Developmental emergence of first- and higher-order thalamic neuron molecular identities.

The thalamus is organized into nuclei that have distinct input and output connectivities with the cortex. Whereas first-order (FO) nuclei - also called core nuclei - relay input from sensory organs on the body surface and project to primary cortical sensory areas, higher-order (HO) nuclei - matrix nuclei - instead receive their driver input from the cortex and project to secondary and associative areas within cortico-thalamo-cortical loops. Input-dependent processes have been shown to play a crucial role in the emergence of FO thalamic neuron identity from a ground-state HO neuron identity, yet how this identity emerges during development remains unknown. Here, using single-cell RNA sequencing of the developing mouse embryonic thalamus, we show that, although they are born together, HO neurons start differentiating earlier than FO neurons. Within the FO visual thalamus, postnatal peripheral input is crucial for the maturation of excitatory, but not inhibitory, neurons. Our findings reveal different differentiation tempos and input sensitivities of HO and FO neurons, and highlight neuron type-specific molecular differentiation programs in the developing thalamus.

Altered thalamocortical tract trajectory growth with undisrupted thalamic parcellation pattern in human lissencephaly brain at mid-gestational stage.

Proper topographically organized neural connections between the thalamus and the cerebral cortex are mandatory for thalamus function. Thalamocortical (TC) fiber growth begins during the embryonic period and completes by the third trimester of gestation, so that human neonates at birth have a thalamus with a near-facsimile of adult functional parcellation. Whether congenital neocortical anomaly (e.g., lissencephaly) affects TC connection in humans is unknown. Here, via diffusion MRI fiber-tractography analysis of long-term formalin-fixed postmortem fetal brain diagnosed as lissencephaly in comparison with an age-matched normal one, we found similar topological patterns of thalamic subregions and of internal capsule parcellated by TC fibers. However, lissencephaly fetal brain showed white matter structural changes, including fewer/less organized TC fibers and optic radiations, and much less cortical plate invasion by TC fibers - particularly around the shallow central sulcus. Diffusion MRI fiber tractography of normal fetal brains at 15, 23, and 26 gestational weeks (GW) revealed dynamic volumetric change of each parcellated thalamic subregion, suggesting coupled developmental progress of the thalamus with the corresponding cortex. Moreover, from GW23 and GW26 normal fetal brains, TC endings in the cortical plate could be delineated to reflect cumulative progressive TC invasion of cortical plate. By contrast, lissencephaly brain showed a dramatic decrease in TC invasion of the cortical plate. Our study thus shows the feasibility of diffusion MRI fiber tractography in postmortem long-term formalin-fixed fetal brains to disclose the developmental progress of TC tracts coordinating with thalamic and neocortical growth both in normal and lissencephaly fetal brains at mid-gestational stage.

Thalamic activity-dependent specification of sensory input neurons in the developing chick entopallium.

During development, cell-intrinsic and cell-extrinsic factors play important roles in neuronal differentiation; however, the underlying mechanisms in nonmammalian species remain largely unknown. We here investigated the mechanisms responsible for the differentiation of sensory input neurons in the chick entopallium, which receives its primary visual input via the tectofugal pathway from the nucleus rotundus. The results obtained revealed that input neurons in the entopallium expressed Potassium Voltage-Gated Channel Subfamily H Member 5 (KCNH5/EAG2) mRNA from embryonic day (E) 11. On the other hand, the onset of protein expression was E20, which was 1 day before hatching. We confirm that entopallium input neurons in chicks were generated during early neurogenesis in the lateral and ventral ventricular zones. Notably, neurons derived from the lateral (LP) and ventral pallium (VP) exhibited a spatially distinct distribution along the rostro-caudal axis. We further demonstrated that the expression of EAG2 was directly regulated by input activity from thalamic axons. Collectively, the present results reveal that thalamic input activity is essential for specifying input neurons among LP- and VP-derived early-generated neurons in the developing chick entopallium.

Higher-order thalamocortical circuits are specified by embryonic cortical progenitor types in the mouse brain.

The sensory cortex receives synaptic inputs from both first-order and higher-order thalamic nuclei. First-order inputs relay simple stimulus properties from the periphery, whereas higher-order inputs relay more complex response properties, provide contextual feedback, and modulate plasticity. Here, we reveal that a cortical neuron's higher-order input is determined by the type of progenitor from which it is derived during embryonic development. Within layer 4 (L4) of the mouse primary somatosensory cortex, neurons derived from intermediate progenitors receive stronger higher-order thalamic input and exhibit greater higher-order sensory responses. These effects result from differences in dendritic morphology and levels of the transcription factor Lhx2, which are specified by the L4 neuron's progenitor type. When this mechanism is disrupted, cortical circuits exhibit altered higher-order responses and sensory-evoked plasticity. Therefore, by following distinct trajectories, progenitor types generate diversity in thalamocortical circuitry and may provide a general mechanism for differentially routing information through the cortex.

Thalamocortical axons regulate neurogenesis and laminar fates in the early sensory cortex.

Area-specific axonal projections from the mammalian thalamus shape unique cellular organization in target areas in the adult neocortex. How these axons control neurogenesis and early neuronal fate specification is poorly understood. By using mutant mice lacking the majority of thalamocortical axons, we show that these axons are required for the production and specification of the proper number of layer 4 neurons in primary sensory areas by the neonatal stage. Part of these area-specific roles is played by the thalamus-derived molecule, VGF. Our work reveals that extrinsic cues from sensory thalamic projections have an early role in the formation of cortical cytoarchitecture by enhancing the production and specification of layer 4 neurons.

LacZ-reporter mapping of Dlx5/6 expression and genoarchitectural analysis of the postnatal mouse prethalamus.

We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.

Generation of Regionally Specified Human Brain Organoids Resembling Thalamus Development.

Thalamus is a critical information relay hub in the cortex; its malfunction causes multiple neurological and psychiatric disorders. However, there are no model systems to study the development and function of human thalamus. Here, we present a protocol to generate regionally specified human brain organoids that recapitulate the development of the thalamus using human pluripotent stem cells (hPSCs). Thalamic organoids can be used to study human thalamus development, to model related diseases, and to discover potential therapeutics. For complete information on human thalamic organoids and their application, please refer to the paper by Xiang et al. (2019).

The maternal microbiome modulates fetal neurodevelopment in mice.

'Dysbiosis' of the maternal gut microbiome, in response to challenges such as infection1, altered diet2 and stress3 during pregnancy, has been increasingly associated with abnormalities in brain function and behaviour of the offspring4. However, it is unclear whether the maternal gut microbiome influences neurodevelopment during critical prenatal periods and in the absence of environmental challenges. Here we investigate how depletion and selective reconstitution of the maternal gut microbiome influences fetal neurodevelopment in mice. Embryos from antibiotic-treated and germ-free dams exhibited reduced brain expression of genes related to axonogenesis, deficient thalamocortical axons and impaired outgrowth of thalamic axons in response to cell-extrinsic factors. Gnotobiotic colonization of microbiome-depleted dams with a limited consortium of bacteria prevented abnormalities in fetal brain gene expression and thalamocortical axonogenesis. Metabolomic profiling revealed that the maternal microbiome regulates numerous small molecules in the maternal serum and the brains of fetal offspring. Select microbiota-dependent metabolites promoted axon outgrowth from fetal thalamic explants. Moreover, maternal supplementation with these metabolites abrogated deficiencies in fetal thalamocortical axons. Manipulation of the maternal microbiome and microbial metabolites during pregnancy yielded adult offspring with altered tactile sensitivity in two aversive somatosensory behavioural tasks, but no overt differences in many other sensorimotor behaviours. Together, our findings show that the maternal gut microbiome promotes fetal thalamocortical axonogenesis, probably through signalling by microbially modulated metabolites to neurons in the developing brain.

TCF7L2 regulates postmitotic differentiation programmes and excitability patterns in the thalamus.

Neuronal phenotypes are controlled by terminal selector transcription factors in invertebrates, but only a few examples of such regulators have been provided in vertebrates. We hypothesised that TCF7L2 regulates different stages of postmitotic differentiation in the thalamus, and functions as a thalamic terminal selector. To investigate this hypothesis, we used complete and conditional knockouts of Tcf7l2 in mice. The connectivity and clustering of neurons were disrupted in the thalamo-habenular region in Tcf7l2-/- embryos. The expression of subregional thalamic and habenular transcription factors was lost and region-specific cell migration and axon guidance genes were downregulated. In mice with a postnatal Tcf7l2 knockout, the induction of genes that confer thalamic terminal electrophysiological features was impaired. Many of these genes proved to be direct targets of TCF7L2. The role of TCF7L2 in terminal selection was functionally confirmed by impaired firing modes in thalamic neurons in the mutant mice. These data corroborate the existence of master regulators in the vertebrate brain that control stage-specific genetic programmes and regional subroutines, maintain regional transcriptional network during embryonic development, and induce terminal selection postnatally.

The enigmatic fetal subplate compartment forms an early tangential cortical nexus and provides the framework for construction of cortical connectivity.

The most prominent transient compartment of the primate fetal cortex is the deep, cell-sparse, synapse-containing subplate compartment (SPC). The developmental role of the SPC and its extraordinary size in humans remain enigmatic. This paper evaluates evidence on the development and connectivity of the SPC and discusses its role in the pathogenesis of neurodevelopmental disorders. A synthesis of data shows that the subplate becomes a prominent compartment by its expansion from the deep cortical plate (CP), appearing well-delineated on MR scans and forming a tangential nexus across the hemisphere, consisting of an extracellular matrix, randomly distributed postmigratory neurons, multiple branches of thalamic and long corticocortical axons. The SPC generates early spontaneous non-synaptic and synaptic activity and mediates cortical response upon thalamic stimulation. The subplate nexus provides large-scale interareal connectivity possibly underlying fMR resting-state activity, before corticocortical pathways are established. In late fetal phase, when synapses appear within the CP, transient the SPC coexists with permanent circuitry. The histogenetic role of the SPC is to provide interactive milieu and capacity for guidance, sorting, "waiting" and target selection of thalamocortical and corticocortical pathways. The new evolutionary role of the SPC and its remnant white matter neurons is linked to the increasing number of associative pathways in the human neocortex. These roles attributed to the SPC are regulated using a spatiotemporal gene expression during critical periods, when pathogenic factors may disturb vulnerable circuitry of the SPC, causing neurodevelopmental cognitive circuitry disorders.

FGF10 regulates thalamocortical axon guidance in the developing thalamus.

Thalamocortical axons (TCAs) transmit sensory information to the neocortex by responding to a variety of guidance cues in the environment. Similar to classical guidance cues (ephrins, slits, semaphorins and netrins), morphogens of FGFs can also help axons navigate to their targets. Here, expression analyses reveal that FGF10 is expressed in the chick prethalamus during the navigation of TCAs. Then, using ex vivo analyses in chick explants, we demonstrate a dose-dependent effect of FGF10 on thalamic axons: low concentration of FGF10 attracts thalamic axons, while high concentration FGF10 repels thalamic axons. Moreover, inhibition of FGF10 function indicates that FGF10 exerts a direct effect on thalamic axons. Together, these studies reveal a direct role for the member of FGF7 subfamily, FGF10, in the axonal navigation of TCAs.

FGF3 from the Hypothalamus Regulates the Guidance of Thalamocortical Axons.

Thalamus is an important sensory relay station: afferent sensory information, except olfactory signals, is transmitted by thalamocortical axons (TCAs) to the cerebral cortex. The pathway choice of TCAs depends on diverse diffusible or substrate-bound guidance cues in the environment. Not only classical guidance cues (ephrins, slits, semaphorins, and netrins), morphogens, which exerts patterning effects during early embryonic development, can also help axons navigate to their targets at later development stages. Here, expression analyses reveal that morphogen Fibroblast growth factor (FGF)-3 is expressed in the chick ventral diencephalon, hypothalamus, during the pathfinding of TCAs. Then, using in vitro analyses in chick explants, we identify a concentration-dependent effect of FGF3 on thalamic axons: attractant 100 ng/mL FGF3 transforms to a repellent at high concentration 500 ng/mL. Moreover, inhibition of FGF3 guidance functions indicates that FGF3 signaling is necessary for the correct navigation of thalamic axons. Together, these studies demonstrate a direct effect for the member of FGF7 subfamily, FGF3, in the axonal pathfinding of TCAs.

The p75 neurotrophin receptor is required for the survival of neuronal progenitors and normal formation of the basal forebrain, striatum, thalamus and neocortex.

During development, the p75 neurotrophin receptor (p75NTR) is widely expressed in the nervous system where it regulates neuronal differentiation, migration and axonal outgrowth. p75NTR also mediates the survival and death of newly born neurons, with functional outcomes being dependent on both timing and cellular context. Here, we show that knockout of p75NTR from embryonic day 10 (E10) in neural progenitors using a conditional Nestin-Cre p75NTR floxed mouse causes increased apoptosis of progenitor cells. By E14.5, the number of Tbr2-positive progenitor cells was significantly reduced and the rate of neurogenesis was halved. Furthermore, in adult knockout mice, there were fewer cortical pyramidal neurons, interneurons, cholinergic basal forebrain neurons and striatal neurons, corresponding to a relative reduction in volume of these structures. Thalamic midline fusion during early postnatal development was also impaired in Nestin-Cre p75NTR floxed mice, indicating a novel role for p75NTR in the formation of this structure. The phenotype of this strain demonstrates that p75NTR regulates multiple aspects of brain development, including cortical progenitor cell survival, and that expression during early neurogenesis is required for appropriate formation of telencephalic structures.

Development and Arealization of the Cerebral Cortex.

Adult cortical areas consist of specialized cell types and circuits that support unique higher-order cognitive functions. How this regional diversity develops from an initially uniform neuroepithelium has been the subject of decades of seminal research, and emerging technologies, including single-cell transcriptomics, provide a new perspective on area-specific molecular diversity. Here, we review the early developmental processes that underlie cortical arealization, including both cortex intrinsic and extrinsic mechanisms as embodied by the protomap and protocortex hypotheses, respectively. We propose an integrated model of serial homology whereby intrinsic genetic programs and local factors establish early transcriptomic differences between excitatory neurons destined to give rise to broad "proto-regions," and activity-dependent mechanisms lead to progressive refinement and formation of sharp boundaries between functional areas. Finally, we explore the potential of these basic developmental processes to inform our understanding of the emergence of functional neural networks and circuit abnormalities in neurodevelopmental disorders.

Prenatal activity from thalamic neurons governs the emergence of functional cortical maps in mice.

The mammalian brain's somatosensory cortex is a topographic map of the body's sensory experience. In mice, cortical barrels reflect whisker input. We asked whether these cortical structures require sensory input to develop or are driven by intrinsic activity. Thalamocortical columns, connecting the thalamus to the cortex, emerge before sensory input and concur with calcium waves in the embryonic thalamus. We show that the columnar organization of the thalamocortical somatotopic map exists in the mouse embryo before sensory input, thus linking spontaneous embryonic thalamic activity to somatosensory map formation. Without thalamic calcium waves, cortical circuits become hyperexcitable, columnar and barrel organization does not emerge, and the somatosensory map lacks anatomical and functional structure. Thus, a self-organized protomap in the embryonic thalamus drives the functional assembly of murine thalamocortical sensory circuits.

Development of the thalamus: From early patterning to regulation of cortical functions.

The thalamus is a brain structure of the vertebrate diencephalon that plays a central role in regulating diverse functions of the cerebral cortex. In traditional view of vertebrate neuroanatomy, the thalamus includes three regions, dorsal thalamus, ventral thalamus, and epithalamus. Recent molecular embryological studies have redefined the thalamus and the associated axial nomenclature of the diencephalon in the context of forebrain patterning. This new view has provided a useful conceptual framework for studies on molecular mechanisms of patterning, neurogenesis and fate specification in the thalamus as well as the guidance mechanisms for thalamocortical axons. Additionally, the availability of genetic tools in mice has led to important findings on how thalamic development is linked to the development of other brain regions, particularly the cerebral cortex. This article will give an overview of the organization of the embryonic thalamus and how progenitor cells in the thalamus generate neurons that are organized into discrete nuclei. I will then discuss how thalamic development is orchestrated with the development of the cerebral cortex and other brain regions. This article is categorized under: Nervous System Development > Vertebrates: Regional Development Nervous System Development > Vertebrates: General Principles.

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.

Defining developmental diversification of diencephalon neurons through single cell gene expression profiling.

The embryonic diencephalon forms integration centers and relay stations in the forebrain. Anecdotal expression studies suggest that the diencephalon contains multiple developmental compartments and subdivisions. Here, we utilized single cell RNA sequencing to profile transcriptomes of dissociated cells from the diencephalon of E12.5 mouse embryos. We identified the divergence of different progenitors, intermediate progenitors, and emerging neurons. By mapping the identified cell groups to their spatial origins, we characterized the molecular features of cell types and cell states arising from various diencephalic domains. Furthermore, we reconstructed the developmental trajectory of distinct cell lineages, and thereby identified the genetic cascades and gene regulatory networks underlying the progression of the cell cycle, neurogenesis and cellular diversification. The analysis provides new insights into the molecular mechanisms underlying the amplification of intermediate progenitor cells in the thalamus. The single cell-resolved trajectories not only confirm a close relationship between the rostral thalamus and prethalamus, but also uncover an unexpected close relationship between the caudal thalamus, epithalamus and rostral pretectum. Our data provide a useful resource for systematic studies of cell heterogeneity and differentiation kinetics within the diencephalon.

Polygenic risk for neuropsychiatric disease and vulnerability to abnormal deep grey matter development.

Neuropsychiatric disease has polygenic determinants but is often precipitated by environmental pressures, including adverse perinatal events. However, the way in which genetic vulnerability and early-life adversity interact remains obscure. We hypothesised that the extreme environmental stress of prematurity would promote neuroanatomic abnormality in individuals genetically vulnerable to psychiatric disorders. In 194 unrelated infants (104 males, 90 females), born before 33 weeks of gestation (mean gestational age 29.7 weeks), we combined Magnetic Resonance Imaging with a polygenic risk score (PRS) for five psychiatric pathologies to test the prediction that: deep grey matter abnormalities frequently seen in preterm infants are associated with increased polygenic risk for psychiatric illness. The variance explained by the PRS in the relative volumes of four deep grey matter structures (caudate nucleus, thalamus, subthalamic nucleus and lentiform nucleus) was estimated using linear regression both for the full, mixed ancestral, cohort and a subsample of European infants. Psychiatric PRS was negatively associated with lentiform volume in the full cohort (ß = -0.24, p = 8 × 10-4) and a European subsample (ß = -0.24, p = 8 × 10-3). Genetic variants associated with neuropsychiatric disease increase vulnerability to abnormal lentiform development after perinatal stress and are associated with neuroanatomic changes in the perinatal period.

The Ciliopathy Gene Ftm/Rpgrip1l Controls Mouse Forebrain Patterning via Region-Specific Modulation of Hedgehog/Gli Signaling.

Primary cilia are essential for CNS development. In the mouse, they play a critical role in patterning the spinal cord and telencephalon via the regulation of Hedgehog/Gli signaling. However, despite the frequent disruption of this signaling pathway in human forebrain malformations, the role of primary cilia in forebrain morphogenesis has been little investigated outside the telencephalon. Here we studied development of the diencephalon, hypothalamus and eyes in mutant mice in which the Ftm/Rpgrip1l ciliopathy gene is disrupted. At the end of gestation, Ftm-/- fetuses displayed anophthalmia, a reduction of the ventral hypothalamus and a disorganization of diencephalic nuclei and axonal tracts. In Ftm-/- embryos, we found that the ventral forebrain structures and the rostral thalamus were missing. Optic vesicles formed but lacked the optic cups. In Ftm-/- embryos, Sonic hedgehog (Shh) expression was virtually lost in the ventral forebrain but maintained in the zona limitans intrathalamica (ZLI), the mid-diencephalic organizer. Gli activity was severely downregulated but not lost in the ventral forebrain and in regions adjacent to the Shh-expressing ZLI. Reintroduction of the repressor form of Gli3 into the Ftm-/- background restored optic cup formation. Our data thus uncover a complex role of cilia in development of the diencephalon, hypothalamus and eyes via the region-specific control of the ratio of activator and repressor forms of the Gli transcription factors. They call for a closer examination of forebrain defects in severe ciliopathies and for a search for ciliopathy genes as modifiers in other human conditions with forebrain defects.SIGNIFICANCE STATEMENT The Hedgehog (Hh) signaling pathway is essential for proper forebrain development as illustrated by a human condition called holoprosencephaly. The Hh pathway relies on primary cilia, cellular organelles that receive and transduce extracellular signals and whose dysfunctions lead to rare inherited diseases called ciliopathies. To date, the role of cilia in the forebrain has been poorly studied outside the telencephalon. In this paper we study the role of the Ftm/Rpgrip1l ciliopathy gene in mouse forebrain development. We uncover complex functions of primary cilia in forebrain morphogenesis through region-specific modulation of the Hh pathway. Our data call for further examination of forebrain defects in ciliopathies and for a search for ciliopathy genes as modifiers in human conditions affecting forebrain development.