear unique phenotype due to the double null condition. These data suggest that TGF-ß and GDNF synergise to promote neuron survival during development of lumbar motoneurons as well as SCG neurons. Both TGF-ß as well as GDNF are well known for their survival-promoting effect of midbrain dopaminergic neurons. Therefore, special attention was given to the analysis of their development. At embryonic stage 12.5 (E12.5), the total number of midbrain dopaminergic neurons was significantly decreased in mice deficient for TGF-ß2 (Tgfß2-/-) compared to wild-type (Tgfß2+/+) mouse embryos. This may give an indication that TGF- ß2 plays a role in the early induction of the dopaminergic neurons. Surprisingly, at E14.5 and E18.5, our analysis failed to reveal significant differences in the total number of THpositive cells in the substantia nigra pars compacta (SNpc) and the vental tegmental area (VTA) in Tgfß2-/-Gdnf +/-, Tgfß2+/-Gdnf -/-, Tgfß2-/-Gdnf -/- mutant mouse embryos compared to the controls (Tgfß2+/+Gdnf +/+). This may indicate that these genes seem to have a marginal effect on the development of the midbrain dopaminergic neurons at these stages in vivo, but may be more important in postnatal maturation of the system. Moreover, the one year old Tgfß2+/- and double heterozygous (Tgfß2+/-Gdnf +/-) mice showed a marginal decrease (10% and 13%, respectively) in the dopaminergic neurons compared to the controls (Tgfß2+/+Gdnf +/+). The locus coeruleus (LC) is the noradrenergic nucleus that is severely affected in neurodegenerative disorders. In this study, at E14.5 there were no significant differences observed in the total number of neurons within the LC between Tgfß2-/-, Gdnf -/-, Tgfß2-/- Gdnf +/-, Tgfß2+/-Gdnf -/- mouse embryos and the controls (Tgfß2+/+Gdnf +/+). 2 The total number of serotonergic neurons at E12.5 was significantly decreased in mice deficient for TGF-ß2 (Tgfß2-/-) compared with wild-type mouse embryos (Tgfß2+/+). This may indicate that TGF-ß2 plays a role in the early induction of the serotonergic neurons. Moreover, there was a significant decrease in the total number of serotonergic neurons in Tgfß2-/- and Tgfß2-/-Gdnf +/- in the paramedian raphe (PMR) at E18.5 compared with wildtype mouse embryos. On the other hand, quantification of rostral 5-HT-positive cells showed a decrease at E14.5 in the double mutant mice (Tgfß2-/-Gdnf -/-) compared with wild-type mouse embryos, but differences did not reach statistical significance. One out of three double knockout mice at E14.5 and E18.5 showed a severe defect (reduction) in the number of the rostral 5-HT-positive neurons which may be due to genetic penetrance. Chromaffin cells are thought to develop from the same progenitors as sympathetic neurons. In the present study, effects of TGF-ß2 and GDNF on proliferation and differentiation of chromaffin cells in mouse adrenal chromaffin cells were investigated in a genetic mouse model. We observed a significant increase in the total number of tyrosine hydroxylasepositive cells (TH+ ) in Tgfß2-/- and Tgfß2-/-Gdnf -/- double knockout mouse embryos at E14.5 and E14.5 compared to wild-type animals (Tgfß2+/+ ), but no changes in the number of TH-immunoreactive cells were observed in GDNF mouse mutants. At E15.5 but not at E18.5, there was a marked increase in the number of proliferative cell nuclear antigen (PCNA+ ) positive chromaffin cells in Tgfß2-/- knockout embryos compared to the wild type group. On the other hand, there was a clear decrease in the ratio of total number of phenylethanolamine-N-methyltransferase-positive cells (PNMT+ ) to the total TH+ in Tgfß2- /- mouse embryos at E18.5 compared to wild type animals. This is the first documentation of the physiological significance of TGF-ß2, an isoform that has been suggested to play a role in the regulation of chromaffin cell proliferation and differentiation based on in vitro experiments. Tgfß2+/+Gdnf -/- and Tgfß2-/-Gdnf -/- double mutant mouse embryos lack most of the enteric neurons using neurofilament (NF) antibody as a neuronal marker. This result is consistent with the results from GDNF knockout mice, Gdnf -/- mice showed an absence of the enteric nervous system (ENS) neurons. In addition to the hypothesis-based analysis and results there were some obvious additional phenotypes detectable in TGF-ß2/GDNF double mutant mice. At E14.5 and E18.5 the entire neural retina of Tgfß2-/-Gdnf -/- double mutant mouse embryos and Tgfß2-/-Gdnf+/- littermates was significantly thicker than wild-type retina. Interestingly, the double mutant mice (Tgfß2-/-Gdnf -/-) showed a complete detachment of the retina from the underlying pigment epithelium at E18.5 and the retina was folded (coloboma formation) at E18.5. Furthermore, Tgfß2-/-Gdnf -/- double mutant mice showed some phenotypes outside the central and peripheral nervous system. Tgfß2-/-Gdnf -/- double mutant embryos showed a reduction in the thickness of the ventral body wall and muscle development, defects in extracellular matrix formation (ECM), and acceleration in molar tooth development. In summary, TGF-ß2/GDNF mutant mice show a unique pattern of phenotypes that partly may be due to a synergism in regulating neuronal survival e.g. MN and SCG. Furthermore, they may also cooperate in other places by regulating proliferation, differentiation as well as production and composition of extracellular matrix. The data obtained also suggest a wide array of potential clinical implications, ranging from the understanding to the treatment of motoneuron to eye disease.