Dendritic Inhibition by Shh Signaling-Dependent Stellate Cell Pool Is Critical for Motor Learning.

Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and nonmotor functions. The molecular layer interneurons (MLIs), consisting of basket cells (BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2+ immature interneurons, which migrate from the prospective white matter to the ML of the cortex. However, little is known about how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Sonic hedgehog (Shh) signal. Conditional abrogation of Shh signaling of either sex inhibited proliferation of GABAergic progenitors and reduced the number of Pax2+ cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2+ cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.SIGNIFICANCE STATEMENT The cerebellar circuit that enables fine motor learning involves MLIs of BCs and SCs, which provide dendritic and somatic inhibitory synapses onto Purkinje cells. Little is known about how their identities and numbers are determined, nor are their specific contributions to motor learning well understood. We show that MLI subtypes are specified independent of Shh signaling and their birth orders but appear to occur in their terminal laminar positions according to the inside-out sequence. This finding challenges the current view that MLI subtypes are specified sequentially at the progenitor level. We also demonstrate that dendritic inhibition by Shh signaling-dependent SC pool is necessary for motor learning.

Insight into the Etiology of Undifferentiated Soft Tissue Sarcomas from a Novel Mouse Model.

Aberrant activation of the Hedgehog signaling pathway has been linked to the formation of numerous cancer types, including the myogenic soft tissue sarcoma, embryonal rhabdomyosarcoma (eRMS). Here, we report PCG2, a novel mouse model in which human GLI2A, a constitutive activator of Hedgehog signaling, induced undifferentiated sarcomas that were phenotypically divergent from eRMS. Rather, sarcomas arising in PCG2 mice featured some characteristics that were reminiscent of Ewing sarcoma. Even though it is widely understood that Ewing sarcoma formation is driven by EWS-ETS gene fusions, a genetically defined mouse model is not well-established. While EWS-ETS gene fusions were not present in PCG2 sarcomas, precluding their designation as Ewing sarcoma, we did find that GLI2A induced expression of known EWS-ETS gene targets essential to Ewing pathogenesis, most notably, Nkx2.2. Moreover, we found that naïve mesenchymal progenitors originate tumors in PCG2 mice. Altogether, our work provides a novel genetic mouse model, which directly connects oncogenic Hedgehog activity to the etiology of undifferentiated soft tissue sarcomas for the first time. IMPLICATIONS: The finding that activation of Gli2 transcription factor is sufficient to induce Ewing-like sarcomas provides a direct transformative role of the Hedgehog signaling pathway in undifferentiated soft tissue sarcoma.

Bergmann glial Sonic hedgehog signaling activity is required for proper cerebellar cortical expansion and architecture.

Neuronal-glial relationships play a critical role in the maintenance of central nervous system architecture and neuronal specification. A deeper understanding of these relationships can elucidate cellular cross-talk capable of sustaining proper development of neural tissues. In the cerebellum, cerebellar granule neuron precursors (CGNPs) proliferate in response to Purkinje neuron-derived Sonic hedgehog (Shh) before ultimately exiting the cell cycle and migrating radially along Bergmann glial fibers. However, the function of Bergmann glia in CGNP proliferation remains not well defined. Interestingly, the Hh pathway is also activated in Bergmann glia, but the role of Shh signaling in these cells is unknown. In this study, we show that specific ablation of Shh signaling using the tamoxifen-inducible TNCYFP-CreER line to eliminate Shh pathway activator Smoothened in Bergmann glia is sufficient to cause severe cerebellar hypoplasia and a significant reduction in CGNP proliferation. TNCYFP-CreER; SmoF/- (SmoCKO) mice demonstrate an obvious reduction in cerebellar size within two days of ablation of Shh signaling. Mutant cerebella have severely reduced proliferation and increased differentiation of CGNPs due to a significant decrease in Shh activity and concomitant activation of Wnt signaling in SmoCKO CGNPs, suggesting that this pathway is involved in cross-talk with the Shh pathway in regulating CGNP proliferation. In addition, Purkinje cells are ectopically located, their dendrites stunted, and the Bergmann glial network disorganized. Collectively, these data demonstrate a previously unappreciated role for Bergmann glial Shh signaling activity in the proliferation of CGNPs and proper maintenance of cerebellar architecture.

Lkb1 regulates granule cell migration and cortical folding of the cerebellar cortex.

Cerebellar growth and foliation require the Hedgehog-driven proliferation of granule cell precursors (GCPs) in the external granule layer (EGL). However, that increased or extended GCP proliferation generally does not elicit ectopic folds suggests that additional determinants control cortical expansion and foliation during cerebellar development. Here, we find that genetic loss of the serine-threonine kinase Liver Kinase B1 (Lkb1) in GCPs increased cerebellar cortical size and foliation independent of changes in proliferation or Hedgehog signaling. This finding is unexpected given that Lkb1 has previously shown to be critical for Hedgehog pathway activation in cultured cells. Consistent with unchanged proliferation rate of GCPs, the cortical expansion of Lkb1 mutants is accompanied by thinning of the EGL. The plane of cell division, which has been implicated in diverse processes from epithelial surface expansions to gyrification of the human cortex, remains unchanged in the mutants when compared to wild-type controls. However, we find that Lkb1 mutants display delayed radial migration of post-mitotic GCPs that coincides with increased cortical size, suggesting that aberrant cell migration may contribute to the cortical expansion and increase foliation. Taken together, our results reveal an important role for Lkb1 in regulating cerebellar cortical size and foliation in a Hedgehog-independent manner.

BET bromodomain inhibitors suppress EWS-FLI1-dependent transcription and the IGF1 autocrine mechanism in Ewing sarcoma.

Ewing sarcoma is driven by characteristic chromosomal translocations between the EWSR1 gene with genes encoding ETS family transcription factors (EWS-ETS), most commonly FLI1. However, direct pharmacological inhibition of transcription factors like EWS-FLI1 remains largely unsuccessful. Active gene transcription requires orchestrated actions of many epigenetic regulators, such as the bromodomain and extra-terminal domain (BET) family proteins. Emerging BET bromodomain inhibitors have exhibited promising antineoplastic activities via suppression of oncogenic transcription factors in various cancers. We reasoned that EWS-FLI1-mediated transcription activation might be susceptible to BET inhibition. In this study, we demonstrated that small molecule BET bromodomain inhibitors repressed EWS-FLI1-driven gene signatures and downregulated important target genes. However, expression of EWS-FLI1 was not significantly affected. Repression of autocrine IGF1 by BET inhibitors led to significant inhibition of the IGF1R/AKT pathway critical to Ewing sarcoma cell proliferation and survival. Consistently, BET inhibitors impaired viability and clonogenic survival of Ewing sarcoma cell lines and blocked EWS-FLI1-induced transformation of mouse NIH3T3 fibroblast cells. Selective depletion of individual BET genes partially phenocopied the actions of BET inhibitors. Finally, the prototypical BET inhibitor, JQ1, significantly repressed Ewing sarcoma xenograft tumor growth. These findings suggest therapeutic potential of BET inhibitors in Ewing sarcoma and highlight an emerging paradigm of using epigenetic agents to treat cancers driven by fusion transcription factors.

An in vivo chemical genetic screen identifies phosphodiesterase 4 as a pharmacological target for hedgehog signaling inhibition.

Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been accepted widely as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain-of-function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen, which induced an Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase 4 (PDE4), leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy, and identifies a unique probe of downstream-of-Smo Hh modulation.

The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors.

The prospective white matter (PWM) in the nascent cerebellum contains a transient germinal compartment that produces all postnatally born GABAergic inhibitory interneurons and astrocytes. However, little is known about the molecular identity and developmental potential of resident progenitors or key regulatory niche signals. Here, we show that neural stem-cell-like primary progenitors (Tnc(YFP-low) CD133(+)) generate intermediate astrocyte (Tnc(YFP-low) CD15(+)) precursors and GABAergic transient amplifying (Ptf1a(+)) cells. Interestingly, these lineally related but functionally divergent progenitors commonly respond to Sonic hedgehog (Shh), and blockade of reception in TNC(YFP-low) cells attenuates proliferation in the PWM, reducing both intermediate progenitor classes. Furthermore, we show that Shh produced from distant Purkinje neurons maintains the PWM niche independently of its classical role in regulating granule cell precursor proliferation. Our results indicate that Purkinje neurons maintain a bidirectional signaling axis, driving the production of spatially and functionally opposed inhibitory and excitatory interneurons important for motor learning and cognition.

Expression and localization of myosin-1d in the developing nervous system.

Myosin-1d is a monomeric actin-based motor found in a wide range of tissues, but highly expressed in the nervous system. Previous microarray studies suggest that myosin-1d is found in oligodendrocytes where transcripts are upregulated during the maturation of these cells. Myosin-1d was also identified as a component of myelin-containing subcellular fractions in proteomic studies and mutations in MYO1D have been linked to autism. Despite the potential implications of these previous studies, there is little information on the expression and localization of myosin-1d in the developing nervous system. Therefore, we analyzed myosin-1d expression patterns in the peripheral and central nervous systems during postnatal development. In mouse sciatic nerve, myosin-1d is expressed along the axon and in the ensheathing myelin compartment. Analysis of mouse cerebellum prior to myelination at day 3 reveals that myosin-1d is present in the Purkinje cell layer, granule cell layer, and region of the cerebellar nuclei. Upon the onset of myelination, myosin-1d enrichment expands along axonal tracts, while still present in the Purkinje and granule cell layers. However, myosin-1d was undetectable in oligodendrocyte progenitor cells at early and late time points. We also show that myosin-1d interacts and is co-expressed with aspartoacylase, an enzyme that plays a key role in fatty acid synthesis throughout the nervous system. Together, these studies provide a foundation for understanding the role of myosin-1d in neurodevelopment and neurological disorders.

Transventricular delivery of Sonic hedgehog is essential to cerebellar ventricular zone development.

Cerebellar neurons are generated from two germinal neuroepithelia: the ventricular zone (VZ) and rhombic lip. Signaling mechanisms that maintain the proliferative capacity of VZ resident progenitors remain elusive. We reveal that Sonic hedgehog (Shh) signaling is active in the cerebellar VZ and essential to radial glial cell proliferation and expansion of GABAergic interneurons. We demonstrate that the cerebellum is not the source of Shh that signals to the early VZ, and suggest a transventricular path for Shh ligand delivery. In agreement, we detected the presence of Shh protein in the circulating embryonic cerebrospinal fluid. This study identifies Shh as an essential proliferative signal for the cerebellar ventricular germinal zone, underscoring the potential contribution of VZ progenitors in the pathogenesis of cerebellar diseases associated with deregulated Shh signaling, and reveals a transventricular source of Shh in regulating neural development.

Sonic hedgehog signaling regulates a novel epithelial progenitor domain of the hindbrain choroid plexus.

Choroid plexuses (ChPs) are vascularized secretory organs involved in the regulation of brain homeostasis, and function as the blood-cerebrospinal fluid (CSF) barrier. Despite their crucial roles, there is limited understanding of the regulatory mechanism driving ChP development. Sonic hedgehog (Shh), a secreted signal crucial for embryonic development and cancer, is strongly expressed in the differentiated hindbrain ChP epithelium (hChPe). However, we identify a distinct epithelial domain in the hChP that does not express Shh, but displays Shh signaling. We find that this distinct Shh target field that adjoins a germinal zone, the lower rhombic lip (LRL), functions as a progenitor domain by contributing directly to the hChPe. By conditional Shh mutant analysis, we show that Shh signaling regulates hChPe progenitor proliferation and hChPe expansion through late embryonic development, starting around E12.5. Whereas previous studies show that direct contribution to the hChPe by the LRL ceases around E14, our findings reveal a novel tissue-autonomous role for Shh production and signaling in driving the continual growth and expansion of the hindbrain choroid plexus throughout development.