Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator

Abstract

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers.

Differential response of pineal microglia to surgical versus pharmacological stimuli

“…Our previous report showed that pineal microglia are highly phagocytic in the normal adult pineal gland (Ibañez Rodriguez et al, ). Therefore, we determined whether the increased microglial cell density following SCGx or SCGd was accompanied by an enhanced phagocytic capacity.…”

“…We recently showed that microglia are constantly ‘activated’ throughout the whole ontogeny of the pineal gland, and that microglial cells regulate pineal development and function by association and interaction with Pax6 + cells and signaling elements such as pinealocyte neurites, blood vessels, and sympathetic nerve fibers (Ibañez Rodriguez et al, ). Association and apparent phagocytosis of Pax6 + cells by highly phagocytic and proliferative microglial cells (Iba1 + /PCNA + /ED1 + cells) were observed mainly in the healthy adult pineal gland (PG).…”

“…Pax6 + cell proliferation in the SCGx PG could therefore occur at a ZT different than ZT6. The Pax6 + cell population is thought to be a latent cell reservoir in the adult PG (Ibañez Rodriguez et al, ; Rath et al, ). Like other progenitors in the injured adult brain (Dimou & Gotz, ; Nakatomi et al, ), the latency of the Pax6 + cell population in the SCGx PG might be being challenged by several factors such as the lack of a functional sympathetic innervation, the transient and acute release of noradrenaline and the inflammation caused by WD, and microglia‐derived signals.…”

“…The level of ED1 expression in a single cell can be correlated with the phagocytic activity of the respective cell type (Damoiseaux et al, ). We recently used this anti‐CD68 antibody to show that microglia are highly phagocytic during the entire pineal gland ontogeny (Ibañez Rodriguez et al, ). The staining presented here matched that described in our previous report (Ibañez Rodriguez et al, ).…”

“…This antibody recognizes a single ∼50 kDa protein band on WBs (Benitez, Castro, Patterson, Muñoz, & Seltzer, ), and intact and degenerated nerve fibers by IHC (Hegarty, Hermes, Yang, & Aicher, ). We used this anti‐Tuj1 antibody to stain nerve fibers in the postnatal pineal gland (Ibañez Rodriguez et al, ; Yu et al, ). Staining observed here matched that of these previous studies.…”