Vitor H. Sousa

Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons

By combining an inducible genetic fate mapping strategy with electrophysiological analysis, we have systematically characterized the populations of cortical GABAergic interneurons that originate from the caudal ganglionic eminence (CGE). Interestingly, compared with medial ganglionic eminence (MGE)-derived cortical interneuron populations, the initiation [embryonic day 12.5 (E12.5)] and peak production (E16.5) of interneurons from this embryonic structure occurs 3 d later in development. Moreover, unlike either pyramidal cells or MGE-derived cortical interneurons, CGE-derived interneurons do not integrate into the cortex in an inside-out manner but preferentially (75%) occupy superficial cortical layers independent of birthdate. In contrast to previous estimates, CGE-derived interneurons are both considerably greater in number (∼30% of all cortical interneurons) and diversity (comprised by at least nine distinct subtypes). Furthermore, we found that a large proportion of CGE-derived interneurons, including the neurogliaform subtype, express the glycoprotein Reelin. In fact, most CGE-derived cortical interneurons express either Reelin or vasoactive intestinal polypeptide. Thus, in conjunction with previous studies, we have now determined the spatial and temporal origins of the vast majority of cortical interneuron subtypes.

The Requirement of Nkx2-1 in the Temporal Specification of Cortical Interneuron Subtypes

Previous work has demonstrated that the character of mouse cortical interneuron subtypes can be directly related to their embryonic temporal and spatial origins. The relationship between embryonic origin and the character of mature interneurons is likely reflected by the developmental expression of genes that direct cell fate. However, a thorough understanding of the early genetic events that specify subtype identity has been hampered by the perinatal lethality resulting from the loss of genes implicated in the determination of cortical interneurons. Here, we employ a conditional loss-of-function approach to demonstrate that the transcription factor Nkx2-1 is required for the proper specification of specific interneuron subtypes. Removal of this gene at distinct neurogenic time points results in a switch in the subtypes of neurons observed at more mature ages. Our strategy reveals a causal link between the embryonic genetic specification by Nkx2-1 in progenitors and the functional attributes of their neuronal progeny in the mature nervous system.

Characterization of Nkx6-2-Derived Neocortical Interneuron Lineages

Ventral telencephalic progenitors expressing the homeodomain transcription factor Nkx6-2 have been shown to give rise to a multitude of cortical interneuron subtypes usually associated with origin in either the medial ganglionic eminence or the caudal ganglionic eminence. The function of Nkx6-2 in directing the fate of those progenitors has, however, not been thoroughly analyzed. We used a combination of genetic inducible fate mapping and in vivo loss-of-function to analyze the requirement of Nkx6-2 in determining the fate of cortical interneurons. We have found that interneuron subtypes are born with a characteristic temporal pattern. Furthermore, we extend the characterization of interneurons from the Nkx6-2 lineage through the application of electrophysiological methods. Analysis of these populations in Nkx6-2 null mice suggests that there is a small and partially penetrant loss of delayed non-fast spiking somatostatin/calretinin double positive cortical interneurons in the absence of Nkx6-2 gene function.

Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis

Nephronophthisis (NPHP), an autosomal recessive kidney disease, is the most frequent genetic cause of end-stage renal failure in the first three decades of life. Positional cloning of the six known NPHP genes has linked its pathogenesis to primary cilia function. Here we identify mutation of GLIS2 as causing an NPHP-like phenotype in humans and mice, using positional cloning and mouse transgenics, respectively. Kidneys of Glis2 mutant mice show severe renal atrophy and fibrosis starting at 8 weeks of age. Differential gene expression studies on Glis2 mutant kidneys demonstrate that genes promoting epithelial-to-mesenchymal transition and fibrosis are upregulated in the absence of Glis2. Thus, we identify Glis2 as a transcription factor mutated in NPHP and demonstrate its essential role for the maintenance of renal tissue architecture through prevention of apoptosis and fibrosis.

Sonic hedgehog functions through dynamic changes in temporal competence in the developing forebrain.

Morphogens act during development to provide graded spatial information that controls patterning and cell lineage specification in the nervous system. The role of morphogen signaling in instructing the expression of downstream effector transcription factors has been well established. However, a key requirement for morphogen signaling is the existence of functional intracellular machinery able to mediate the appropriate response in target cells. Here we suggest that dynamic changes in the temporal responses to Shh in the developing ventral telencephalon occur through alterations in progenitor competence. We suggest these developmental changes in competence are mediated by a transcriptional mechanism that intrinsically integrates information from the distinct signaling pathways that act to pattern the telencephalic neuroepithelium.

Emx1-Lineage Progenitors Differentially Contribute to Neural Diversity in the Striatum and Amygdala

In the developing mammalian basal telencephalon, neural progenitors from the subpallium generate the majority of inhibitory medium spiny neurons (MSNs) in the striatum, while both pallial- and subpallial-derived progenitors contribute to excitatory and inhibitory neuronal diversity in the amygdala. Using a combination of approaches, including genetic fate mapping, cell birth dating, cell migration assays, and electrophysiology, we find that cells derived from the Emx1 lineage contribute to two distinct neuronal populations in the mature basal forebrain: inhibitory MSNs in the striatum and functionally distinct subclasses of excitatory neurons in the amygdala. Our cell birth-dating studies reveal that these two populations are born at different times during early neurogenesis, with the amygdala population born before the MSNs. In the striatum, Emx1-lineage neurons represent a unique subpopulation of MSNs: they are disproportionately localized to the dorsal striatum, are found in dopamine receiving, reelin-positive patches, and are born throughout striatal neurogenesis. In addition, our data suggest that a subpopulation of these Emx1-lineage cells originate in the pallium and subsequently migrate to the developing striatum and amygdala. Our intersectional fate-mapping analysis further reveals that Emx1-lineage cells that coexpress Dlx exclusively generate MSNs but do not contribute to the excitatory neurons in the amygdala. Thus, both the timing of neurogenesis and differential combinatorial gene expression appear to be key determinants of striatal versus amygdala fate decisions of Emx1-lineage cells.

Sonic hedgehog expressing and responding cells generate neuronal diversity in the medial amygdala

BackgroundThe mammalian amygdala is composed of two primary functional subdivisions, classified according to whether the major output projection of each nucleus is excitatory or inhibitory. The posterior dorsal and ventral subdivisions of the medial amygdala, which primarily contain inhibitory output neurons, modulate specific aspects of innate socio-sexual and aggressive behaviors. However, the development of the neuronal diversity of this complex and important structure remains to be fully elucidated.ResultsUsing a combination of genetic fate-mapping and loss-of-function analyses, we examined the contribution and function of Sonic hedgehog (Shh)-expressing and Shh-responsive (Nkx2-1+ and Gli1+) neurons in the medial amygdala. Specifically, we found that Shh- and Nkx2-1-lineage cells contribute differentially to the dorsal and ventral subdivisions of the postnatal medial amygdala. These Shh- and Nkx2-1-lineage neurons express overlapping and non-overlapping inhibitory neuronal markers, such as Calbindin, FoxP2, nNOS and Somatostatin, revealing diverse fate contributions in discrete medial amygdala nuclear subdivisions. Electrophysiological analysis of the Shh-derived neurons additionally reveals an important functional diversity within this lineage in the medial amygdala. Moreover, inducible Gli1CreER(T2) temporal fate mapping shows that early-generated progenitors that respond to Shh signaling also contribute to medial amygdala neuronal diversity. Lastly, analysis of Nkx2-1 mutant mice demonstrates a genetic requirement for Nkx2-1 in inhibitory neuronal specification in the medial amygdala distinct from the requirement for Nkx2-1 in cerebral cortical development.ConclusionsTaken together, these data reveal a differential contribution of Shh-expressing and Shh-responding cells to medial amygdala neuronal diversity as well as the function of Nkx2-1 in the development of this important limbic system structure.

A developmental cell-type switch in cortical interneurons leads to a selective defect in cortical oscillations

The cellular diversity of interneurons in the neocortex is thought to reflect subtype-specific roles of cortical inhibition. Here we ask whether perturbations to two subtypes—parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons—can be compensated for with respect to their contributions to cortical development. We use a genetic cell fate switch to delete both PV+ and SST+ interneurons selectively in cortical layers 2–4 without numerically changing the total interneuron population. This manipulation is compensated for at the level of synaptic currents and receptive fields (RFs) in the somatosensory cortex. By contrast, we identify a deficit in inhibitory synchronization in vitro and a large reduction in cortical gamma oscillations in vivo. This reveals that, while the roles of inhibition in establishing cortical inhibitory/excitatory balance and RFs can be subserved by multiple interneuron subtypes, gamma oscillations depend on cellular properties that cannot be compensated for—likely, the fast signalling properties of PV+ interneurons.

The integration of CGE-derived interneurons into the developing cortex: Challenging the temporal matching hypothesis

Genome-level knowledge promises a substantial increase in our power to understand the genetic factors that underlie the development of higher cognitive functions, such as speech and language, or social cognition. The mechanisms and evolution of these key aspects of higher cognitive functions have significant implications for many human neurodevelopmental diseases, including autism. However, organizing the vast quantities of data generated in genomic explorations of human brain using microarrays and NextGen sequencing whose systematic analysis poses challenges to even the most sophisticated bio-informaticians. Furthermore, differentiating a neutral and non-neutral model at the individual transcript level is not possible using standard analyses of differential gene expression. We have worked to unravel the underlying structure of the brain transcriptome in a systematic way, by the application of graph theory, and robust statistical methods imbedded in weighted gene co-expression network analysis (WGCNA) developed by Steve Horvath and collaborators at UCLA: (http://www.genetics.ucla.edu/labs/horvath/CoexpressionNetwork/). Several studies from our group have now demonstrated that such networks are robust and that a gene’s network position has significant functional implications. In Oldham et al. (PNAS, 2006), we showed that we can use WGCNA to identify transcriptional targets of human brain evolution in the context of the neutral model of evolution for the transcriptome, as any changes in a gene’s network position between the species indicates a functional alteration. We subsequently elucidated the structure of a core human cerebral cortex transcriptome that we used to drive functional understanding, including the identification of a module of genes related that can be used as markers for human adult neural stem cells (Oldham et al., 2008. Nat. Neurosci.). Here we describe the application of these methods to understanding of the role of FoxP2 in brain evolution and language. By comparing chimpanzee and human forms of FoxP2 we can see differences in the transcriptional networks that may be relevant to the development of human speech, as well as genes related to neuropsychiatric disease. We will also describe how these methods can be used to explore gene expression data across species based on NextGen sequences approaches.