3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

Experience-dependent functional plasticity and visual response selectivity of surviving subplate neurons in the mouse visual cortex.

Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca2+ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

Altered synaptic connectivity in an in vitro human model of STXBP1 encephalopathy.

Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, at the relevant time period in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain by using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post-conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically and form new synaptic networks. This preparation thus permits the study of genetic manipulations and their downstream effects on an intact developing human cortical network. We focused on STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a reduced density of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

Abnormal development of transient fetal zones in mild isolated fetal ventriculomegaly.

Mild isolated fetal ventriculomegaly (iFVM) is the most common abnormality of the fetal central nervous system. It is characterized by enlargement of one or both of the lateral ventricles (defined as ventricular width greater than 10 mm, but less than 12 mm). Despite its high prevalence, the pathophysiology of iFVM during fetal brain development and the neurobiological substrate beyond ventricular enlargement remain unexplored. In this work, we aimed to establish the relationships between the structural development of transient fetal brain zones/compartments and increased cerebrospinal fluid volume. For this purpose, we used in vivo structural T2-weighted magnetic resonance imaging of 89 fetuses (48 controls and 41 cases with iFVM). Our results indicate abnormal development of transient zones/compartments belonging to both hemispheres (i.e. on the side with and also on the contralateral side without a dilated ventricle) in fetuses with iFVM. Specifically, compared to controls, we observed enlargement of proliferative zones and overgrowth of the cortical plate in iFVM with associated reduction of volumes of central structures, subplate, and fetal white matter. These results indicate that enlarged lateral ventricles might be linked to the development of transient fetal zones and that global brain development should be taken into consideration when evaluating iFVM.

Geodesic theory of long association fibers arrangement in the human fetal cortex.

Association fibers connect different areas of the cerebral cortex over long distances and integrate information to achieve higher brain functions, particularly in humans. Prototyped association fibers are developed to the respective tangential direction throughout the cerebral hemispheres along the deepest border of the subplate during the fetal period. However, how guidance to remote areas is achieved is not known. Because the subplate is located below the cortical surface, the tangential direction of the fibers may be biased by the curved surface geometry due to Sylvian fissure and cortical poles. The fiber length can be minimized if the tracts follow the shortest paths (geodesics) of the curved surface. Here, we propose and examine a theory that geodesics guide the tangential direction of long association fibers by analyzing how geodesics are spatially distributed on the fetal human brains. We found that the geodesics were dense on the saddle-shaped surface of the perisylvian region and sparse on the dome-shaped cortical poles. The geodesics corresponded with the arrangement of five typical association fibers, supporting the theory. Thus, the geodesic theory provides directional guidance information for wiring remote areas and suggests that long association fibers emerge from minimizing their tangential length in fetal brains.

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in the adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity," which has been examined during or after the classic "critical period." Because peripheral perturbations can alter the auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters the ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo widefield imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activities in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs, shifting the excitation-inhibition balance toward excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Fetal indusium griseum is a possible biomarker of the regularity of brain midline development in 3T MR imaging: A retrospective observational study.

INTRODUCTION: This study aimed to assess the visibility of the indusium griseum (IG) in magnetic resonance (MR) scans of the human fetal brain and to evaluate its reliability as an imaging biomarker of the normality of brain midline development. MATERIAL AND METHODS: The retrospective observational study encompassed T2-w 3T MR images from 90 post-mortem fetal brains and immunohistochemical sections from 41 fetal brains (16-40 gestational weeks) without cerebral pathology. Three raters independently inspected and evaluated the visibility of IG in post-mortem and in vivo MR scans. Weighted kappa statistics and regression analysis were used to determine inter- and intra-rater agreement and the type and strength of the association of IG visibility with gestational age. RESULTS: The visibility of the IG was the highest between the 25 and 30 gestational week period, with a very good inter-rater variability (kappa 0.623-0.709) and excellent intra-rater variability (kappa 0.81-0.93). The immunochemical analysis of the histoarchitecture of IG discloses the expression of highly hydrated extracellular molecules in IG as the substrate of higher signal intensity and best visibility of IG during the mid-fetal period. CONCLUSIONS: The knowledge of developmental brain histology and fetal age allows us to predict the IG-visibility in magnetic resonance imaging (MRI) and use it as a biomarker to evaluate the morphogenesis of the brain midline. As a biomarker, IG is significant for post-mortem pathological examination by MRI. Therefore, in the clinical in vivo imaging examination, IG should be anticipated when an assessment of the brain midline structures is needed in mid-gestation, including corpus callosum thickness measurements.

Earliest Experience of a Relatively Rare Sound But Not a Frequent Sound Causes Long-Term Changes in the Adult Auditory Cortex.

Sensory experience during a critical period alters sensory cortical responses and organization. We find that the earliest sound-driven activity in the mouse auditory cortex (ACX) starts before ear-canal opening (ECO). The effects of auditory experience before ECO on ACX development are unknown. We find that in mouse ACX subplate neurons (SPNs), crucial in thalamocortical maturation, respond to sounds before ECO showing oddball selectivity. Before ECO, SPNs are more selective to oddball sounds in auditory streams than thalamo-recipient layer 4 (L4) neurons and not after ECO. We hypothesize that SPN's oddball selectivity can direct the development of L4 responses before ECO. Exposing mice, of either sex, before ECO to a rarely occurring tone in a stream of another tone occurring frequently leads to strengthening the adult cortical representation of the rare tone, but not that of the frequent tone. Results of control exposure experiments at multiple developmental windows that also use only a single tone corroborate the observations. We further explain the strengthening of deviant inputs before ECO and not after ECO using a binary network model mimicking the hierarchical structure of subplate and L4 neurons and response properties derived from our data, with synapses following Hebbian spike time-dependent plasticity learning rule. Information-theoretic analysis with sparse coding assumptions also predicts the observations. Thus, relatively salient low probability sounds in the earliest auditory environment cause long-term changes in the ACX.SIGNIFICANCE STATEMENT Early auditory experience can change the organization and responses of the auditory cortex in adulthood. However, little is known about how auditory experience at prenatal ages changes neural circuits and response properties. In mice at equivalent early developmental stages, we find that auditory experience of a particular kind, with a less frequently occurring sound in a stream of another sound, alters adult cortical responsiveness, specifically of the less frequent sound. However, at the previously known critical period of development, the opposite is observed, where the more frequent sound's representation is strengthened in the adult compared with the less frequent sound. We thus show that a specific type of auditory environment can influence adult auditory processing at the earliest ages.

Transient Coupling between Infragranular and Subplate Layers to Layer 1 Neurons Before Ear Opening and throughout the Critical Period Depends on Peripheral Activity.

Cortical layer 1 (L1) contains a diverse population of interneurons that can modulate processing in superficial cortical layers, but the intracortical sources of synaptic input to these neurons and how these inputs change over development and with sensory experience is unknown. We here investigated the changing intracortical connectivity to L1 in the primary auditory cortex (A1) of mice of both sexes in in vitro slices across development using laser-scanning photostimulation. Before postnatal day (P)10, L1 cells receive excitatory input from within L1, L2/3, L4, and L5/6 as well as from subplate. Excitatory inputs from all layers increase, especially from L4, and peak during P10-P16, around the peak of the critical period for tonotopy. Inhibitory inputs followed a similar pattern. Functional circuit diversity in L1 emerges after P16. In adults, L1 neurons receive ascending inputs from L2/3 and L5/6, but only few inputs from L4. The transient hyperconnectivity from deep layers but not L2/3 is absent in deaf mice. Our results demonstrate that deep excitatory and superficial inhibitory circuits are tightly linked in early development and might provide a functional scaffold for the layers in between. These results suggest that early thalamically driven spontaneous and sensory activity in subplate can be relayed to L1 from the earliest ages on and shape L1 connectivity from deep layers. Our results also reveal a period of high transient columnar hyperconnectivity after ear opening coinciding with the critical period, suggesting that circuits originating in deep layers might play a key role in this process.SIGNIFICANCE STATEMENT L1 contains a diverse population of interneurons that can modulate processing in superficial cortical layers but the sources of synaptic input to these neurons and how these inputs change over development is unknown. We found that during the critical period a large fraction of excitatory inputs to L1 originated in L5/6 and the cortical subplate. This hyperconnectivity is absent in deaf mice. Our results directly demonstrate that deep excitatory and superficial inhibitory circuits are tightly linked in early development and might provide a functional scaffold for the layers in between.

Microglial Characterization in Transient Human Neurodevelopmental Structures.

Human neurodevelopment is characterized by the appearance, development, and disappearance or transformation of various transient structures that underlie the establishment of connectivity within and between future cortical and subcortical areas. Examples of transient structures in the forebrain (among many others) include the subpial granular layer and the subplate zone. We have previously characterized the precise spatiotemporal dynamics of microglia in the human telencephalon. Here, we describe the diversity of microglial morphologies in the subpial granular layer and the subplate zone. Where possible, we couple the predominant morphological phenotype with functional characterizations to infer tentative roles for microglia in a changing neurodevelopmental landscape. We interpret these findings within the context of relevant morphogenetic and neurogenetic events in humans. Due to the unique genetic, molecular, and anatomical features of the human brain and because many human neurological and psychiatric diseases have their origins during development, these structures deserve special attention.

In Utero MRI Identifies Impaired Second Trimester Subplate Growth in Fetuses with Congenital Heart Disease.

The subplate is a transient brain structure which plays a key role in the maturation of the cerebral cortex. Altered brain growth and cortical development have been suggested in fetuses with complex congenital heart disease (CHD) in the third trimester. However, at an earlier gestation, the putative role of the subplate in altered brain development in CHD fetuses is poorly understood. This study aims to examine subplate growth (i.e., volume and thickness) and its relationship to cortical sulcal development in CHD fetuses compared with healthy fetuses by using 3D reconstructed fetal magnetic resonance imaging. We studied 260 fetuses, including 100 CHD fetuses (22.3-32 gestational weeks) and 160 healthy fetuses (19.6-31.9 gestational weeks). Compared with healthy fetuses, CHD fetuses had 1) decreased global and regional subplate volumes and 2) decreased subplate thickness in the right hemisphere overall, in frontal and temporal lobes, and insula. Compared with fetuses with two-ventricle CHD, those with single-ventricle CHD had reduced subplate volume and thickness in right occipital and temporal lobes. Finally, impaired subplate growth was associated with disturbances in cortical sulcal development in CHD fetuses. These findings suggested a potential mechanistic pathway and early biomarker for the third-trimester failure of brain development in fetuses with complex CHD. SIGNIFICANCE STATEMENT: Our findings provide an early biomarker for brain maturational failure in fetuses with congenital heart disease, which may guide the development of future prenatal interventions aimed at reducing neurological compromise of prenatal origin in this high-risk population.

Impaired Hearing and Altered Subplate Circuits During the First and Second Postnatal Weeks of Otoferlin-Deficient Mice.

Sensory deprivation from the periphery impacts cortical development. Otoferlin deficiency leads to impaired cochlear synaptic transmission and is associated with progressive hearing loss in adults. However, it remains elusive how sensory deprivation due to otoferlin deficiency impacts the early development of the auditory cortex (ACX) especially before the onset of low threshold hearing. To test that, we performed in vivo imaging of the ACX in awake mice lacking otoferlin (Otof-/-) during the first and second postnatal weeks and found that spontaneous and sound-driven cortical activity were progressively impaired. We then characterized the effects on developing auditory cortical circuits by performing in vitro recordings from subplate neurons (SPN), the first primary targets of thalamocortical inputs. We found that in Otof-/- pups, SPNs received exuberant connections from excitatory and inhibitory neurons. Moreover, as a population, SPNs showed higher similarity with respect to their circuit topology in the absence of otoferlin. Together, our results show that otoferlin deficiency results in impaired hearing and has a powerful influence on cortical connections and spontaneous activity in early development even before complete deafness. Therefore, peripheral activity has the potential to sculpt cortical structures from the earliest ages, even before hearing impairment is diagnosed.

Partial ablation of frontal cortical subplate leads to developmental abnormalities in KCC2 in the prefrontal cortex.

During early brain development, the subplate relays thalamocortical afferents to the overlying cortex. Disconnection of thalamic inputs to the prefrontal cortex by lesions of the subplate of the developing prefrontal cortex at early neonatal periods result in adult-onset behavioral abnormalities reminiscent of positive, negative, and cognitive symptoms of schizophrenia. Delayed maturation of γ-amino butyric acid (GABA) function may contribute to certain abnormalities of the prefrontal cortex and clinical manifestations of schizophrenia. Lesions to the subplate have also been implicated in developmental abnormalities of GABA neurotransmission in somatosensory and visual cortices. Therefore, we sought to examine the effects of subplate lesions in the developing prefrontal cortex of rats on the expression of GABA markers [parvalbumin and glutamic acid decarboxylase (GAD67)] and proteins responsible for GABAergic synaptic maturation [potassium-chloride cotransporter (KCC2) and sodium­potassium-chloride cotransporter (NKCC1)]. Lesioned and control rats were sacrificed between postnatal days (P) 5 and 90 and immunolabeled for parvalbumin, GAD67, KCC2, and NKCC1 in the prelimbic area of the prefrontal cortex. We found decreased immunoreactivity of KCC2 on neuronal cell membranes at P11 compared to control rats. However, the overall immunoreactivity of KCC2 and NKCC1 did not differ between lesion and control animals at all time points studied. Lesioned rats also showed decreased expression of parvalbumin, but not GAD67. Our results indicate that mechanisms underlying trafficking and membrane binding of KCC2 may contribute to altered GABA receptor function during development in schizophrenia.

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.

Association between Quantitative MR Markers of Cortical Evolving Organization and Gene Expression during Human Prenatal Brain Development.

The relationship between structural changes of the cerebral cortex revealed by Magnetic Resonance Imaging (MRI) and gene expression in the human fetal brain has not been explored. In this study, we aimed to test the hypothesis that relative regional thickness (a measure of cortical evolving organization) of fetal cortical compartments (cortical plate [CP] and subplate [SP]) is associated with expression levels of genes with known cortical phenotype. Mean regional SP/CP thickness ratios across age measured on in utero MRI of 25 healthy fetuses (20-33 gestational weeks [GWs]) were correlated with publicly available regional gene expression levels (23-24 GW fetuses). Larger SP/CP thickness ratios (more pronounced cortical evolving organization) was found in perisylvian regions. Furthermore, we found a significant association between SP/CP thickness ratio and expression levels of the FLNA gene (mutated in periventricular heterotopia, congenital heart disease, and vascular malformations). Further work is needed to identify early MRI biomarkers of gene expression that lead to abnormal cortical development.

Transient Subplate Sublayer Forms Unique Corridor for Differential Ingrowth of Associative Pulvinar and Primary Visual Projection in the Prospective Visual Cortical Areas of the Human Fetal Occipital Lobe.

Cytoarchitectonical parcellation of the visual cortex into the striate and extrastriate cortex requires complex histogenetic events within a precise spatio-temporal frame to attain the specification of areal domains and associated thalamocortical connections during the fetal brain development. We analyzed a deep subplate cellular monolayer (subplate "corridor" cells) present during a restricted period of 13-15 postconceptional weeks, showing the 3D caudo-ventro-medial position in the human fetal occipital lobe, corresponding to the segregation point of pulvinocortical and geniculocortical fibers at the prospective area 17/18 border. Immunofluorescence stainings revealed subplate "corridor" cells as the specific class of the deepest subplate neurons (NeuN+, Tbr1+, Cplx3+) expressing axon guidance molecules (Sema-3A+, EphA6+), presumably for the attraction of pulvinocortical axons and the repulsion of geniculocortical axons growing at that time (SNAP25+, Syn+, FN+). Furthermore, quantitative analysis of the subplate "corridor" region of interest, considering cell number, immunofluorescence signal intensity per cell and per region, revealed significant differences to other regions across the tangential circumference of the developing cerebral wall. Thus, our study sheds new light on the deepest subplate sublayer, strategically aligned along the growing axon systems in the prospective visual system, suggesting the establishment of the area 17/18 border by differential thalamocortical input during the fetal brain development.

Aberrant Claustrum Microstructure in Humans after Premature Birth.

Several observations suggest an impact of prematurity on the claustrum. First, the claustrum's development appears to depend on transient subplate neurons of intra-uterine brain development, which are affected by prematurity. Second, the claustrum is the most densely connected region of the mammalian forebrain relative to its volume; due to its effect on pre-oligodendrocytes, prematurity impacts white matter connections and thereby the development of sources and targets of such connections, potentially including the claustrum. Third, due to its high connection degree, the claustrum contributes to general cognitive functioning (e.g., selective attention and task switching/maintaining); general cognitive functioning, however, is at risk in prematurity. Thus, we hypothesized altered claustrum structure after premature birth, with these alterations being associated with impaired general cognitive performance in premature born persons. Using T1-weighted and diffusion-weighted magnetic resonance imaging in 70 very preterm/very low-birth-weight (VP/VLBW) born adults and 87 term-born adults, we found specifically increased mean diffusivity in the claustrum of VP/VLBW adults, associated both with low birth weight and at-trend with reduced IQ. This result demonstrates altered claustrum microstructure after premature birth. Data suggest aberrant claustrum development, which is potentially related with aberrant subplate neuron and forebrain connection development of prematurity.

The Subplate Layers: The Superficial and Deep Subplate Can be Discriminated on 3 Tesla Human Fetal Postmortem MRI.

The subplate (SP) is a transient structure of the human fetal brain that becomes the most prominent layer of the developing pallium during the late second trimester. It is important in the formation of thalamocortical and cortico-cortical connections. The SP is vulnerable in perinatal brain injury and may play a role in complex neurodevelopmental disorders, such as schizophrenia and autism. Nine postmortem fetal human brains (19-24 GW) were imaged on a 3 Tesla MR scanner and the T2-w images in the frontal and temporal lobes were compared, in each case, with the histological slices of the same brain. The brains were confirmed to be without any brain pathology. The purpose of this study was to demonstrate that the superficial SP (sSP) and deep SP (dSP) can be discriminated on postmortem MR images. More specifically, we aimed to clarify that the observable, thin, hyperintense layer below the cortical plate in the upper SP portion on T2-weighted MR images has an anatomical correspondence to the histologically established sSP. Therefore, the distinction between the sSP and dSP layers, using clinically available MR imaging methodology, is possible in postmortem MRI and can help in the imaging interpretation of the fetal cerebral layers.

Loss of Dmrt5 Affects the Formation of the Subplate and Early Corticogenesis.

Dmrt5 (Dmrta2) and Dmrt3 are key regulators of cortical patterning and progenitor proliferation and differentiation. In this study, we show an altered apical to intermediate progenitor transition, with a delay in SP neurogenesis and premature birth of Ctip2+ cortical neurons in Dmrt5-/- mice. In addition to the cortical progenitors, DMRT5 protein appears present in postmitotic subplate (SP) and marginal zone neurons together with some migrating cortical neurons. We observed the altered split of preplate and the reduced SP and disturbed radial migration of cortical neurons into cortical plate in Dmrt5-/- brains and demonstrated an increase in the proportion of multipolar cells in primary neuronal cultures from Dmrt5-/- embryonic brains. Dmrt5 affects cortical development with specific time sensitivity that we described in two conditional mice with slightly different deletion time. We only observed a transient SP phenotype at E15.5, but not by E18.5 after early (Dmrt5lox/lox;Emx1Cre), but not late (Dmrt5lox/lox;NestinCre) deletion of Dmrt5. SP was less disturbed in Dmrt5lox/lox;Emx1Cre and Dmrt3-/- brains than in Dmrt5-/- and affects dorsomedial cortex more than lateral and caudal cortex. Our study demonstrates a novel function of Dmrt5 in the regulation of early SP formation and radial cortical neuron migration. SUMMARY STATEMENT: Our study demonstrates a novel function of Dmrt5 in regulating marginal zone and subplate formation and migration of cortical neurons to cortical plate.

Spatiotemporal Differences in the Regional Cortical Plate and Subplate Volume Growth during Fetal Development.

The regional specification of the cerebral cortex can be described by protomap and protocortex hypotheses. The protomap hypothesis suggests that the regional destiny of cortical neurons and the relative size of the cortical area are genetically determined early during embryonic development. The protocortex hypothesis suggests that the regional growth rate is predominantly shaped by external influences. In order to determine regional volumes of cortical compartments (cortical plate (CP) or subplate (SP)) and estimate their growth rates, we acquired T2-weighted in utero MRIs of 40 healthy fetuses and grouped them into early (<25.5 GW), mid- (25.5-31.6 GW), and late (>31.6 GW) prenatal periods. MRIs were segmented into CP and SP and further parcellated into 22 gyral regions. No significant difference was found between periods in regional volume fractions of the CP or SP. However, during the early and mid-prenatal periods, we found significant differences in relative growth rates (% increase per GW) between regions of cortical compartments. Thus, the relative size of these regions are most likely conserved and determined early during development whereas more subtle growth differences between regions are fine-tuned later, during periods of peak thalamocortical growth. This is in agreement with both the protomap and protocortex hypothesis.