Mammalian forebrain cholinergic neurons are composed of local circuit neurons in the striatum and projection neurons in the basal forebrain. These neurons are known to arise from a common pool of progenitors that primarily resides in the medial ganglionic eminence (MGE). However, little is known about the genetic programs that differentiate these two types of cholinergic neurons. Using inducible genetic fate mapping, here we examined the developmental fate of cells that express the homeodomain transcription factor Gbx2 in the MGE. We show that the Gbx2 lineage-derived cells that undergo tangential migration exclusively give rise to almost all cholinergic interneurons in the striatum, whereas those undergoing radial migration mainly produce noncholinergic neurons in the basal forebrain. Deletion of Gbx2 throughout the mouse embryo or specifically in the MGE results in abnormal distribution and significant reduction of cholinergic neurons in the striatum. We show that early-born (before embryonic day 12.5) cholinergic interneurons preferentially populate the lateral aspect of the striatum and mature earlier than late-born (after embryonic day 12.5) neurons, which normally reside in the medial part of the striatum. In the absence of Gbx2, early-born striatal cholinergic precursors display abnormal neurite outgrowth and increased complexity, and abnormally contribute to the medial part of the caudate-putamen, whereas late-born striatal cholinergic interneurons are mostly missing. Together, our data demonstrate that Gbx2 is required for the development of striatal cholinergic interneurons, perhaps by regulating tangential migration of the striatal cholinergic precursors.

BackgroundThe habenula and the thalamus are two critical nodes in the forebrain circuitry and they connect the midbrain and the cerebral cortex in vertebrates. The habenula is derived from the epithalamus and rests dorsally to the thalamus. Both epithalamus and thalamus arise from a single diencephalon segment called prosomere (p)2. Shh is expressed in the ventral midline of the neural tube and in the mid-diencephalic organizer (MDO) at the zona limitans intrathalamica between thalamus and prethalamus. Acting as a morphogen, Shh plays an important role in regulating cell proliferation and survival in the diencephalon and thalamic patterning. The molecular regulation of the MDO Shh expression and the potential role of Shh in development of the habenula remain largely unclear.ResultsWe show that deleting paired-box and homeobox-containing gene Pax6 results in precocious and expanded expression of Shh in the prospective MDO in fish and mice, whereas gain-of-function of pax6 inhibits MDO shh expression in fish. Using gene expression and genetic fate mapping, we have characterized the expression of molecular markers that demarcate the progenitors and precursors of habenular neurons. We show that the thalamic domain is shifted dorsally and the epithalamus is missing in the alar plate of p2 in the Pax6 mutant mouse. Conversely, the epithalamus is expanded ventrally at the expense of the thalamus in mouse embryos with reduced Shh activity. Significantly, attenuating Shh signaling largely rescues the patterning of p2 and restores the epithalamus in Pax6 mouse mutants, suggesting that Shh acts downstream of Pax6 in controlling the formation of the habenula. Similar to that found in the mouse, we show that pax6 controls the formation of the epithalamus mostly via the regulation of MDO shh expression in zebrafish.ConclusionsOur findings demonstrate that Pax6 has an evolutionarily conserved function in establishing the temporospatial expression of Shh in the MDO in vertebrates. Furthermore, Shh mediates Pax6 function in regulating the partition of the p2 domain into the epithalamus and thalamus.

SUMMARYCombinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCAspecific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs.

The diencephalon gives rise to structures that play an important role in connecting the anterior forebrain with the rest of the central nervous system. The thalamus is the major diencephalic derivative that functions as a relay station between the cortex and other lower order sensory systems. Almost two decades ago, neuromeric/prosomeric models were proposed describing the subdivision and potential segmentation of the diencephalon. Unlike the laminar structure of the cortex, the diencephalon is progressively divided into distinct functional compartments consisting principally of thalamus, epithalamus, pretectum, and hypothalamus. Neurons generated within these domains further aggregate to form clusters called nuclei, which form specific structural and functional units. We review the recent advances in understanding the genetic mechanisms that are involved in the patterning and compartment formation of the diencephalon.

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 β-CATENIN are expressed in the mouse and human embryonic choroid plexus epithelium indicating that this pathway is active. Point mutations in human β-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 β-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.

The thalamus and habenula, two important nodes of the forebrain circuitry, are derived from a single developmental compartment, called prosomere 2, in the diencephalon. Habenular and thalamic neurons display distinct molecular identity, neurochemistry, and connectivity. Furthermore, their progenitors exhibit distinctive neurogenic patterns with a marked delay in the onset of neurogenesis in the thalamus. However, the progenitors in prosomere 2 express many common developmental regulators and the mechanism underlying the specification and differentiation of these two populations of neurons remains unknown. Gbx2, coding for a homeodomain transcription factor, is initially expressed in thalamic neuronal precursors that have just exited the cell cycle, and its expression is maintained in many mature thalamic neurons in adults. Deletion of Gbx2 severely disrupts histogenesis of the thalamus and abolishes thalamocortical projections in mice. Here, by using genome-wide transcriptional profiling, we show that Gbx2 promotes thalamic but inhibits habenular molecular characters. Remarkably, although Gbx2 is expressed in postmitotic neuronal precursors, deletion of Gbx2 changes gene expression and cell proliferation in dividing progenitors in the developing thalamus. These defects are partially rescued by the mosaic presence of wild-type cells, demonstrating a cell non-autonomous role of Gbx2 in regulating the development of thalamic progenitors. Our results suggest that Gbx2 is essential for the acquisition of the thalamic neuronal identity by repressing habenular identity through a feedback signaling from postmitotic neurons to progenitors.

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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. Since the hem expresses multiple Wnt genes, we examined whether β-CATENIN, which mediates canonical Wnt signaling and also participates in cell adhesion, is necessary for the development of hem-derived lineages. We report that the fimbrial glial scaffold is disorganized and CR cells are mispositioned upon hem-specific disruption of β-CATENIN. Consequently, the dentate migratory stream is severely affected, and the dentate gyrus fails to form. Using selective Cre drivers, we further determined that β-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 β-CATENIN for the organization of the fimbrial scaffold and a secondary role for this factor in dentate gyrus morphogenesis.