neural-stem-cell-reactivation-in-cultured-drosophila-brain-explants

Neural Stem Cell Reactivation in Cultured <em>Drosophila</em> Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

University of Virginia

Correct positioning of stem cells within their niche is essential for tissue morphogenesis and homeostasis. How stem cells acquire and maintain niche position remains largely unknown. Here, we show that a subset of brain neuroblasts (NBs) in  utilize Phosphoinositide 3-kinase (PI3-kinase) and DE-cadherin to build adhesive contact for NB niche positioning. NBs remain within their native microenvironment when levels of PI3-kinase activity and DE-cadherin are elevated in NBs. This occurs through PI3-kinase-dependent regulation of DE-Cadherin-mediated cell adhesion between NBs and neighboring cortex glia, and between NBs and their ganglion mother cell daughters. When levels of PI3-kinase activity and/or DE-Cadherin are reduced in NBs, NBs lose niche position and relocate to a non-native brain region that is rich in neurosecretory neurons, including those that secrete some of the  insulin-like peptides. Linking levels of PI3-kinase activity to the strength of adhesive attachment could provide cancer stem cells and hematopoietic stem cells with a means to cycle from trophic-poor to trophic-rich microenvironments.

Neuroblast niche position is controlled by Phosphoinositide 3-kinase-dependent DE-Cadherin adhesion.

How the developmental potential of differentiating stem cell progeny becomes rapidly and stably restricted following asymmetric stem cell division is unclear. In the fly larval brain, earmuff (erm) uniquely functions to restrict the developmental potential of intermediate neural progenitors (INPs) generated by asymmetrically dividing neural stem cells (neuroblasts). Here we demonstrate that the histone deacetylase Hdac1/Rpd3 functions together with self-renewal transcriptional repressors to maintain the erm immature INP enhancer in an inactive but poised state in neuroblasts. Within 2 hr of immature INP birth, downregulation of repressor activities alleviates Rpd3-mediated repression on the erm enhancer, enabling acetylation of multiple histone proteins and activating Erm expression. Erm restricts the developmental potential in immature INPs by repressing genes encoding neuroblast transcriptional activators. We propose that poising the fast-activating enhancers of master regulators of differentiation through continual histone deacetylation in stem cells enables self-renewal and rapid restriction of developmental potential following asymmetric division.

An Hdac1/Rpd3-Poised Circuit Balances Continual Self-Renewal and Rapid Restriction of Developmental Potential during Asymmetric Stem Cell Division.

Cell proliferation is coupled with nutrient availability. If nutrients become limited, proliferation ceases, because growth factor and/or PI3-kinase activity levels become attenuated. Here, we report an exception to this generality within a subpopulation of  neural stem cells (neuroblasts). We find that most neuroblasts enter and exit cell cycle in a nutrient-dependent manner that is reversible and regulated by PI3-kinase. However, a small subset, the mushroom body neuroblasts, which generate neurons important for memory and learning, divide independent of dietary nutrient conditions and PI3-kinase activity. This nutrient-independent proliferation is regulated by Eyeless, a Pax-6 orthologue, expressed in mushroom body neuroblasts. When Eyeless is knocked down, mushroom body neuroblasts exit cell cycle when nutrients are withdrawn. Conversely, when Eyeless is ectopically expressed, some non-mushroom body neuroblasts divide independent of dietary nutrient conditions. Therefore, Eyeless uncouples MB neuroblast proliferation from nutrient availability, allowing preferential neurogenesis in brain subregions during nutrient poor conditions.

Eyeless uncouples mushroom body neuroblast proliferation from dietary amino acids in .

Most neurogenesis occurs during development, driven by the cell divisions of neural stem cells (NSCs). We use Drosophila to understand how neurogenesis terminates once development is complete, a process critical for neural circuit formation. We identified E93, a steroid-hormone-induced transcription factor that downregulates phosphatidylinositol 3-kinase (PI3K) levels to activate autophagy for elimination of mushroom body (MB) neuroblasts. MB neuroblasts are a subset of Drosophila NSCs that generate neurons important for memory and learning. MB neurogenesis extends into adulthood when E93 is reduced and terminates prematurely when E93 is overexpressed. E93 is expressed in MB neuroblasts during later stages of pupal development only, which includes the time when MB neuroblasts normally terminate their divisions. Cell intrinsic Imp and Syp temporal factors regulate timing of E93 expression in MB neuroblasts, and extrinsic steroid hormone receptor (EcR) activation boosts E93 levels high for termination. Imp inhibits premature expression of E93 in a Syp-dependent manner, and Syp positively regulates E93 to promote neurogenesis termination. Imp and Syp together with E93 form a temporal cassette, which consequently links early developmental neurogenesis with termination. Altogether, E93 functions as a late-acting temporal factor integrating extrinsic hormonal cues linked to developmental timing with neuroblast intrinsic temporal cues to precisely time neurogenesis ending during development.

E93 Integrates Neuroblast Intrinsic State with Developmental Time to Terminate MB Neurogenesis via Autophagy.

Termination of Drosophila mushroom body neurogenesis via autophagy and apoptosis.

Dietary nutrients provide macromolecules necessary for organism growth and development. In response to animal feeding, evolutionarily conserved growth signaling pathways are activated, leading to increased rates of cell proliferation and tissue growth. It remains unclear how different cell types within developing tissues coordinate growth in response to dietary nutrients and whether coordinated growth of different cell types is necessary for proper tissue function. Using the early Drosophila larval brain, we asked whether nutrient-dependent growth of neural stem cells (neuroblasts), glia, and trachea is coordinated and whether coordinated growth among these major brain cell types is required for neural development. It is known that in response to dietary nutrients and PI3-kinase activation, brain and ventral nerve cord neuroblasts reactivate from quiescence and ventral nerve cord glia expand their membranes. Here, we assay growth in a cell-type specific manner at short time intervals in the brain and determine that growth is coordinated among different cell types and that coordinated growth is mediated in part through activation of PI3-kinase signaling. Of the 7 Drosophila insulin-like peptides (Dilps), we find that Dilp-2 is required for PI3-kinase activation and growth coordination between neuroblasts and glia in the brain. Dilp-2 induces brain cortex glia to initiate membrane growth and make first contact with quiescent neuroblasts. Once reactivated, neuroblasts promote cortex glia growth to ultimately form a selective membrane barrier. Our results highlight the importance of bidirectional growth signaling between neural stem cells and surrounding cell types in the brain in response to nutrition and demonstrate how coordinated growth among different cell types drives tissue morphogenesis and function.

Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.

Temporal patterning of neural progenitors, in which different factors are sequentially expressed, is an evolutionarily conserved strategy for generating neuronal diversity during development. In the Drosophila embryo, mechanisms that mediate temporal patterning of neural stem cells (neuroblasts) are largely cell-intrinsic. However, after embryogenesis, neuroblast temporal patterning relies on extrinsic cues as well, as freshly hatched larvae seek out nutrients and other key resources in varying natural environments. We recap current understanding of neuroblast-intrinsic temporal programs and discuss how neuroblast extrinsic cues integrate and coordinate with neuroblast intrinsic programs to control numbers and types of neurons produced. One key emerging extrinsic factor that impacts temporal patterning of neuroblasts and their daughters as well as termination of neurogenesis is the steroid hormone, ecdysone, a known regulator of large-scale developmental transitions in insects and arthropods. Lastly, we consider evolutionary conservation and discuss recent work on thyroid hormone signaling in early vertebrate brain development.

Steroid hormones, dietary nutrients, and temporal progression of neurogenesis.

Stem cells enter and exit quiescence as part of normal developmental programs and to maintain tissue homeostasis in adulthood. Although it is clear that stem cell intrinsic and extrinsic cues, local and systemic, regulate quiescence, it remains unclear whether intrinsic and extrinsic cues coordinate to control quiescence and how cue coordination is achieved. Here, we report that Notch signaling coordinates neuroblast intrinsic temporal programs with extrinsic nutrient cues to regulate quiescence in Drosophila. When Notch activity is reduced, quiescence is delayed or altogether bypassed, with some neuroblasts dividing continuously during the embryonic-to-larval transition. During embryogenesis before quiescence, neuroblasts express Notch and the Notch ligand Delta. After division, Delta is partitioned to adjacent GMC daughters where it transactivates Notch in neuroblasts. Over time, in response to intrinsic temporal cues and increasing numbers of Delta-expressing daughters, neuroblast Notch activity increases, leading to cell cycle exit and consequently, attenuation of Notch pathway activity. Quiescent neuroblasts have low to no active Notch, which is required for exit from quiescence in response to nutrient cues. Thus, Notch signaling coordinates proliferation versus quiescence decisions.

Sarah E. Siegrist

Biology Department

University of Virginia

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants