Numerous past studies have been successful at proving the fact that Notch signalling, a tightly regulated signalling system existent in majority of multicellular organisms can play a vital role in the muscle regeneration of satellite cells by catalysing the proliferation and renewal of the cell. These satellite cells are skeletal muscle stem cells which are directly related in the growth and repair of skeletal muscle. However, the function of Notch3, a gene that instructs the production of the Notch3 receptor protein, which is expressed in the adult satellite cells and is thought to play regulatory roles in muscle growth, has never been subject to thorough investigation.
In a bid to recognise the possible role of Notch3 expression in muscle regeneration, scientists at the embryology department of Kitasato University of Japan designed a set of experiments in which the rate and extent of muscle regeneration and growth was measured in Notch3-deficient mutant mice. However since they did not observe any evident phenotypical expression in the musculature of the mutant mice population, on a second study they focused on Notch3-deficient mice that suffered induced repetitive muscle injuries. This choice of experiment was based on the fact that satellite cells reach their peak activity at times of injury, in a quest to regenerate muscle replacing the damaged tissue; this is the ideal situation to observe any change in muscle regeneration pattern.
Mice which were known to lack the Notch3 gene (Notch-/-) were crossed (made to reproduce) with mice which had the Notch3 gene but also carried a loss-of-function mutation which lead to cycles of severe muscle degeneration followed by regeneration throughout their life. This is somehow advantageous over human imposed muscle injuries as there is already existent muscle degeneration and injury occurring due to gene dysfunction. These newly reproduced mice were injected with 5-bromo-2-deoxyuridine, a synthetic nucleoside that is commonly used in the detection of proliferating cells in living tissues every day for four days. The muscle segments were then frozen in isopentane (solution used in tissue freezing) and examined by histological and immunohistochemical methods. There were no significant changes in the samples analysed four weeks after the birth of the mice, but at the four month mark, there was an astounding observation made which suggested the Notch3 mice were larger than the control group and there was an increase in the body weight (1.3 – 1.6 times) of these mice throughout the rest of the first year of life.
Figure1| A photograph showing the effect of repetitive muscle (hind limb) injury on mutant and control mice.
In order to be thoroughly convinced that the muscle hyperplasia (muscle overgrowth) witnessed in the bred mice was derived from repeated muscle injury, the Notch3-deficient mice went under direct injury induced experiments as a part of which the hind limb muscle was exposed to incision and cardiotoxin (CTX) (a toxin that causes muscle cell death and impairment). Between the 2nd and 3rd months after the procedure, muscles were isolated from the mutant mice, as for the delight of researchers the muscles were a remarkable 1.3-1.6 fold heavier in weight than the control mice, depending on the dose and duration of exposure to CTX. The muscle that was subject to incisions followed by injection of three doses of CTX showed a greater increase in mass than the muscle treated with only one dose of CTX. Hence longer durations and higher exposures to CTX coincide with increased muscle mass in mutant mice.
In an attempt to further endorse the role of Notch3 in muscle proliferation, a set of notch3-deficient myoblasts (cell that gives rise to muscle cells) where mixed with an active form of Notch 3 and were cultivated with EdU (an indicator of myoblast proliferation) in an attempt to see the effect that Notch 3 may have on the Notch3-deficient muscles which as previously stated show significant growth when subject to muscle damage. Predictably the results indicated that Notch3 negatively enhances the proliferation of myoblasts and shows hindering properties.
Notch3 being structurally different (lacking a gene division) from its pathway counterparts Notch1 and Notch2, could be a sign as of why this gene is distinct in behaviour and why it exhibits different functionality within the pathway. However this is subject to further research.
Further investigation discovered that upon using Western blot and immunocytochemical methods of analysis, the Notch3 pathway was very much distinctive in these newly bred mice than in the control group. It was further speculated that the self-regeneration of the cells are positively regulated by Notch1 whereas Notch3 is directly reflective of a negative regulation in the cells. Data indicates Notch3 is responsible for regulating the self-regeneration of cells after muscle injury, thus, absence of Notch3 should mean up- regulation of Notch1 gene which is assumed to result in heightened proliferation of postnatal satellite cells.
Amid thoughts that Notch1 gene expression results in amplified numbers of satellite cells and eventually more muscle formation, a set of data from previous experiments involving the mutant mice did not show a noticeable escalation in Notch1 activity in the myoblast that lacked Notch3 gene despite muscle hyperplasia. Subsequently these ambiguous regulatory relations between Notch3 and Notch1 genes remain to be a motif for further investigation. In addition it has been proposed that interaction of Notch3 protein receptors with other regulatory elements (e.g. Delta-1 ligand) reverses the negative regulatory presence of Notch3, so the mechanism by which Notch3 regulates Notch1, Notch2 and skeletal muscle satellite cells is yet to be elucidated and is believed to be independent.
The consensus amongst the researchers in this study is that Notch3 deficiency leads to a clear visible rise in the quantity of satellite cells during muscle self-renewal. This demonstration of self-regeneration is highly insightful for the future of cell research as it gives solid evidence of the mechanisms behind self-regeneration of satellite cells, which may prompt a revolutionary breakthrough in molecular embryology, as well as therapeutic applications concerning degenerative muscle diseases – where manipulation of the Notch3 signalling may well be favourable in transplantation therapy.
References
Kitamoto, T & Hanaoka, K , ‘Notch3 null mutation in mice causes muscle hyperplasia by repetitive muscle regeneration’, Laboratory of Molecular Embryology, Department of Bioscience, Kitasato University School of Science, Japan.
