Emx1 Is Required for Neocortical Area Patterning.

Establishing appropriate area patterning in the neocortex is a critical developmental event, and transcription factors whose expression is graded across the developing neural axes have been implicated in this process. While previous reports suggested that the transcription factor Emx1 does not contribute to neocortical area patterning, those studies were performed at perinatal ages prior to the emergence of primary areas. We therefore examined two different Emx1 deletion mouse lines once primary areas possess mature features. Following the deletion of Emx1, the frontal and motor areas were expanded while the primary visual area was reduced, and overall the areas shifted posterio-medially. This patterning phenotype was consistent between the two Emx1 deletion strategies. The present study demonstrates that Emx1 is an area patterning transcription factor and is required for the specification of the primary visual area.

Generation and analysis of an improved Foxg1-IRES-Cre driver mouse line.

Foxg1 expression is highly restricted to the telencephalon and other head structures in the early embryo. This expression pattern has been exploited to generate conditional knockout mice, based on a widely used Foxg1-Cre knock-in line (Foxg1(tm1(cre)Skm)), in which the Foxg1 coding region was replaced by the Cre gene. The utility of this line, however, is severely hampered for two reasons: (1) Foxg1-Cre mice display ectopic and unpredictable Cre activity, and (2) Foxg1 haploinsufficiency can produce neurodevelopmental phenotypes. To overcome these issues, we have generated a new Foxg1-IRES-Cre knock-in mouse line, in which an IRES-Cre cassette was inserted in the 3'UTR of Foxg1 locus, thus preserving the endogenous Foxg1 coding region and un-translated gene regulatory sequences in the 3'UTR, including recently discovered microRNA target sites. We further demonstrate that the new Foxg1-IRES-Cre line displays consistent Cre activity patterns that recapitulated the endogenous Foxg1 expression at embryonic and postnatal stages without causing defects in cortical development. We conclude that the new Foxg1-IRES-Cre mouse line is a unique and advanced tool for studying genes involved in the development of the telencephalon and other Foxg1-expressing regions starting from early embryonic stages.

Formation of the Cortical Subventricular Zone Requires MDGA1-Mediated Aggregation of Basal Progenitors.

The subventricular zone (SVZ) provides a specialized neurogenic microenvironment for proliferation and aggregation of basal progenitors (BPs). Our study reveals a mechanism for the aggregation of BPs within the SVZ required for their proliferation and generation of cortical layer neurons. The autism-related IgCAM, MDGA1, is locally expressed in the BP cell membrane where it co-localizes and complexes with the gap junction protein Connexin43. To address MDGA1 function, we created a floxed allele of MDGA1 and deleted it from BPs. MDGA1 deletion results in reduced BP proliferation and size of the SVZ, with an aberrant population of BPs ectopically positioned in the cortical plate. These defects are manifested in diminished production of cortical layer neurons and a significant reduction of the cortical layers. We conclude that MDGA1 functions to aggregate and maintain BPs within the SVZ providing the neurogenic niche required for their proliferation and generation of cortical layer neurons.

Postmitotic regulation of sensory area patterning in the mammalian neocortex by Lhx2.

Current knowledge suggests that cortical sensory area identity is controlled by transcription factors (TFs) that specify area features in progenitor cells and subsequently their progeny in a one-step process. However, how neurons acquire and maintain these features is unclear. We have used conditional inactivation restricted to postmitotic cortical neurons in mice to investigate the role of the TF LIM homeobox 2 (Lhx2) in this process and report that in conditional mutant cortices area patterning is normal in progenitors but strongly affected in cortical plate (CP) neurons. We show that Lhx2 controls neocortical area patterning by regulating downstream genetic and epigenetic regulators that drive the acquisition of molecular properties in CP neurons. Our results question a strict hierarchy in which progenitors dominate area identity, suggesting a novel and more comprehensive two-step model of area patterning: In progenitors, patterning TFs prespecify sensory area blueprints. Sequentially, sustained function of alignment TFs, including Lhx2, is essential to maintain and to translate the blueprints into functional sensory area properties in cortical neurons postmitotically. Our results reemphasize critical roles for Lhx2 that acts as one of the terminal selector genes in controlling principal properties of neurons.

Multiple EphB receptors mediate dorsal-ventral retinotopic mapping via similar bi-functional responses to ephrin-B1.

The projection from the retina to the superior colliculus in mice is organized in a retinotopic map that develops through the formation and guidance of interstitial branches extended by retinal ganglion cell axons. Bidirectional branch guidance along the lateral-medial collicular axis is critical to mapping the dorsal-ventral retinal axis. EphB receptor tyrosine kinases expressed in an overall low to high dorsal-ventral retinal gradient have been implicated in this mapping in response to the graded low to high lateral-medial expression of a ligand, ephrin-B1, in the superior colliculus. However, the relative contributions of EphBs and ephrin-B1 are not well understood. We examined EphB1, EphB2, and EphB3 mutant mice and find that each has ectopic arborizations of retinal axon branches lateral to their appropriate termination zone, with no qualitative differences in aberrant mapping, suggesting a similar role for each EphB. However, the frequency of cases with map defects progressively rises in compound EphB mutants coincident with the number of EphB null alleles from one to five of the six total alleles indicating that EphB level is critical. We analyzed branch extension in vitro and find that dorsal branches, with low EphB levels, exhibit a negative response to ephrin-B1, whereas ventral branches, with high EphB levels, exhibit a positive response to ephrin-B1. Using EphB mutant retina, we show that both of these differential branch extension responses are dependent on EphB level. Our findings show a bifunctional action of ephrin-B1 regulated by EphB levels that can account for the bidirectional extension of interstitial branches required to establish a retinotopic map.

Lhx1 maintains synchrony among circadian oscillator neurons of the SCN.

The robustness and limited plasticity of the master circadian clock in the suprachiasmatic nucleus (SCN) is attributed to strong intercellular communication among its constituent neurons. However, factors that specify this characteristic feature of the SCN are unknown. Here, we identified Lhx1 as a regulator of SCN coupling. A phase-shifting light pulse causes acute reduction in Lhx1 expression and of its target genes that participate in SCN coupling. Mice lacking Lhx1 in the SCN have intact circadian oscillators, but reduced levels of coupling factors. Consequently, the mice rapidly phase shift under a jet lag paradigm and their behavior rhythms gradually deteriorate under constant condition. Ex vivo recordings of the SCN from these mice showed rapid desynchronization of unit oscillators. Therefore, by regulating expression of genes mediating intercellular communication, Lhx1 imparts synchrony among SCN neurons and ensures consolidated rhythms of activity and rest that is resistant to photic noise.

Genetic analysis reveals that amyloid precursor protein and death receptor 6 function in the same pathway to control axonal pruning independent of ß-secretase.

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that ß-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that ß-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning.

Role of BRCA1 in brain development.

Breast cancer susceptibility gene 1 (BRCA1) is a breast and ovarian cancer tumor suppressor whose loss leads to DNA damage and defective centrosome functions. Despite its tumor suppression functions, BRCA1 is most highly expressed in the embryonic neuroepithelium when the neural progenitors are highly proliferative. To determine its functional significance, we deleted BRCA1 in the developing brain using a neural progenitor-specific driver. The phenotype is characterized by severe agenesis of multiple laminated cerebral structures affecting most notably the neocortex, hippocampus, cerebellum, and olfactory bulbs. Major phenotypes are caused by excess apoptosis, as these could be significantly suppressed by the concomitant deletion of p53. Certain phenotypes attributable to centrosomal and cell polarity functions could not be rescued by p53 deletion. A double KO with the DNA damage sensor kinase ATM was able to rescue BRCA1 loss to a greater extent than p53. Our results suggest distinct apoptotic and centrosomal functions of BRCA1 in neural progenitors, with important implications to understand the sensitivity of the embryonic brain to DNA damage, as well as the developmental regulation of brain size.

A transcription factor code defines nine sensory interneuron subtypes in the mechanosensory area of the spinal cord.

Interneurons in the dorsal spinal cord process and relay innocuous and nociceptive somatosensory information from cutaneous receptors that sense touch, temperature and pain. These neurons display a well-defined organization with respect to their afferent innervation. Nociceptive afferents innervate lamina I and II, while cutaneous mechanosensory afferents primarily innervate sensory interneurons that are located in lamina III-IV. In this study, we outline a combinatorial transcription factor code that defines nine different inhibitory and excitatory interneuron populations in laminae III-IV of the postnatal cord. This transcription factor code reveals a high degree of molecular diversity in the neurons that make up laminae III-IV, and it lays the foundation for systematically analyzing and manipulating these different neuronal populations to assess their function. In addition, we find that many of the transcription factors that are expressed in the dorsal spinal cord at early postnatal times continue to be expressed in the adult, raising questions about their function in mature neurons and opening the door to their genetic manipulation in adult animals.

In vivo expression of a light-activatable potassium channel using unnatural amino acids.

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K(+) current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation.

Sensory cortex limits cortical maps and drives top-down plasticity in thalamocortical circuits.

The primary somatosensory cortex (S1) contains a complete body map that mirrors the subcortical maps developed by peripheral sensory input projecting to the sensory hindbrain, the thalamus and then S1. Peripheral changes during development alter these maps through 'bottom-up' plasticity. Unknown is how S1 size influences map organization and whether an altered S1 map feeds back to affect subcortical maps. We show that the size of S1 in mice is significantly reduced by cortex-specific deletion of Pax6, resulting in a reduced body map and loss of body representations by an exclusion of later-differentiating sensory thalamocortical input. An initially normal sensory thalamus was repatterned to match the aberrant S1 map by apoptotic deletion of thalamic neurons representing body parts with axons excluded from S1. Deleted representations were rescued by altering competition between thalamocortical axons using sensory deprivation or increasing the size of S1. Thus, S1 size determined the resolution and completeness of body maps and engaged 'top-down' plasticity that repatterned the sensory thalamus to match S1.

Geniculocortical input drives genetic distinctions between primary and higher-order visual areas.

Studies of area patterning of the neocortex have focused on primary areas, concluding that the primary visual area, V1, is specified by transcription factors (TFs) expressed by progenitors. Mechanisms that determine higher-order visual areas (V(HO)) and distinguish them from V1 are unknown. We demonstrated a requirement for thalamocortical axon (TCA) input by genetically deleting geniculocortical TCAs and showed that they drive differentiation of patterned gene expression that distinguishes V1 and V(HO). Our findings suggest a multistage process for area patterning: TFs expressed by progenitors specify an occipital visual cortical field that differentiates into V1 and V(HO); this latter phase requires geniculocortical TCA input to the nascent V1 that determines genetic distinctions between V1 and V(HO) for all layers and ultimately determines their area-specific functional properties.

Role for Lhx2 in corticogenesis through regulation of progenitor differentiation.

The neocortex represents the brain region that has undergone a major increase in its relative size during the course of mammalian evolution. The larger cortex results from a corresponding increase in progenitor cell number. The progenitors giving rise to neocortex are located in the ventricular zone of the dorsal telencephalon and highly express Lhx2, a LIM-homeodomain transcription factor. The neocortex fails to form in the Lhx2 constitutive knockout, indicating a role for Lhx2 in corticogenesis, but mid-embryonic lethality of the Lhx2 knockout requires the use of conditional strategies for further studies. Therefore, to explore Lhx2 function in neocortical progenitors, we generated mice with Lhx2 conditionally deleted from cortical progenitors at the onset of neurogenesis. We find that Lhx2 is critical for maintaining the proliferative state of neocortical progenitors during corticogenesis. In the conditional knockouts, the neocortex is formed but is significantly smaller than wild type. We find that deletion of Lhx2 leads to significantly decreased numbers of cortical progenitors and premature neuronal differentiation. A likely mechanism is indicated by our findings that Lhx2 is required for the expression of Hes1 in cortical progenitors, a key effector in the Notch signaling pathway that maintains the proliferative progenitor state. We conclude that Lhx2 regulates the balance between proliferation and differentiation in cortical progenitors and through this mechanism Lhx2 controls cortical size.

A caspase cascade regulating developmental axon degeneration.

Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other "upstream" caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.

The fraction of cortical GABAergic neurons is constant from near the start of cortical neurogenesis to adulthood.

Approximately one in five neurons is GABAergic in many neocortical areas and species, forming a critical balance between inhibition and excitation in adult circuits. During development, cortical GABAergic neurons are generated in ventral telencephalon and migrate up to developing cortex where the excitatory glutamatergic neurons are born. We ask here: when during development is the adult GABAergic/glutamatergic neuron ratio first established? To answer this question, we have determined the fraction of all neocortical GABAergic neurons that will become inhibitory (GAD67(+)) in mice from embryonic day 10.5 (E10.5) to postnatal day 28 (P28). We find that this fraction is close to 1/5, the adult value, starting from early in corticogenesis (E14.5, when GAD67(+) neurons are still migrating tangentially to the cortex) and continuing at the same 1/5 value throughout the remainder of brain development. Thus our data indicate the one-in-five fraction of GABAergic neurons is already established during their neuronal migration and well before significant synapse formation.

Neuregulin repellent signaling via ErbB4 restricts GABAergic interneurons to migratory paths from ganglionic eminence to cortical destinations.

BACKGROUND: Cortical GABAergic interneurons (INs) are generated in the medial ganglionic eminence (MGE) and migrate tangentially into cortex. Because most, if not all, migrating MGE-derived INs express the neuregulin (NRG) receptor, ErbB4, we investigated influences of Nrg1 isoforms and Nrg3 on IN migration through ventral telencephalon (vTel) and within cortex. RESULTS: During IN migration, NRG expression domains and distributions of ErbB4-expressing, MGE-derived INs are complementary with minimal overlap, both in vTel and cortex. In wild-type mice, within fields of NRG expression, these INs are focused at positions of low or absent NRG expression. However, in ErbB4-/- HER4(heart) mutant mice in which INs lack ErbB4, these complementary patterns are degraded with considerable overlap evident between IN distribution and NRG expression domains. These findings suggest that NRGs are repellents for migrating ErbB4-expressing INs, a function supported by in vitro and in vivo experiments. First, in collagen co-cultures, MGE-derived cells preferentially migrate away from a source of secreted NRGs. Second, cells migrating from wild-type MGE explants on living forebrain slices from wild-type embryonic mice tend to avoid endogenous NRG expression domains, whereas this avoidance behavior is not exhibited by ErbB4-deficient cells migrating from MGE explants and instead they have a radial pattern with a more uniform distribution. Third, ectopic NRG expression in the IN migration pathway produced by in utero electroporation blocks IN migration and results in cortex distal to the blockade being largely devoid of INs. Finally, fewer INs reach cortex in ErbB4 mutants, indicating that NRG-ErbB4 signaling is required for directing IN migration from the MGE to cortex. CONCLUSIONS: Our results show that NRGs act as repellents for migrating ErbB4-expressing, MGE-derived GABAergic INs and that the patterned expression of NRGs funnels INs as they migrate from the MGE to their cortical destinations.

Islet1-mediated activation of the ß-catenin pathway is necessary for hindlimb initiation in mice.

The transcriptional basis of vertebrate limb initiation, which is a well-studied system for the initiation of organogenesis, remains elusive. Specifically, involvement of the ß-catenin pathway in limb initiation, as well as its role in hindlimb-specific transcriptional regulation, are under debate. Here, we show that the ß-catenin pathway is active in the limb-forming area in mouse embryos. Furthermore, conditional inactivation of ß-catenin as well as Islet1, a hindlimb-specific factor, in the lateral plate mesoderm results in a failure to induce hindlimb outgrowth. We further show that Islet1 is required for the nuclear accumulation of ß-catenin and hence for activation of the ß-catenin pathway, and that the ß-catenin pathway maintains Islet1 expression. These two factors influence each other and function upstream of active proliferation of hindlimb progenitors in the lateral plate mesoderm and the expression of a common factor, Fgf10. Our data demonstrate that Islet1 and ß-catenin regulate outgrowth and Fgf10-Fgf8 feedback loop formation during vertebrate hindlimb initiation. Our study identifies Islet1 as a hindlimb-specific transcriptional regulator of initiation, and clarifies the controversy regarding the requirement of ß-catenin for limb initiation.

Antiapoptotic protein Lifeguard is required for survival and maintenance of Purkinje and granular cells.

Lifeguard (LFG) is an inhibitor of Fas-mediated cell death and is highly expressed in the cerebellum. We investigated the biological role of LFG in the cerebellum in vivo, using mice with reduced LFG expression generated by shRNA lentiviral transgenesis (shLFG mice) as well as LFG null mice. We found that LFG plays a role in cerebellar development by affecting cerebellar size, internal granular layer (IGL) thickness, and Purkinje cell (PC) development. All these features are more severe in early developmental stages and show substantial recovery overtime, providing a remarkable example of cerebellar plasticity. In adult mice, LFG plays a role in PC maintenance shown by reduced cellular density and abnormal morphology with increased active caspase 8 and caspase 3 immunostaining in shLFG and knockout (KO) PCs. We studied the mechanism of action of LFG as an inhibitor of the Fas pathway and provided evidence of the neuroprotective role of LFG in cerebellar granule neurons (CGNs) and PCs in an organotypic cerebellar culture system. Biochemical analysis of the Fas pathway revealed that LFG inhibits Fas-mediated cell death by interfering with caspase 8 activation. This result is supported by the increased number of active caspase 8-positive PCs in adult mice lacking LFG. These data demonstrate that LFG is required for proper development and survival of granular and Purkinje cells and suggest LFG may play a role in cerebellar disorders.

A forward genetic screen with a thalamocortical axon reporter mouse yields novel neurodevelopment mutants and a distinct emx2 mutant phenotype.

BACKGROUND: The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. RESULTS: To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs), but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. CONCLUSIONS: These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the mouse brain, identifying mutant phenotypes at multiple steps of the pathway, and revealing a new aspect of the Emx2 mutant. The phenotypes highlight vulnerable choice points and latent tendencies of TCAs, and will lead to a refined understanding of the elements and interactions required to form the thalamocortical system.