Estimation of nonproliferating cells in the neural retina of embryonic chicks by flow cytometry

Abstract

Nonproliferating cells (N cells) in the neural retina of embryonic chicks were estimated after isolating them in the 2C peak of a DNA distribution by exposure to the cell-cycle inhibitor ICRF 159. ICRF 159 inhibits cell division but not DNA synthesis, so proliferating cells can leave the 2C peak but not reenter it. Cells left in the 2C peak after exposure to ICRF 159 were assumed to be N cells. The effectiveness of ICRF 159 in inhibiting cell division but not inhibiting DNA synthesis was demonstrated in neural retinae from stage 17 embryos which showed no evidence of a 2C peak after 6-h exposure to ICRF 159 and which were thus shown to have no N cells. A test to detect escape from the cell division block in older embryos with an N cell population in the neural retina showed some escape after longer exposures to ICRF 159. The escape was suppressed by a second dose of ICRF 159, given some hours after the first. Key terms: Nonproliferating cells, embryo, chick, DNA distribution, ICRF 159, cytostatic, in vivo methodThis report is part of a study of the growth of an embryonic cell population being carried out in vivo on the neural retina of embryonic chicks. A growth curve of the increase in total numbers of cells in the neural retina has been measured from a time shortly after the optic vesicle invaginates to a time when cell numbers reach a plateau (9). The increase in cell numbers observed in the growth curve is a result of cell production, which occurs when a proliferating cell (P cell) divides into two at the end of a cell cycle, and one or more factors which stabilize cell numbers. One of these factors, if autoradiographic evidence of the end of cell proliferation in the neural retina (6) is accepted, is a change to a nonproliferative state. In a previous explanation of the stabilization of cell numbers (91, it was proposed that progeny of P cells became nonproliferating cells (N cells) at various predetermined generations of cell cycles after the start of growth. To investigate whether such a conversion to a nonproliferative state is the way cell numbers are controlled requires a method of estimating N cells at various times during the growth of the neural retina. Here, we describe a procedure, using cycle inhibitor ICRF 159, to isolate and estimate N cells in a DNA distribution, and we report preliminary estimates of their proportions.The neural retina at times contains both P cells and N cells. Our assumption is that P cells have DNA values ranging from 2C to 4C and N cells have a 2C value of DNA. The 2C peak of a DNA distribution will then be made up of P cells in the G1 phase of the cell cycle and N cells. N cells could be isolated in the 2C peak by exposure to a cytostatic agent which allows P cells to leave the 2C peak but prevents their reentry at the end of a cell cycle. The requirements are that the agent should not inhibit DNA synthesis but should inhibit cell division. The agent chosen was ICRF 159. It has been reported to arrest cycling cells in prophase (11,14) and G2 (1). Some slowing of cell...