Cheng Yu Lee

klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division

Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. We used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. We identify the transcription factor Klumpfuss (Klu) as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led us to propose that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division.

Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/Armadillo activity

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification.

Earmuff restricts progenitor cell potential by attenuating the competence to respond to self-renewal factors

Despite expressing stem cell self-renewal factors, intermediate progenitor cells possess restricted developmental potential, which allows them to give rise exclusively to differentiated progeny rather than stem cell progeny. Failure to restrict the developmental potential can allow intermediate progenitor cells to revert into aberrant stem cells that might contribute to tumorigenesis. Insight into stable restriction of the developmental potential in intermediate progenitor cells could improve our understanding of the development and growth of tumors, but the mechanisms involved remain largely unknown. Intermediate neural progenitors (INPs), generated by type II neural stem cells (neuroblasts) in fly larval brains, provide an in vivo model for investigating the mechanisms that stably restrict the developmental potential of intermediate progenitor cells. Here, we report that the transcriptional repressor protein Earmuff (Erm) functions temporally after Brain tumor (Brat) and Numb to restrict the developmental potential of uncommitted (immature) INPs. Consistently, endogenous Erm is detected in immature INPs but undetectable in INPs. Erm-dependent restriction of the developmental potential in immature INPs leads to attenuated competence to respond to all known neuroblast self-renewal factors in INPs. We also identified that the BAP chromatin-remodeling complex probably functions cooperatively with Erm to restrict the developmental potential of immature INPs. Together, these data led us to conclude that the Erm-BAP-dependent mechanism stably restricts the developmental potential of immature INPs by attenuating their genomic responses to stem cell self-renewal factors. We propose that restriction of developmental potential by the Erm-BAP-dependent mechanism functionally distinguishes intermediate progenitor cells from stem cells, ensuring the generation of differentiated cells and preventing the formation of progenitor cell-derived tumor-initiating stem cells.

Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead

The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.03502.001

It takes two to tango, a dance between the cells of origin and cancer stem cells in the Drosophila larval brain

During malignant transformation the cells of origin give rise to cancer stem cells which possess the capacity to undergo limitless rounds of self-renewing division, regenerating themselves while producing more tumor cells. Within normal tissues, a limitless self-renewal capacity is unique to the stem cells, which divide asymmetrically to produce more restricted progenitors. Accumulating evidence suggests that misregulation of the self-renewal machinery in stem cell progeny can lead to tumorigenesis, but how it influences the properties of the resulting tumors remains unclear. Studies of the type II neural stem cell (neuroblast) lineages in the Drosophila larval brain have identified a regulatory cascade that promotes commitment to a progenitor cell identity by restricting their response to the self-renewal machinery. Brain tumor (Brat) and Numb initiate this cascade by asymmetrically extinguishing the activity of the self-renewal factors. Subsequently, Earmuff (Erm) and the SWI/SNF complex stably restrict the competence of the progenitor cell to respond to reactivation of self-renewal mechanisms. Together, this cascade programs the progenitor cell to undergo limited rounds of division, generating exclusive differentiated progeny. Here we review how defects in this cascade lead to tumor initiation and how inhibiting the self-renewal mechanisms may be an effective strategy to block CSC expansion.

Immunofluorescent Staining of Drosophila Larval Brain Tissue

INTRODUCTION The Drosophila larval brain is a well-established model for investigating the role of stem cells in development. Neuroblasts (neural stem cells) must be competent to generate many thousands of differentiated neurons through asymmetric divisions during normal development. Studies in fly neuroblasts have been instrumental in identifying how the establishment and maintenance of cell polarity influence cell fate, and they have produced a wide array of molecular cell-polarity markers. Moreover, neuroblasts and their progeny can be positively identified using a variety of cell-fate markers. This article describes procedures for the collection and processing of Drosophila larval brains for examination by immunolocalization of cell-fate and cell-polarity markers. The protocol can be used for dissecting, fixing, and staining brains from larvae at any developmental stage. The number of brains processed using this method is limited only by how many brains can be dissected in 20 min, which is the maximum amount of time dissected tissues should remain in buffer before fixation. This protocol can be used for simultaneous costaining of multiple proteins.

Identification of neural stem cells in the Drosophila larval brain

The balance between self-renewal and differentiation must be tightly regulated in somatic stem cells to ensure proper tissue generation and to prevent tumorlike overgrowth. A Drosophila larval brain lobe consists of the central brain and the optic lobe and possesses three well-defined neural stem cell lineages that generate differentiated cells in a highly reproducible pattern. Unambiguous identification of various cell types in these stem cell lineages is pivotal for studying the regulation of neural stem cells and progenitor cells at a single-cell resolution. This chapter will describe the methodology for collection and processing of larval brains for examination by fluorescence confocal microscopy.

Multicolor fluorescence rna in situ hybridization of Drosophila brain tissue

INTRODUCTION RNA in situ hybridization is a useful method for determining the transcriptional expression pattern of a gene when antibodies are not available. Using this technique, it is possible to assay the expression of multiple RNA species using distinct labels on RNA probes, or simultaneously examine RNA and protein localization within larval tissues. This protocol describes RNA in situ hybridization of Drosophila brain tissue. It utilizes a fluorophore-conjugated tyramide that is easily made in the laboratory for a fraction of the cost of the commercially produced product.