Ashwin S. Shetty

Lhx2 Selector Activity Specifies Cortical Identity and Suppresses Hippocampal Organizer Fate

The earliest step in creating the cerebral cortex is the specification of neuroepithelium to a cortical fate. Using mouse genetic mosaics and timed inactivations, we demonstrated that Lhx2 acts as a classic selector gene and essential intrinsic determinant of cortical identity. Lhx2 selector activity is restricted to an early critical period when stem cells comprise the cortical neuroepithelium, where it acts cell-autonomously to specify cortical identity and suppress alternative fates in a spatially dependent manner. Laterally, Lhx2 null cells adopt antihem identity, whereas medially they become cortical hem cells, which can induce and organize ectopic hippocampal fields. In addition to providing functional evidence for Lhx2 selector activity, these findings show that the cortical hem is a hippocampal organizer.

Signals from the edges: The cortical hem and antihem in telencephalic development

The early cortical primordium develops from a sheet of neuroepithelium that is flanked by distinct signaling centers. Of these, the hem and the antihem are positioned as longitudinal stripes, running rostro-caudally along the medial and lateral faces, respectively, of each telencepahlic hemisphere. In this review we examine the similarities and differences in how these two signaling centers arise, their roles in patterning adjacent tissues, and the cells and structures they contribute to. Since both the hem and the antihem have been identified across many vertebrate phyla, they appear to be part of an evolutionary conserved set of mechanisms that play fundamental roles in forebrain development.

Lhx2 regulates a cortex-specific mechanism for barrel formation

Significance The somatosensory barrels are a unique feature of the rodent cortex. Each barrel represents a functional unit in which clustered innervation from an individual whisker connects with a ring of cortical neurons. This study reports that when a single transcription factor, LIM homeobox 2, is deleted specifically in the cortex, neither the barrel cores nor the cortical barrel walls are able to form, although a rudimentary functional mapping of the somatosensory innervation does occur. Understanding how barrels form will shed light on how functional neurocircuitry is assembled in its final stages, and this insight may be broadly applicable in the nervous system. LIM homeodomain transcription factors are critical regulators of early development in multiple systems but have yet to be examined for a role in circuit formation. The LIM homeobox gene Lhx2 is expressed in cortical progenitors during development and also in the superficial layers of the neocortex in maturity. However, analysis of Lhx2 function at later stages of cortical development has been hampered by severe phenotypes associated with early loss of function. We identified a particular Cre-recombinase line that acts in the cortical primordium after its specification is complete, permitting an analysis of Lhx2 function in neocortical lamination, regionalization, and circuit formation by selective elimination of Lhx2 in the dorsal telencephalon. We report a profound disruption of cortical neuroanatomical and molecular features upon loss of Lhx2 in the cortex from embryonic day 11.5. A unique feature of cortical circuitry, the somatosensory barrels, is undetectable, and molecular patterning of cortical regions appears disrupted. Surprisingly, thalamocortical afferents innervate the mutant cortex with apparently normal regional specificity. Electrophysiological recordings reveal a loss of responses evoked by stimulation of individual whiskers, but responses to simultaneous stimulation of multiple whiskers were present, suggesting that thalamic afferents are unable to organize the neurocircuitry for barrel formation because of a cortex-specific requirement of Lhx2. We report that Lhx2 is required for the expression of transcription factor paired box gene 6, axon guidance molecule Ephrin A5, and the receptor NMDA receptor 1. These genes may mediate Lhx2 function in the formation of specialized neurocircuitry necessary for neocortical function.

Dmrt5, a novel neurogenic factor, reciprocally regulates Lhx2 to control the neuron-glia cell fate switch in the developing hippocampus

Regulation of the neuron–glia cell-fate switch is a critical step in the development of the CNS. Previously, we demonstrated that Lhx2 is a necessary and sufficient regulator of this process in the mouse hippocampal primordium, such that Lhx2 overexpression promotes neurogenesis and suppresses gliogenesis, whereas loss of Lhx2 has the opposite effect. We tested a series of transcription factors for their ability to mimic Lhx2 overexpression and suppress baseline gliogenesis, and also to compensate for loss of Lhx2 and suppress the resulting enhanced level of gliogenesis in the hippocampus. Here, we demonstrate a novel function of Dmrt5/Dmrta2 as a neurogenic factor in the developing hippocampus. We show that Dmrt5, as well as known neurogenic factors Neurog2 and Pax6, can each not only mimic Lhx2 overexpression, but also can compensate for loss of Lhx2 to different extents. We further uncover a reciprocal regulatory relationship between Dmrt5 and Lhx2, such that each can compensate for loss of the other. Dmrt5 and Lhx2 also have opposing regulatory control on Pax6 and Neurog2, indicating a complex bidirectionally regulated network that controls the neuron–glia cell-fate switch. SIGNIFICANCE STATEMENT We identify Dmrt5 as a novel regulator of the neuron–glia cell-fate switch in the developing hippocampus. We demonstrate Dmrt5 to be neurogenic, and reciprocally regulated by Lhx2: loss of either factor promotes gliogenesis; overexpression of either factor suppresses gliogenesis and promotes neurogenesis; each can substitute for loss of the other. Furthermore, each factor has opposing effects on established neurogenic genes Neurog2 and Pax6. Dmrt5 is known to suppress their expression, and we show that Lhx2 is required to maintain it. Our study reveals a complex regulatory network with bidirectional control of a fundamental feature of CNS development, the control of the production of neurons versus astroglia in the developing hippocampus. Finally, we confirm that Lhx2 binds a highly conserved putative enhancer of Dmrt5, suggesting an evolutionarily conserved regulatory relationship between these factors. Our findings uncover a complex network that involves Lhx2, Dmrt5, Neurog2, and Pax6, and that ensures the appropriate amount and timing of neurogenesis and gliogenesis in the developing hippocampus.

Hierarchical genetic interactions between FOXG1 and LHX2 regulate the formation of the cortical hem in the developing telencephalon

ABSTRACT During forebrain development, a telencephalic organizer called the cortical hem is crucial for inducing hippocampal fate in adjacent cortical neuroepithelium. How the hem is restricted to its medial position is therefore a fundamental patterning issue. Here, we demonstrate that Foxg1-Lhx2 interactions are crucial for the formation of the hem. Loss of either gene causes a region of the cortical neuroepithelium to transform into hem. We show that FOXG1 regulates Lhx2 expression in the cortical primordium. In the absence of Foxg1, the presence of Lhx2 is sufficient to suppress hem fate, and hippocampal markers appear selectively in Lhx2-expressing regions. FOXG1 also restricts the temporal window in which loss of Lhx2 results in a transformation of cortical primordium into hem. Therefore, Foxg1 and Lhx2 form a genetic hierarchy in the spatiotemporal regulation of cortical hem specification and positioning, and together ensure the normal development of this hippocampal organizer. Summary: The cortical hem, a telencephalic organizer that induces the hippocampus, is positionally restricted by the transcription factor FOXG1 acting directly and also by positively regulating LHX2.

PAX6 can substitute for LHX2 and override NFIA-induced astrogliogenesis in developing hippocampus in vivo

In the developing central nervous system, transcription factors play a crucial role in the regulation of cell fate. Previously we demonstrated that LHX2 is a critical regulator of the neuron-glia cell fate switch in the developing mouse hippocampus. Here, we test LHX2 target gene Pax6 for a role in this process. We report that Pax6 overexpression is able to suppress the enhanced astrogliogenesis arising due to loss of functional LHX2. Furthermore, we show that like Lhx2, Pax6 is also able to suppress induced astrogliogenesis caused by overexpression of progliogenic factor Nfia. This demonstrates that overexpression of Pax6 can substitute for Lhx2 in the regulation of the neuronal versus glial cell fate in the developing hippocampus, and therefore, supports a role for PAX6 as a mediator of LHX2 function in this process.

Novel functions of LHX2 and PAX6 in the developing telencephalon revealed upon combined loss of both genes

Patterning of the telencephalic neuroepithelium is a tightly regulated process controlled by transcription factors and signalling molecules. The cortical primordium is flanked by two signalling centres, the hem medially, and the antihem laterally. The hem induces the formation of the hippocampus in adjacent neuroepithelium. Therefore, the position of the hem defines the position of the hippocampus in the brain. The antihem is positioned at the boundary between the dorsal and ventral telencephalon and proposed to provide patterning cues during development.LIM-homeodomain (LIM-HD) transcription factor LHX2 suppresses both hem and antihem fate in the cortical neuroepithelium. Upon loss of Lhx2, medial cortical neuroepithelium is transformed into hem, whereas lateral cortical neuroepithelium is transformed into antihem. Here, we show that transcription factor PAX6, known to regulate patterning of the lateral telencephalon, restricts this tissue from transforming into hem upon loss of Lhx2. When Lhx2 and Pax6 are both deleted, the cortical hem expands to occupy almost the complete extent of the cortical primordium, indicating that both factors act to suppress hem fate in the lateral telencephalon. Furthermore, the shift in the pallial-subpallial boundary and absence of the antihem, observed in the Pax6 mutant, are both restored in the Lhx2; Pax6 double mutant.Together, these results not only reveal a novel function for LHX2 in regulating dorsoventral patterning in the telencephalon, but also identify PAX6 as a fundamental regulator of where the hem can form, and therefore implicate this molecule as a determinant of hippocampal positioning.

Seizure evoked regulation of LIM-HD genes and co-factors in the postnatal and adult hippocampus

The LIM-homeodomain (LIM-HD) family of transcription factors is well known for its functions during several developmental processes including cell fate specification, cell migration and axon guidance, and its members play fundamental roles in hippocampal development. The hippocampus is a structure that displays striking activity dependent plasticity. We examined whether LIM-HD genes and their co-factors are regulated during kainic acid induced seizure in the adult rat hippocampus as well as in early postnatal rats, when the hippocampal circuitry is not fully developed. We report a distinct and field-specific regulation of LIM-HD genes Lhx1, Lhx2, and Lhx9, LIM-only gene Lmo4, and cofactor Clim1a in the adult hippocampus after seizure induction. In contrast none of these genes displayed altered levels upon induction of seizure in postnatal animals. Our results provide evidence of temporal and spatial seizure mediated regulation of LIM-HD family members and suggest that LIM-HD gene function may be involved in activity dependent plasticity in the adult hippocampus

Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus

Lakshmi Subramanian 1, Anindita Sarkar 1, Ashwin S. Shetty 1, Bhavana Muralidharan 1, Hari Padmanabhan 1, Michael Piper 2, Edwin S. Monuki 3, Ingolf Bach 4, Richard M. Gronostajski 5,6, Linda J. Richards 2, Shubha Tole 2 1 Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India 2 Queensland Brain Institute and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia 3 Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Irvine, CA 92697, United States 4 Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States 5 Department of Biochemistry, State University of New York, Buffalo, NY 14203, United States 6 Developmental Genomics Group, New York State Centre of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14203, United States The sequential production of neurons and astrocytes from neuroepithelial precursors is a fundamental feature of central nervous system development. We report that LIM-homeodomain (LIM-HD) transcription factor Lhx2 regulates this transition in the developing hippocampus. Disrupting Lhx2 function in the embryonic hippocampus by in utero electroporation and in organotypic slice culture caused the premature production of astrocytes at stages when neurons are normally generated. Lhx2 function is therefore necessary to suppress astrogliogenesis during the neurogenic period. Furthermore, Lhx2 overexpression was sufficient to suppress astrogliogenesis and prolong the neurogenic period. We provide evidence that Lhx2 overexpression can counteract the instructive astrogliogenic effect of Notch activation. Lhx2 overexpression was also able to override and suppress the activation of the GFAP promoter by Nfia, a Notch-regulated transcription factor that is required for gliogenesis. Thus, Lhx2 appears to act as a “brake” on Notch/Nfia-mediated astrogliogenesis. This critical role for Lhx2 is spatially restricted to the hippocampus, because loss of Lhx2 function in the neocortex did not result in premature astrogliogenesis at the expense of neurogenesis. Our results therefore place Lhx2 as a central regulator of the neuron-glia cell fate decision in the hippocampus and reveal a striking regional specificity of this fundamental function within the dorsal telencephalon.