Alexandra Joyner, PhD

The mammalian brain is an extremely complex structure both anatomically and functionally, that simultaneously integrates multiple inputs to produce an appropriate response (Sillitoe, 2007; Joyner & Bayin, 2022). Our studies are aimed at bringing together studies of neural development with analysis of the final circuitry and function of the adult brain. The neurons responsible for each circuit in the brain have a specific spatial organization that is optimized not only for the function of individual circuits, but also to allow long range circuits to interact with precision. How neurons become organized in 3D during development is a critical unanswered question. We have two main projects. In one we are identifying the signals that regulate scaling of the numbers of different cerebellar neurons during development to generate robust circuits, particularly those that communicate with the neocortex. In the second project we are exploring the degree to which the brain can be repaired after neuron loss and identify factors that can stimulate stem cells to regenerate neurons.

The cerebellum: complex morphogenesis yet simple circuitry

The cerebellum, consisting of 80% of the neurons in the human brain, is critical for skilled motor performance, and also participates in cognitive and social functions. As the cerebellum is the only brain region in rodents with a foliated structure (Fig. 1A), one aspect of our research is to identify cellular and genetic events that regulate the highly stereotyped process of foliation, or gyrification seen in the human neocortex (Sgaier, 2005; Sudarov, 2007; Legué, 2015, 2016; Lawton 2019). At the cellular level, the cerebellum consists of a dozen major cell types organized into a layered folded cortex with 3 pairs of cerebellar nuclei (CN) at its base (Fig. 1B). The developing cerebellum is particularly vulnerable to clinical and environmental factors, since much of growth in humans occurs in the third trimester and continues for a year after birth (Wang, 2014), and for three weeks after birth in mouse (Movie 1). Unlike other brain regions, the cerebellum has multiple progenitor zones (Leto, 2015; Joyner & Bayin, 2022)(Fig. 1C-E). The ventricular zone gives rise to all the GABAergic (inhibitory) cells including Purkinje cells, whereas the glutamatergic (excitatory) neurons, including the output neurons of the cerebellar nuclei and granule cells, are derived from the upper rhombic lip. Unique to the cerebellum, interneurons and astrocytes are generated from Nestin-expressing progenitors (NEPs) that leave the ventricular zone and continue to proliferate in the cortex after birth. The rhombic lip first generates neurons of the cerebellar nuclei at embryonic day (E) 10-13 in mouse, and then granule cell progenitors that migrate over the surface of the cerebellum to form a transient amplifying stem cell population of granule cell progenitors that undergo tremendous expansion of cell numbers for two weeks in mouse through symmetric cell divisions (Legué, 2015). The Purkinje cells express sonic hedgehog (SHH), a mitogen necessary for granule cell precursor expansion and also for expansion NEPs and production of interneurons and glia. Purkinje cells also integrate all the incoming information to the cerebellum, modulated by granule cells and interneurons, and relay the information to the cerebellar nuclei neurons that instruct the rest of the brain to modify behaviors. The Purkinje cells thus act as a critical hub that developmentally scales production of all the cells in the cerebellar cortex and functionally links all the cells of local and long-range circuits.

Principals underlying proportional scaling of neurons

In one project, we have utilized the genes encoding the engrailed homeobox transcription factors (En1 and En2) as an entry point to study cerebellar growth, circuit formation and patterning, as conditional mutants have defects in all these processes (Joyner, 1991; Sillitoe, 2008, 2010; Joyner, 1991; Sgaier, 2007; Orvis, 2012; Cheng, 2010; Legué, 2016). Our current studies are based on our finding that while growth of particular folds are reduced in En1/2 mutants, the basic cytoarchitecture and scaling of different neurons is normal (Fig. 2). Consequently, there are regionally defined reductions in the number of cerebellar nuclei neurons and Purkinje cells before birth, and a subsequent reduction in SHH signaling and generation of granule cells (Willett, 2019). We are currently testing whether experimental ablation of particular subsets of cerebellar nuclei neurons results in loss of circuit-related Purkinje cells, whether neurotrophic factors expressed by cerebellar nuclei underlie the Purkinje cell phenotype, and defining the circuits linking the cerebellum and neocortex, and are generating mice expressing tagged-EN2 protein for molecular studies to identify key target genes. We also have developed a method for live imaging of fluorescently labeled cells in slices of the developing cerebellum. In one set of experiments, we are using the approach to study aspects of proliferation and migration of granule cell precursors that underlie foliation (Movie 2). Our long-term goal is to understand how cerebellar circuits regulate neocortex initiated behaviors. Since sequence changes in human EN2 have been linked to autism (Benayed, 2005), and En2 mutant mice have deficits in social behaviors (Brielmaier, 2012; Crawley, 2012), a better understanding of how the En1/2 genes regulate cerebellum development and modulate behaviors should provide insight into human cerebellar malformation syndromes with gross morphological changes, as well as diseases such as autism that could have significant cerebrocerebellar circuit dysfunction.

Adaptive reprogramming of Nestin-expressing stem cells during regeneration

In a second project, we have uncovered that the mouse neonatal cerebellum has a large capacity to regenerate neurons killed around birth. In one study we demonstrated that Purkinje cells killed a day after birth can be replaced by proliferation of a rare population of immature Purkinje cells (Bayin, 2018). In a second series of studies we showed that ventricular zone-derived cells expressing the stem cell marker Nestin in the cerebellar cortex have the ability to change their normal fate (glia) and become rhombic lip-derived granule cells in response to injury (Wojcinski, 2017; Wojcinski, 2019; Yang & Joyner, 2019). Using genetic ablation (Cre/loxP) or irradiation of the early postnatal mouse brain to eliminate most of the granule cell precursors, we found a remarkable regeneration of the granule cells. Moreover, using genetic labeling and fate mapping (with FlpoER; see Fig. 4) we identified Nestin-expressing progenitors (NEPs) in the cortex that rapidly respond to the injury by expanding, migrating to the granule cell progenitor niche and adopting a granule cell molecular profile. We developed a method for live imaging of fluorescently labeled cells in slices of the developing cerebellum and used the approach to study aspects of proliferation and migration of granule cell precursors (Movie 2) and to image fluorescently labeled-NEPs to prove that NEPs migrate to the granule cell progenitor layer (Movie 3). A second population of NEPs responds to the injury by delaying production of interneurons and astrocytes, thus maintaining circuit cell proportions, and rescuing motor behaviors. Most recently, single cell sequencing (scRNA-seq) was used to identify genes that underlie adaptive reprogramming of NEPs. We uncovered that the neurogenic transcription factor ASCl1 must be upregulated by gliogenic NEPs for them to change their fate to neurons (Bayin, 2021). Additional projects include determining whether NEPs in the adult cerebellum can be stimulated to replenish cells lost due to injury and testing the requirement of ROS signaling for driving adaptive reprogramming.

Videos

• MRI surface rendering of developing mouse brain viewed from the back showing how the cerebellum folds and grows from 1 to 11 days after birth (adapted from Szulc, 2015).

• Live imaging of a cerebellar slice from an E18.5 Atoh1-CreER; R26LSL-tdTom mouse given tamoxifen two days earlier showing movement of fluorescently labeled granule cell precursors (green) in the external granule cell layer (EGL). The white line marks the outer edge of the cerebellum. Images taken every 3 minutes for 1.5 hours. See Figure 4 for explanation of fate mapping using R26LSL-tdTom mice and tamoxifen.

• Live imaging of a cerebellar slice from a P6 Nestin-Cfp/+ transgenic mouse. Cyan florescent protein (CFP; white) labels the cytoplasm of NEPs. A NEP in the Purkinje cell layer is marked with a blue dot and migrates to the external granule cell layer (EGL) and undergoes cell division during adaptive reprogramming following ablation of granule cell precursors in the EGL. Normal NEPs stay in the Purkinje cell layer (Wojcinski, 2017). The green line indicates outer edge of the cerebellum (under the EGL). The magenta line indicates outer edge of the Purkinje cell layer where the NEPs reside. Images taken every 3 minutes for 4.5 hours.

A critical aspect of growth control is to ensue that all organs attain sizes that have the correct proportions with other organs. The developmental signaling pathways and cellular processes that control organ size also can be involved in recover from injury (Roselló-Díez, 2015). Furthermore, during evolution the degree to which these mechanisms are active has likely changed and account for the distinct body sizes and organ proportions observed in different species. On the other hand, when growth-signaling pathways are unchecked, cancers can develop. We recently developed a novel model to study growth regulation pathways using the bilateral limbs as an example, as by altering growth of only the left limb the right limb serves as an internal control (Roselló-Díez, 2017, 2018; Ahmadzadeh, 2020). We have also studied regeneration and cancer of the prostate (Peng, 2012; Yang, 2017). Currently, we have two main projects focused on determining the roles of SHH-signaling in cerebellar growth regulation and cancer.

Sonic hedgehog regulates developmental growth and adult stem cell behaviors

The secreted factor sonic hedgehog (SHH) that mediates signaling through three GLI transcription factors (Fig. 3) is critical for development of most mammalian organs and maintains the pluripotent state of several adult stem cell populations. Human mutations that reduce SHH signaling cause a variety of developmental defects, whereas inappropriate activation of signaling contributes to cancer (Petrova, 2014; Peng, 2015). To study the in vivo functions of the GLI transcription factors, we generated an array of mouse targeted conditional and knock-in alleles. By analyzing the embryonic phenotypes of our mutants in the limb and nervous system, we uncovered a universal rule for GLI function: HH signaling modifies GLI2 to form an activator and GLI3 to attenuate formation of a repressor (Fig. 3). Moreover, high level SHH signaling induces transcription of Gli1 (Bai & Joyner, 2001; Bai, 2002, 2004). We have taken advantage of Gli1 being a HH target gene to identify cells undergoing high HH signaling, and follow the fate of their progeny using genetic inducible fate mapping (Fig. 4; Joyner, 2006). In particular, we used our Gli1^CreER allele to identify and fate map stem cells in a variety of adult tissues (forebrain, skin and prostate) (Ahn, 2004, 2005; Brownell, 2011; Peng, 2012). For example, we discovered that in the skin, SHH is secreted by peripheral nerves, and the nerves are required to maintain hair follicle bulge stem cells in a plastic state, able to be reprogrammed into interfollicular skin stem cells (Brownell, 2011). Other groups have since found nerves are a critical source of HH ligands in several adult organs.

SHH coordinates expansion of multiple cerebellar progenitors

In the cerebellum, SHH drives the tremendous expansion of progenitors after birth in mouse that is responsible for folding of the cerebellar surface (Fig. 1D and Movie 1). Accordingly, we found that ablation of SHH signaling results in a hypoplastic cerebellum, as SHH is required for expansion of granule cell progenitors, the most numerous cell type in the brain (Corrales, 2004, 2006). On the other hand, SHH signaling is elevated in the SHH subgroup of medulloblastoma for which the cerebellar granule cell progenitors, which proliferate for almost 1 year after birth in man, are the main cell of origin. SHH also regulates expansion of ventricular zone-derived progenitors (NEPs) that give rise to interneurons and astrocytes. Thus, SHH is a key growth regulator of the postnatal cerebellum. One aspect of our research addresses how SHH coordinates proliferation and differentiation of several distinct progenitor populations in the cerebellar cortex. Using our novel mosaic mutant analysis technique (MASTR; Lao, 2012), we uncovered that a primary role of SHH in NEPs is to maintain them in an undifferentiated state (Wojcinski, 2017, 2018; Tan, 2018), similar to our finding for adult stem cells in the subventricular zone of the forebrain (Petrova, 2013). Similar studies are underway for the other progenitor populations.

Timing of and type of initiating mutation stratify SHH-medulloblastomas

Medulloblastoma (MB) is the most common malignant brain tumor in children. Although the majority of children with MB survive to adulthood, the quality of life of survivors is greatly compromised due to the toxicity of combined surgery, radiation, and chemotherapy. MB is a heterogeneous disease that was divided into 4 subgroups based on their molecular signatures. As the current SHH inhibitors in the clinic are only effective in a minority of SHH-MB patients and all patients develop resistance, new drug targets are needed to improve survival and minimize the significant side effects of current therapies. A recent study further subdivided SHH-MB into 3 subtypes based on age of presentation, as particular genetic lesions are specific to infants (SUFU), children (TP53 associated with amplification of GLI2/MYCN) and adults (SMO)(Kool, 2012; and see Fig. 3). However, patients of all ages have mutations in the Gorlin syndrome gene PTCH1. The most recent classification of MB divides SHH-MB into 4 subtypes, with TP53 mutations defining a group with poor prognosis (Cavalli, 2014). We have developed mouse models of SHH-MB (Suero-Abreu, 2015; Tan, 2018), including using MASTR to initiate mutations in SMO or PTCH1, with or without mutations in Trp53, and identified differences in the biology of the tumors. Microarray analysis of genes expressed in GFP-labeled mutant granule cell precursors that progress to form tumors, compared to those that regress or wild type cells have identified candidate genes that underlie tumor progression. We have teamed up with leading experts in the genetics and pathology of human MBs, to determine the extent to which human tumors with the same mutations have similar tumor characteristics (Vladoiu, 2019). In addition, we are using functional assays in granule cell precursors with MB activating mutations to determine which of the genes identified are required for tumor formation and/or progression, with the goal of identifying candidates for novel therapies specific to SHH-MB subtype-specific patients. We are also using similar approaches to determine the role of immune cells in the microenvironment of mouse and human SHH-MBs on tumor progression (Rallapalli, 2020; Tan, 2021). Our studies should uncover which mice best model particular subtypes of human SHH-MBs for preclinical testing, and contribute to stratification of patients for targeted therapy.