PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

Regulation of chromatin accessibility and gene expression in the developing hippocampal primordium by LIM-HD transcription factor LHX2.

In the mammalian cerebral cortex, the hippocampal primordium (Hcp) occupies a discrete position in the dorsal telencephalic neuroepithelium adjacent to the neocortical primordium (Ncp). We examined transcriptomic and chromatin-level features that distinguish the Hcp from the Ncp in the mouse during the early neurogenic period, embryonic day (E)12.5. ATAC-seq revealed that the Hcp was more accessible than the Ncp at this stage. Motif analysis of the differentially accessible loci in these tissues revealed LHX2 as a candidate transcription factor for modulating gene regulatory networks (GRNs). We analyzed LHX2 occupancy profiles and compared these with transcriptomic data from control and Lhx2 mutant Hcp and Ncp at E12.5. Our results revealed that LHX2 directly regulates distinct genes in the Hcp and Ncp within a set of common pathways that control fundamental aspects of development namely pluripotency, axon pathfinding, Wnt, and Hippo signaling. Loss of Lhx2 caused a decrease in accessibility, specifically in hippocampal chromatin, suggesting that this factor may play a unique role in hippocampal development. We identified 14 genes that were preferentially enriched in the Hcp, for which LHX2 regulates both chromatin accessibility and mRNA expression, which have not thus far been examined in hippocampal development. Together, these results provide mechanistic insight into how LHX2 function in the Hcp may contribute to the process by which the hippocampus acquires features distinct from the neocortex.

Dentate gyrus morphogenesis is regulated by ß-catenin function in hem-derived fimbrial glia.

The dentate gyrus, a gateway for input to the hippocampal formation, arises from progenitors in the medial telencephalic neuroepithelium adjacent to the cortical hem. Dentate progenitors navigate a complex migratory path guided by two cell populations that arise from the hem, the fimbrial glia and Cajal-Retzius (CR) cells. As the hem expresses multiple Wnt genes, we examined whether ß-catenin, which mediates canonical Wnt signaling and also participates in cell adhesion, is necessary for the development of hem-derived lineages. We report that, in mice, the fimbrial glial scaffold is disorganized and CR cells are mispositioned upon hem-specific disruption of ß-catenin. Consequently, the dentate migratory stream is severely affected, and the dentate gyrus fails to form. Using selective Cre drivers, we further determined that ß-catenin function is required in the fimbrial glial scaffold, but not in the CR cells, for guiding the dentate migration. Our findings highlight a primary requirement for ß-catenin for the organization of the fimbrial scaffold and a secondary role for this factor in dentate gyrus morphogenesis.

BAF (mSWI/SNF) complex regulates mediolateral cortical patterning in the developing forebrain.

Early forebrain patterning entails the correct regional designation of the neuroepithelium, and appropriate specification, generation, and distribution of neural cells during brain development. Specific signaling and transcription factors are known to tightly regulate patterning of the dorsal telencephalon to afford proper structural/functional cortical arealization and morphogenesis. Nevertheless, whether and how changes of the chromatin structure link to the transcriptional program(s) that control cortical patterning remains elusive. Here, we report that the BAF chromatin remodeling complex regulates the spatiotemporal patterning of the mouse dorsal telencephalon. To determine whether and how the BAF complex regulates cortical patterning, we conditionally deleted the BAF complex scaffolding subunits BAF155 and BAF170 in the mouse dorsal telencephalic neuroepithelium. Morphological and cellular changes in the BAF mutant forebrain were examined using immunohistochemistry and in situ hybridization. RNA sequencing, Co-immunoprecipitation, and mass spectrometry were used to investigate the molecular basis of BAF complex involvement in forebrain patterning. We found that conditional ablation of BAF complex in the dorsal telencephalon neuroepithelium caused expansion of the cortical hem and medial cortex beyond their developmental boundaries. Consequently, the hippocampal primordium is not specified, the mediolateral cortical patterning is compromised, and the cortical identity is disturbed in the absence of BAF complex. The BAF complex was found to interact with the cortical hem suppressor LHX2. The BAF complex suppresses cortical hem fate to permit proper forebrain patterning. We provide evidence that BAF complex modulates mediolateral cortical patterning possibly by interacting with the transcription factor LHX2 to drive the LHX2-dependent transcriptional program essential for dorsal telencephalon patterning. Our data suggest a putative mechanistic synergy between BAF chromatin remodeling complex and LHX2 in regulating forebrain patterning and ontogeny.

Tbr1 Misexpression Alters Neuronal Development in the Cerebral Cortex.

Changes in the transcription factor (TF) expression are critical for brain development, and they may also underlie neurodevelopmental disorders. Indeed, T-box brain1 (Tbr1) is a TF crucial for the formation of neocortical layer VI, and mutations and microdeletions in that gene are associated with malformations in the human cerebral cortex, alterations that accompany autism spectrum disorder (ASD). Interestingly, Tbr1 upregulation has also been related to the occurrence of ASD-like symptoms, although limited studies have addressed the effect of increased Tbr1 levels during neocortical development. Here, we analysed the impact of Tbr1 misexpression in mouse neural progenitor cells (NPCs) at embryonic day 14.5 (E14.5), when they mainly generate neuronal layers II-IV. By E18.5, cells accumulated in the intermediate zone and in the deep cortical layers, whereas they became less abundant in the upper cortical layers. In accordance with this, the proportion of Sox5+ cells in layers V-VI increased, while that of Cux1+ cells in layers II-IV decreased. On postnatal day 7, fewer defects in migration were evident, although a higher proportion of Sox5+ cells were seen in the upper and deep layers. The abnormal neuronal migration could be partially due to the altered multipolar-bipolar neuron morphologies induced by Tbr1 misexpression, which also reduced dendrite growth and branching, and disrupted the corpus callosum. Our results indicate that Tbr1 misexpression in cortical NPCs delays or disrupts neuronal migration, neuronal specification, dendrite development and the formation of the callosal tract. Hence, genetic changes that provoke ectopic Tbr1 upregulation during development could provoke cortical brain malformations.

Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate.

The choroid plexus secretes cerebrospinal fluid and is critical for the development and function of the brain. In the telencephalon, the choroid plexus epithelium arises from the Wnt- expressing cortical hem. Canonical Wnt signaling pathway molecules such as nuclear ß-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human ß-CATENIN are known to result in the constitutive activation of canonical Wnt signaling. In a mouse model that recapitulates this perturbation, we report a loss of choroid plexus epithelial identity and an apparent transformation of this tissue to a neuronal identity. Aspects of this phenomenon are recapitulated in human embryonic stem cell derived organoids. The choroid plexus is also disrupted when ß-Catenin is conditionally inactivated. Together, our results indicate that canonical Wnt signaling is required in a precise and regulated manner for normal choroid plexus development in the mammalian brain.

An Early Cortical Progenitor-Specific Mechanism Regulates Thalamocortical Innervation.

The cortical subplate is critical in regulating the entry of thalamocortical sensory afferents into the cortex. These afferents reach the subplate at embryonic day (E)15.5 in the mouse, but "wait" for several days, entering the cortical plate postnatally. We report that when transcription factor LHX2 is lost in E11.5 cortical progenitors, which give rise to subplate neurons, thalamocortical afferents display premature, exuberant ingrowth into the E15.5 cortex. Embryonic mutant subplate neurons are correctly positioned below the cortical plate, but they display an altered transcriptome and immature electrophysiological properties during the waiting period. The sensory thalamus in these cortex-specific Lhx2 mutants displays atrophy and by postnatal day (P) 7, sensory innervation to the cortex is nearly eliminated leading to a loss of the somatosensory barrels. Strikingly, these phenotypes do not manifest if LHX2 is lost in postmitotic subplate neurons, and the transcriptomic dysregulation in the subplate resulting from postmitotic loss of LHX2 is vastly distinct from that seen when LHX2 is lost in progenitors. These results demonstrate a mechanism operating in subplate progenitors that has profound consequences on the growth of thalamocortical axons into the cortex.SIGNIFICANCE STATEMENT Thalamocortical nerves carry sensory information from the periphery to the cortex. When they first grow into the embryonic cortex, they "wait" at the subplate, a structure critical for the guidance and eventual connectivity of thalamic axons with their cortical targets. How the properties of subplate neurons are regulated is unclear. We report that transcription factor LHX2 is required in the progenitor "mother" cells of the cortical primordium when they are producing their "daughter" subplate neurons, in order for the thalamocortical pathway to wait at the subplate. Without LHX2 function in subplate progenitors, thalamocortical axons grow past the subplate, entering the cortical plate prematurely. This is followed by their eventual attrition and, consequently, a profound loss of sensory innervation of the mature cortex.

An evolutionarily conserved Lhx2-Ldb1 interaction regulates the acquisition of hippocampal cell fate and regional identity.

The protein co-factor Ldb1 regulates cell fate specification by interacting with LIM-homeodomain (LIM-HD) proteins in a tetrameric complex consisting of an LDB:LDB dimer that bridges two LIM-HD molecules, a mechanism first demonstrated in the Drosophila wing disc. Here, we demonstrate conservation of this interaction in the regulation of mammalian hippocampal development, which is profoundly defective upon loss of either Lhx2 or Ldb1 Electroporation of a chimeric construct that encodes the Lhx2-HD and Ldb1-DD (dimerization domain) in a single transcript cell-autonomously rescues a comprehensive range of hippocampal deficits in the mouse Ldb1 mutant, including the acquisition of field-specific molecular identity and the regulation of the neuron-glia cell fate switch. This demonstrates that the LHX:LDB complex is an evolutionarily conserved molecular regulatory device that controls complex aspects of regional cell identity in the developing brain.

Neuronal diversity and reciprocal connectivity between the vertebrate hippocampus and septum.

A hallmark of the nervous system is the precision with which myriad cell types are integrated into functional networks that control complex behaviors. The limbic system governs evolutionarily conserved processes essential for survival. The septum and the hippocampus are central to the limbic system, and control not only emotion-related behaviors but also learning and memory. Here, we provide a developmental and evolutionary perspective of the hippocampus and septum and highlight the neuronal diversity and circuitry that connects these two central components of the limbic system. This article is categorized under: Nervous System Development > Vertebrates: Regional Development Nervous System Development > Vertebrates: General Principles Comparative Development and Evolution > Regulation of Organ Diversity.

Early dorsomedial tissue interactions regulate gyrification of distal neocortex.

The extent of neocortical gyrification is an important determinant of a species' cognitive abilities, yet the mechanisms regulating cortical gyrification are poorly understood. We uncover long-range regulation of this process originating at the telencephalic dorsal midline, where levels of secreted Bmps are maintained by factors in both the neuroepithelium and the overlying mesenchyme. In the mouse, the combined loss of transcription factors Lmx1a and Lmx1b, selectively expressed in the midline neuroepithelium and the mesenchyme respectively, causes dorsal midline Bmp signaling to drop at early neural tube stages. This alters the spatial and temporal Wnt signaling profile of the dorsal midline cortical hem, which in turn causes gyrification of the distal neocortex. Our study uncovers early mesenchymal-neuroepithelial interactions that have long-range effects on neocortical gyrification and shows that lissencephaly in mice is actively maintained via redundant genetic regulation of dorsal midline development and signaling.

LDB1 Is Required for the Early Development of the Dorsal Telencephalon and the Thalamus.

LIM domain binding protein 1 (LDB1) is a protein cofactor that participates in several multiprotein complexes with transcription factors that regulate mouse forebrain development. Since Ldb1 null mutants display early embryonic lethality, we used a conditional knockout strategy to examine the role of LDB1 in early forebrain development using multiple Cre lines. Loss of Ldb1 from E8.75 using Foxg1Cre caused a disruption of midline boundary structures in the dorsal telencephalon. While this Cre line gave the expected pattern of recombination of the floxed Ldb1 locus, unexpectedly, standard Cre lines that act from embryonic day (E)10.5 (Emx1Cre) and E11.5 (NesCre) did not show efficient or complete recombination in the dorsal telencephalon by E12.5. Intriguingly, this effect was specific to the Ldb1 floxed allele, since three other lines including floxed Ai9 and mTmG reporters, and a floxed Lhx2 line, each displayed the expected spatial patterns of recombination. Furthermore, the incomplete recombination of the floxed Ldb1 locus using NesCre was limited to the dorsal telencephalon, while the ventral telencephalon and the diencephalon displayed the expected loss of Ldb1. This permitted us to examine the requirement for LDB1 in the development of the thalamus in a context wherein the cortex continued to express Ldb1. We report that the somatosensory VB nucleus is profoundly shrunken upon loss of LDB1. Our findings highlight the unusual nature of the Ldb1 locus in terms of recombination efficiency, and also report a novel role for LDB1 during the development of the thalamus.

Lhx2, an evolutionarily conserved, multifunctional regulator of forebrain development.

A hundred years after Lhx2 ortholog apterous was identified as a critical regulator of wing development in Drosophila, LIM-HD gene family members have proved to be versatile and powerful components of the molecular machinery that executes the blueprint of embryogenesis across vertebrate and invertebrate species. Here, we focus on the spatio-temporally varied functions of LIM-homeodomain transcription factor LHX2 in the developing mouse forebrain. Right from its earliest known role in telencephalic and eye field patterning, to the control of the neuron-glia cell fate switch, and the regulation of axon pathfinding and dendritic arborization in late embryonic stages, LHX2 has been identified as a fundamental, temporally dynamic, always necessary, and often sufficient factor in a range of critical developmental phenomena. While Lhx2 mutant phenotypes have been characterized in detail in multiple brain structures, only recently have we advanced in our understanding of the molecular mechanisms by which this factor acts. Common themes emerge from how this multifunctional molecule controls a range of developmental steps in distinct forebrain structures. Examining these shared features, and noting unique aspects of LHX2 function is likely to inform our understanding of how a single factor can bring about a diversity of effects and play central and critical roles across systems and stages. The parallels in LHX2 and APTEROUS functions, and the protein complexes they participate in, offer insights into evolutionary strategies that conserve tool kits and deploy them to play new, yet familiar roles in species separated by hundreds of millions of years.

An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture.

We established an efficient cell culture assay that permits combinatorial genetic perturbations in hippocampal progenitors to examine cell-autonomous mechanisms of fate specification. The procedure begins with ex vivo electroporation of isolated, intact embryonic brains, in a manner similar to in utero electroporation but with greatly improved access and targeting. The electroporated region is then dissected and transiently maintained in organotypic explant culture, followed by dissociation and plating of cells on coverslips for in vitro culture. This assay recapitulates data obtained in vivo with respect to the neuron-glia cell fate switch and can be effectively used to test intrinsic or extrinsic factors that regulate this process. The advantages of this ex vivo procedure over in utero electroporation include the fact that distinct combinations of perturbative reagents can be introduced in different embryos from a single litter, and issues related to embryonic lethality of transgenic animals can be circumvented.

Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis.

This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system for the study of developmental processes that include progenitor proliferation, migration and cell fate acquisition.

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.

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

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.

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.

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.