Ronald R. Waclaw

Sonic Hedgehog Signaling Confers Ventral Telencephalic Progenitors with Distinct Cortical Interneuron Fates

Interneurons in the cerebral cortex regulate cortical functions through the actions of distinct subgroups that express parvalbumin, somatostatin, or calretinin. The genesis of the first two subgroups requires the expression of NKX2.1, which is maintained by SHH signaling during neurogenesis. In this paper, we report that mosaic elimination in the medial ganglionic eminence (MGE) of Smo, a key effector of SHH signaling, reveals that MGE progenitors retain a remarkable degree of plasticity during the neurogenic period. SHH signaling prevents the upregulation of GSX2 and conversion of some MGE progenitors to a caudal ganglionic eminence-like, bipolar calretinin-expressing cell fate that is promoted by GSX2. In addition, a higher level of SHH signaling promotes the generation of the somatostatin-expressing interneuron at the expense of parvalbumin-expressing subgroup. These results indicate that cortical interneuron diversity, a major determinant of cortical function, is critically influenced by differential levels of SHH signaling within the ventral telencephalon.

Sp8 is crucial for limb outgrowth and neuropore closure

In this report we describe the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation.

MELK-Dependent FOXM1 Phosphorylation is Essential for Proliferation of Glioma Stem Cells

Glioblastoma multiforme (GBM) is a life‐threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem‐like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase‐dependent manner. This MELK‐dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK‐driven FOXM1 activation is regulated by the binding and subsequent trans‐phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA‐mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK‐mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first‐line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC‐derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1‐dependent manner and thus, this protein complex is a potential therapeutic target for GBM. STEM Cells 2013;31:1051–1063

Distinct Temporal Requirements for the Homeobox Gene Gsx2 in Specifying Striatal and Olfactory Bulb Neuronal Fates

The homeobox gene Gsx2 (formerly Gsh2) is known to be required for striatal and olfactory bulb neurogenesis; however, its specific role in the specification of these two neuronal subtypes remains unclear. To address this, we have employed a temporally regulated gain-of-function approach in transgenic mice and found that misexpression of Gsx2 at early stages of telencephalic neurogenesis favors the specification of striatal projection neuron identity over that of olfactory bulb interneurons. In contrast, delayed activation of the Gsx2 transgene until later stages exclusively promotes olfactory bulb interneuron identity. In a complementary approach, we have conditionally inactivated Gsx2 in a temporally progressive manner. Unlike germline Gsx2 mutants, which exhibit severe alterations in both striatal and olfactory bulb neurogenesis at birth, the conditional mutants exhibited defects restricted to olfactory bulb interneurons. These results demonstrate that Gsx2 specifies striatal projection neuron and olfactory bulb interneuron identity at distinct time points during telencephalic neurogenesis.

Rac1 Controls the Formation of Midline Commissures and the Competency of Tangential Migration in Ventral Telencephalic Neurons

Previous studies using dominant-mutant constructs have implicated Rac1 GTPase in neuritogenesis and neuronal migration. However, overexpression of dominant mutants generally blocks Rho–GTPase activity; thus, it may not reveal the specific or physiological functions of Rac1. To address this issue, we have applied a conditional gene-targeting strategy, using Foxg1–Cre mice to delete Rac1 in the ventricular zone (VZ) of telencephalon and Dlx5/6–Cre–IRES (internal ribosomal entry site)–EGFP (enhanced green fluorescent protein) (Dlx5/6–CIE) in the subventricular zone (SVZ) of ventral telencephalon, respectively. Surprisingly, the deletion of Rac1 in VZ progenitors did not prevent axonal outgrowth of telencephalic neurons. However, the anterior commissure was absent, and the corpus callosal as well as hippocampal commissural axons failed to cross the midline in Rac1/Foxg1–Cre knock-out embryos. The thalamocortical and corticothalamic axons also showed defasciculation or projection defects. These results suggest that Rac1 controls axon guidance rather than neuritogenesis. In addition, although Rac1/Foxg1–Cre knock-out embryos showed delayed radial migration of cortical projection neurons and severe impairment of tangential migration by the ventral telencephalon-derived interneurons, deletion of Rac1 in the SVZ by Dlx5/6–CIE mice produced no discernible defects in tangential migration. These contrasting effects of Rac1 deletion on tangential migration suggest that Rac1 is dispensable for cellular motility per se during neuronal migration. Together, these results underscore the challenge of deciphering the biological functions of Rac1, and Rho–GTPases in general, during mammalian brain development. Moreover, they indicate that Rac1 has a critical role in axon guidance and in acquisition of migratory competency during differentiation of the progenitors for the ventral telencephalon-derived interneurons.

Developmental Origin of the Neuronal Subtypes That Comprise the Amygdalar Fear Circuit in the Mouse

We have taken a genetic-based fate-mapping approach to determine the specific contributions of telencephalic progenitors to the structures that comprise the amygdalar fear circuit including the central (CA), lateral (LA), and basolateral (BLA) amygdala. Our data indicate that progenitors in the ventral pallium (VP) contribute projection neurons to the LA and BLA but not the CA. Rather, the CA appears to derive, at least in part, from progenitors located in the ventral lateral ganglionic eminence (vLGE). Diverse groups of interneurons populate these amygdalar nuclei, and as predicted our data support the notion that they originate from subpallial progenitors. A rather specific population of amygdalar interneurons, the intercalated cells (ITCs), is known to play a fundamental role in fear-related behaviors. However, no information on their specific origin has, as yet, been provided. Our findings suggest that the ITCs arise from the dorsal lateral ganglionic eminence (dLGE) and migrate in the lateral migratory stream to populate the paracapsular regions as well as the main intercalated mass of the amygdala (IA). Germ-line Gsx2 mutants are known to exhibit an expansion of the VP into the LGE and a concomitant reduction in the dLGE and vLGE. Accordingly, Gsx2 conditional mutants display a significantly enlarged LA and a significant reduction in ITCs both within the paracapsular regions and the IA. Additional support for a dLGE origin of the ITCs was obtained in conditional mutants of the dLGE gene Sp8. Thus, our findings indicate diverse origins for the neuronal components that comprise the amygdalar fear circuit.

Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity.

In caudal regions of the diencephalon, sonic hedgehog (Shh) is expressed in the ventral midline of prosomeres 1-3 (p1-p3), which underlie the pretectum, thalamus and prethalamus, respectively. Shh is also expressed in the zona limitans intrathalamica (zli), a dorsally projecting spike that forms at the p2-p3 boundary. The presence of two Shh signaling centers in the thalamus has made it difficult to determine the specific roles of either one in regional patterning and neuronal fate specification. To investigate the requirement of Shh from a focal source of expression in the ventral midline of the diencephalon, we used a newly generated mouse line carrying a targeted deletion of the 525 bp intronic sequence mediating Shh brain enhancer-1 (SBE1) activity. In SBE1 mutant mice, Shh transcription was initiated but not maintained in the ventral midline of the rostral midbrain and caudal diencephalon, yet expression in the zli was unaffected. In the absence of ventral midline Shh, rostral thalamic progenitors (pTH-R) adopted the molecular profile of a more caudal thalamic subtype (pTH-C). Surprisingly, despite their early mis-specification, neurons derived from the pTH-R domain continued to migrate to their proper thalamic nucleus, extended axons along their normal trajectory and expressed some, but not all, of their terminal differentiation markers. Our results, and those of others, suggest a model whereby Shh signaling from distinct spatial and temporal domains in the diencephalon exhibits unique and overlapping functions in the development of discrete classes of thalamic interneurons.

Gsx2 controls region-specific activation of neural stem cells and injury-induced neurogenesis in the adult subventricular zone

Neural stem cells (NSCs) reside in widespread regions along the lateral ventricle and generate diverse olfactory bulb (OB) interneuron subtypes in the adult mouse brain. Molecular mechanisms underlying their regional diversity, however, are not well understood. Here we show that the homeodomain transcription factor Gsx2 plays a crucial role in the region-specific control of adult NSCs in both persistent and injury-induced neurogenesis. In the intact brain, Gsx2 is expressed in a regionally restricted subset of NSCs and promotes the activation and lineage progression of stem cells, thereby controlling the production of selective OB neuron subtypes. Moreover, Gsx2 is ectopically induced in damaged brains outside its normal expression domains and is required for injury-induced neurogenesis in the subventricular zone (SVZ). These results demonstrate that mobilization of adult NSCs is controlled in a region-specific manner and that distinct mechanisms operate in continuous and injury-induced neurogenesis in the adult brain.

Ascl1 is a required downstream effector of Gsx gene function in the embryonic mouse telencephalon.

BackgroundThe homeobox gene Gsx2 (formerly Gsh2) is known to regulate patterning in the lateral ganglionic eminence (LGE) of the embryonic telencephalon. In its absence, the closely related gene Gsx1 (previously known as Gsh1) can partially compensate in the patterning and differentiation of ventral telencephalic structures, such as the striatum. However, the cellular and molecular mechanisms underlying this compensation remain unclear.ResultsWe show here that in the Gsx2 mutants Gsx1 is expressed in only a subset of the ventral telencephalic progenitors that normally express Gsx2. Based on the similarities in the expression of Gsx1 and Ascl1 (Mash1) within the Gsx2 mutant LGE, we examined whether Ascl1 plays an integral part in the Gsx1-based recovery. Ascl1 mutants show only modest alterations in striatal development; however, in Gsx2;Ascl1 double mutants, striatal development is severely affected, similar to that seen in the Gsx1;Gsx2 double mutants. This is despite the fact that Gsx1 is expressed, and even expands, in the Gsx2;Ascl1 mutant LGE, comparable to that seen in the Gsx2 mutant. Finally, Notch signaling has recently been suggested to be required for normal striatal development. In spite of the fact that Notch signaling is severely disrupted in Ascl1 mutants, it actually appears to be improved in the Gsx2;Ascl1 double mutants.ConclusionThese results, therefore, reveal a non-proneural requirement of Ascl1 that together with Gsx1 compensates for the loss of Gsx2 in a subset of LGE progenitors.

The homeobox gene Gsh2 is required for retinoid production in the embryonic mouse telencephalon.

We have examined the role of the homeobox gene Gsh2 in retinoid production and signaling within the ventral telencephalon of mouse embryos. Gsh2 mutants exhibit altered ventral telencephalic development, including a smaller striatum with fewer DARPP-32 neurons than wild types. We show that the expression of the retinoic acid (RA) synthesis enzyme, retinaldehyde dehydrogenase 3 (Raldh3, also known as Aldh1a3), is reduced in the lateral ganglionic eminence (LGE) of Gsh2 mutants. Moreover, using a retinoid reporter cell assay, we found that retinoid production in the Gsh2 mutants is markedly reduced. The striatal defects in Gsh2 mutants are thought to result from ectopic expression of Pax6 in the LGE. Previously, we had shown that removal of Pax6 from the Gsh2 mutant background improves the molecular identity of the LGE in these double mutants; however, Raldh3 expression is not improved. The Pax6;Gsh2 double mutants possess a larger striatum than the Gsh2 mutants, but the disproportionate reduction in DARPP-32 neurons is not improved. These findings suggest that reduced retinoid production in the Gsh2 mutant contributes to the striatal differentiation defects. As RA promotes the expression of DARPP-32 in differentiating LGE cells in vitro, we examined whether exogenous RA can improve striatal neuron differentiation in the Gsh2 mutants. Indeed, RA supplementation of Gsh2 mutants, during the period of striatal neurogenesis, results in a significant increase in DARPP-32 expression. Thus, in addition to the previously described role for Gsh2 to maintain correct molecular identity in the LGE, our results demonstrate a novel requirement of this gene for retinoid production within the ventral telencephalon.