MRG15, a component of HAT and HDAC complexes, is essential for proliferation and differentiation of neural precursor cells

Abstract

Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells (NPCs). Chromatin regulation is a key step in self-renewal activity and fate decision of NPCs. However, the molecular mechanism or mechanisms of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of NPCs. Neuroepithelia from Mrg15-deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated NPCs from Mrg15-deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15-deficient embryo grew less efficiently than those from wild type. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incorporation revealed that Mrg15-deficient NPCs have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15-deficient NPCs most likely accounts for the thinner neuroepithelia in Mrg15-deficient embryonic brain. Moreover, we also demonstrate Mrg15-deficient NPCs are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development.

Figure 1. Expression of progenitor and mitotic markers in E10.5 Mrg15 neural tube

A, B: MPM2 (green)/TUNEL (red) double-staining of coronal section of E10.5 embryonic forebrain from Mrg15 heterozygote (A) and Mrg15 null (B). C, D: Nestin (green)/TUNEL (red) double-staining of coronal section of E10.5 embryonic forebrain from Mrg15 heterozygote (C) and Mrg15 null (D).

Figure 2. Decreased neurosphere formation in Mrg15 null neural precursor cells

A, B: Representative pictures of primary (upper row), secondary (middle row), and tertiary (lower) neurosphere cultures from wild-type (panel A) and Mrg15 null (panel B) cells. Single neural precursor cells were seeded into 96-well plate at 10³, 10² and 10 cells per well cultured in Neurobasal Medium supplemented with EGF and FGF2 for the neurosphere formation assay for 10 days (Primary and Secondary). The same size spheres from both wild-type and null cell that were larger than 100 μm were collected and treated with trypsin to make a single cell suspension for secondary NSFA. Single cell suspension made from one of the largest spheres from each genotype was seeded into a 96-well plate at 500, 50, 5 cells per well and cultured for 14 days (Tertiary). C: Quantitative analyses of neurosphere number and size. After defined time points, sphere size and number were measured under the microscope. Y-axis indicates sphere numbers per 1000 or 500 cells which were initially seeded.

Figure 3. Reduced cell proliferation in Mrg15 null neural precursor cells

A, B: Representative BrdU immunostaining in wild-type (A) and Mrg15 null (B) cells at fifth passage (x4 magnification). Single cells were plated on poly-L-lysine-coated coverslips, grown for 36 h, and incubated with 10 μM BrdU for 4 h. Incorporated BrdU was detected by mouse anti-BrdU antibody followed by incubation with biotinilyted anti-mouse IgG and positive cells demonstrated by DAB staining. C: Quantitative analyses of BrdU positive cells. Percentage of positive cells is shown. BrdU positive cells in Mrg15 null population were fewer than those in wild-type population both in primary culture and at fifth subculture (P<0.001).

Figure 4. Apoptosis in Mrg15 null neural precursor cells does not increase in neurosphere culture

Single cells from wild-type and Mrg15 null neural precursor cells were seeded on poly-L-lysine-coated coverslips and cultured in Neurobasal Medium supplemented with stem cell growth component for 5 days. The cells on coverslips were fixed with 4% paraformaldehyde and TUNEL assay (green) used to detect apoptotic cells. We co-stained these cells with anti-nestin antibody (red) to detect the neural precursor cell population. Cells were counterstained with 0.1 μg/ml of DAPI (blue) for nuclear staining. Wild-type (A) and Mrg15 null (B) neural precursor cells are shown.

Figure 5. Re-expression of MRG15 by adenovirus can rescue the cell proliferation defect in Mrg15 null neural precursor cells

Mrg15 null neural precursor cells were infected with adenovirus expressing MRG15 or EGFP at 1000 moi twice at a one week interval. Four days after the second infection, cells were incubated with 10 μM BrdU for 4 h and BrdU positive cells detected by immunostaining. The percentage of BrdU positive cells in adenovirus MRG15 infected Mrg15 null cells significantly increased compared with Mrg15 null control cells (P<0.01), although the control adenovirus EGFP did not have any effect on BrdU incorporation in these cells.

Figure 6. MRG15 is required for differentiation into neural lineages

A: Representative pictures of primary wild-type and Mrg15 null cells 10 days after exposure to differentiation conditions (upper row) and passaged cells 7 days after differentiation condition (lower row). Wild-type cells (left panel) changed morphology and appeared to be differentiated under this condition, but most of the Mrg15 null cells (right panel) remained as colony-like aggregates. B: Reduced Tuj1 immunoresposive cell number of differentiated cells from passaged Mrg15 null neural precursor cells (lower row) compared with wild-type (upper row). Neurospheres were plated on poly-L-lysine-coated coverslips and cultured under differentiation condition for 7 days. Cells were fixed with 4% paraformaldehyde and stained with antibody against Tuj1 (neurons, green), GFAP (glial, red), followed by incubation with fluorescent conjugated secondary antibodies. Cells were counterstained with 0.1 μg/ml of DAPI (blue). C: Quantitative analyses of Tuj1 and GFAP positive cells. The percentage of positive cells is shown. Tuj1 positive cells in the Mrg15 null population were fewer than those in the wild-type population (P<0.05).