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1.  Sizn1 Is a Novel Protein That Functions as a Transcriptional Coactivator of Bone Morphogenic Protein Signaling▿ § 
Molecular and Cellular Biology  2007;28(5):1565-1572.
Bone morphogenic proteins (BMPs) play pleotrophic roles in nervous system development, and their signaling is highly regulated at virtually every step in the pathway. We have cloned a novel gene, Sizn1 (Smad-interacting zinc finger protein), which functions as a transcriptional coactivator of BMP signaling. It positively modulates BMP signaling by interacting with Smad family members and associating with CBP in the transcription complex. Sizn1 is expressed in the ventral embryonic forebrain, where, as we will show, it contributes to BMP-dependent, cholinergic-neuron-specific gene expression. These data indicate that Sizn1 is a positive modulator of BMP signaling and provide further insight into how BMP signaling can be modulated in neuronal progenitor subsets to influence cell-type-specific gene expression and development.
PMCID: PMC2258788  PMID: 18160706
2.  Evidence that SIZN1 is a Candidate X-Linked Mental Retardation Gene 
An estimated 1-3% of individuals within the United States are diagnosed with mental retardation (MR), yet the cause is unknown in nearly 50% of the patients. While several environmental, genetic and combined teratogenetic etiologies have been identified, many causative genes remain to be identified. Furthermore, the pathogenetic mechanisms underlying MR are known for very few of these genes. Males have a much higher incidence of MR implicating genes on the X-chromosome. We have recently identified a novel gene, SIZN1, on the X-chromosome and showed that it functions in modulating the BMP signaling pathway. Furthermore, we have shown this gene is necessary for basal forebrain cholinergic neuron (BFCN) specific gene expression. Given that cognitive function is impaired when BFCNs are lost or functionally disrupted, we undertook a screen of cognitively impaired males for SIZN1 mutations. We report on four different sequence variants in SIZN1 in 11 individuals with nonsyndromic X-linked mental retardation. Our data implicate SIZN1 as a candidate gene for X-linked mental retardation and/or as a neurocognitive functional modifier.
PMCID: PMC2575800  PMID: 18798319
BMP; SIZN1(ZCCHC12); forebrain cholinergic neuron; X-linked mental retardation
3.  Early B-cell factors 2 and 3 (EBF2/3) regulate early migration of Cajal–Retzius cells from the cortical hem 
Developmental Biology  2012;365(1-6):277-289.
Cajal–Retzius (CR) cells play a crucial role in the formation of the cerebral cortex, yet the molecules that control their development are largely unknown. Here, we show that Ebf transcription factors are expressed in forebrain signalling centres—the septum, cortical hem and the pallial–subpallial boundary—known to generate CR cells. We identified Ebf2, through fate mapping studies, as a novel marker for cortical hem- and septum-derived CR cells. Loss of Ebf2 in vivo causes a transient decrease in CR cell numbers on the cortical surface due to a migratory defect in the cortical hem, and is accompanied by upregulation of Ebf3 in this and other forebrain territories that produce CR cells, without affecting proper cortical lamination. Accordingly, using in vitro preparations, we demonstrated that both Ebf2 and Ebf3, singly or together, control the migration of CR cells arising in the cortical hem. These findings provide evidence that Ebfs directly regulate CR cell development.
► Ebf2 is a novel marker for hem- and septum-derived Cajal–Retzius cells. ► Ebf2 mutant shows a transient migratory defect of hem-derived Cajal–Retzius cells. ► Ebf3 is expressed in similar territories as Ebf2 and rescues Ebf2 mutant phenotype. ► Both Ebf2 and Ebf3 are required for Cajal–Retzius cell migration.
PMCID: PMC3368273  PMID: 22421355
Corticogenesis; Migration; Reelin; Transcription factor
4.  Lhx2 Regulates the Development of the Forebrain Hem System 
Cerebral Cortex (New York, NY)  2013;24(5):1361-1372.
Early brain development is regulated by the coordinated actions of multiple signaling centers at key boundaries between compartments. Three telencephalic midline structures are in a position to play such roles in forebrain patterning: The cortical hem, the septum, and the thalamic eminence at the diencephalic–telencephalic boundary. These structures express unique complements of signaling molecules, and they also produce distinct populations of Cajal–Retzius cells, which are thought to act as “mobile patterning units,” migrating tangentially to cover the telencephalic surface. We show that these 3 structures require the transcription factor Lhx2 to delimit their extent. In the absence of Lhx2 function, all 3 structures are greatly expanded, and the Cajal–Retzius cell population is dramatically increased. We propose that the hem, septum, and thalamic eminence together form a “forebrain hem system” that defines and regulates the formation of the telencephalic midline. Disruptions in the forebrain hem system may be implicated in severe brain malformations such as holoprosencephaly. Lhx2 functions as a central regulator of this system's development. Since all components of the forebrain hem system have been identified across several vertebrate species, the mechanisms that regulate them may have played a fundamental role in driving key aspects of forebrain evolution.
PMCID: PMC3977624  PMID: 23307637
Cajal–Retzius cells; human; Lhx2; midline; mouse; patterning; thalamic eminence
5.  Cajal, Retzius, and Cajal–Retzius cells 
The marginal zone (MZ) of the prenatal cerebral cortex plays a crucial role in cellular migration and laminar patterning in the developing neocortex and its equivalent in the adult brain – layer I, participates in cortical circuitry integration within the adult neocortex. The MZ/layer I, which has also been called the plexiform layer and cell-poor zone of Meynert, among others, is home to several cell populations including glia, neurons, and Cajal–Retzius (CR) cells. Cajal once said that the MZ is one of the oldest formations in the phylogenetic series, and that the characteristics of layer I in human are similar in all vertebrates except fish (Ramon y Cajal, 1899). Despite the presence of CR cells in the MZ/layer I of all developing and adult vertebrate brains, and more than one hundred years of research, the phenotype and function of layer I cells have still not been clearly defined. Recent technological advances have yielded significant progress in functional and developmental studies, but much remains to be understood about neurons in MZ/layer I. Since the time of Retzius and Cajal, and continuing with modern era research from the likes of Marín-Padilla, the study of CR cells has been based on their morphological characteristics in Golgi staining. However, since Cajal’s initial description, the term “CR cell” has been applied differently and now is often used to indicate reelin (Reln)-positive cells in MZ/layer I. Here we review the history of work by Cajal, Retzius, and others pertaining to CR cells. We will establish a link between original descriptions of CR cell morphology by Cajal, Retzius, and others, and current understandings of the cell populations that reside in MZ/layer I based on the use of cellular markers. We propose to use the term “CR cell” for the class of neurons that express Reln in the MZ/layer I in both prenatal, developing and adult cerebral cortex.
PMCID: PMC4060955  PMID: 24987337
Ramón y Cajal; Cajal–Retzius cells; marginal zone; layer I; horizontal cells; short axon cells; Reelin
6.  Inducible Genetic Lineage Tracing of Cortical Hem Derived Cajal-Retzius Cells Reveals Novel Properties 
PLoS ONE  2011;6(12):e28653.
During cortical development, Cajal-Retzius (CR) cells are among the earliest-born subclasses of neurons. These enigmatic neurons play an important role in cortical development through their expression of the extracellular protein, reelin. CR cells arise from discrete sources within the telencephalon, including the pallial-subpallial border and the medial (cortical hem) regions of the pallium. Combined evidence suggests that CR cells derived from distinct origins may have different distributions and functions. By tracing CR cells derived from the cortical hem using the inducible Cre transgenic mouse tool, Frizzled 10-CreER™, we examined the specific properties of hem-derived CR cells during cortical development. Our results show that the progenitor zone for later production of CR cells from the hem can be specifically marked as early as embryonic day 6.5 (E6.5), a pre-neural period. Moreover, using our Cre line, we found that some hem-derived CR cells migrated out along the fimbrial radial glial scaffold, which was also derived from the cortical hem, and preferentially settled in the hippocampal marginal zone, which indicated specific roles for hem-derived CR cells in hippocampal development.
PMCID: PMC3236758  PMID: 22174859
7.  Dynamic expression of calretinin in embryonic and early fetal human cortex 
Calretinin (CR) is one of the earliest neurochemical markers in human corticogenesis. In embryos from Carnegie stages (CS) 17 to 23, calbindin (CB) and CR stain opposite poles of the incipient cortex suggesting early regionalization: CB marks the neuroepithelium of the medial boundary of the cortex with the choroid plexus (cortical hem). By contrast, CR is confined to the subventricular zone (SVZ) of the lateral and caudal ganglionic eminences at the pallial-subpallial boundary (PSB, or antihem), from where CR+/Tbr1- neurons migrate toward piriform cortex and amygdala as a component of the lateral cortical stream. At CS 19, columns of CR+ cells arise in the rostral cortex, and contribute at CS 20 to the “monolayer” of horizontal Tbr1+/CR+ and GAD+ cells in the preplate. At CS 21, the “pioneer cortical plate” appears as a radial aggregation of CR+/Tbr1+ neurons, which cover the entire future neocortex and extend the first corticofugal axons. CR expression in early human corticogenesis is thus not restricted to interneurons, but is also present in the first excitatory projection neurons of the cortex. At CS 21/22, the cortical plate is established following a lateral to medial gradient, when Tbr1+/CR- neurons settle within the pioneer cortical plate, and thus separate superficial and deep pioneer neurons. CR+ pioneer neurons disappear shortly after the formation of the cortical plate. Reelin+ Cajal-Retzius cells begin to express CR around CS21 (7/8 PCW). At CS 21–23, the CR+ SVZ at the PSB is the source of CR+ interneurons migrating into the cortical SVZ. In turn, CB+ interneurons migrate from the subpallium into the intermediate zone following the fibers of the internal capsule. Early CR+ and CB+ interneurons thus have different origins and migratory routes. CR+ cell populations in the embryonic telencephalon take part in a complex sequence of events not analyzed so far in other mammalian species, which may represent a distinctive trait of the initial steps of human corticogenesis.
PMCID: PMC4042362  PMID: 24917793
preplate; pioneer cells; cortical plate; regionalization; lateral migratory stream; ganglionic eminence
8.  Meningeal defects alter the tangential migration of cortical interneurons in Foxc1hith/hith mice 
Neural Development  2012;7:2.
Tangential migration presents the primary mode of migration of cortical interneurons translocating into the cerebral cortex from subpallial domains. This migration takes place in multiple streams with the most superficial one located in the cortical marginal zone. While a number of forebrain-expressed molecules regulating this process have emerged, it remains unclear to what extent structures outside the brain, like the forebrain meninges, are involved.
We studied a unique Foxc1 hypomorph mouse model (Foxc1hith/hith) with meningeal defects and impaired tangential migration of cortical interneurons. We identified a territorial correlation between meningeal defects and disruption of interneuron migration along the adjacent marginal zone in these animals, suggesting that impaired meningeal integrity might be the primary cause for the observed migration defects. Moreover, we postulate that the meningeal factor regulating tangential migration that is affected in homozygote mutants is the chemokine Cxcl12. In addition, by using chromatin immunoprecipitation analysis, we provide evidence that the Cxcl12 gene is a direct transcriptional target of Foxc1 in the meninges. Further, we observe migration defects of a lesser degree in Cajal-Retzius cells migrating within the cortical marginal zone, indicating a less important role for Cxcl12 in their migration. Finally, the developmental migration defects observed in Foxc1hith/hith mutants do not lead to obvious differences in interneuron distribution in the adult if compared to control animals.
Our results suggest a critical role for the forebrain meninges to promote during development the tangential migration of cortical interneurons along the cortical marginal zone and Cxcl12 as the factor responsible for this property.
PMCID: PMC3287125  PMID: 22248045
9.  A Novel Role for Dbx1-Derived Cajal-Retzius Cells in Early Regionalization of the Cerebral Cortical Neuroepithelium 
PLoS Biology  2010;8(7):e1000440.
Patterning of the cerebral cortex during embryogenesis depends not only on passive diffusion of morphogens but also on signal delivery by Cajal-Retzius neurons that migrate over long distances.
Patterning of the cortical neuroepithelium occurs at early stages of embryonic development in response to secreted molecules from signaling centers. These signals have been shown to establish the graded expression of transcription factors in progenitors within the ventricular zone and to control the size and positioning of cortical areas. Cajal-Retzius (CR) cells are among the earliest generated cortical neurons and migrate from the borders of the developing pallium to cover the cortical primordium by E11.5. We show that molecularly distinct CR subtypes distribute in specific combinations in pallial territories at the time of cortical regionalization. By means of genetic ablation experiments in mice, we report that loss of septum Dbx1-derived CR cells in the rostromedial pallium between E10.5 and E11.5 results in the redistribution of CR subtypes. This leads to changes in the expression of transcription factors within the neuroepithelium and in the proliferation properties of medial and dorsal cortical progenitors. Early regionalization defects correlate with shifts in the positioning of cortical areas at postnatal stages in the absence of alterations of gene expression at signaling centers. We show that septum-derived CR neurons express a highly specific repertoire of signaling factors. Our results strongly suggest that these cells, migrating over long distances and positioned in the postmitotic compartment, signal to ventricular zone progenitors and, thus, function as modulators of early cortical patterning.
Author Summary
Patterning of the cerebral cortex occurs early during embryonic development in response to secreted molecules or morphogens produced at signaling centers. These morphogens establish the graded expression of transcription factors (TFs) in progenitor cells and control the size and positioning of cortical areas in the postnatal animal. CR cells are among the earliest born cortical neurons and play a crucial role in cortical lamination. They are generated at signaling centers and migrate over long distances to cover its entire surface. We show that three different CR subtypes distribute in specific proportions in cortical territories. Genetic ablation of one subpopulation leads to a highly dynamic redistribution of the two others. This results in defects in expression of transcription factors and in progenitor cell proliferation, which correlate with the resulting changes in the size and positioning of cortical areas. Given our additional evidence that CR subtypes express specific repertoires of signaling factors, the ablation phenotypes point to a novel early role for CR cells as mediators of cortical patterning and suggest that CR cells are able to signal to progenitor cells. Our data thus add to the conventional model that morphogens act by passive diffusion and point to a strategy of morphogen delivery over long distance by migrating cells.
PMCID: PMC2910656  PMID: 20668538
10.  LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells 
Cajal-Retzius (C-R) cells play important roles in the lamination of the mammalian cortex via reelin secretion. The genetic mechanisms underlying the development of these neurons have just begun to be unraveled. Here we show that two closely related LIM homeobox genes Lhx1 and Lhx5 are expressed in reelin+, cells in various regions in the mouse telencephalon at or adjacent to sites where the C-R cells are generated, including the cortical hem, the mantle region of the septal/retrobulbar area and the ventral pallium. Whereas Lhx5 is expressed in all of these reelin-expressing domains, Lhx1 is preferentially expressed in the septal area and in a continuous domain spanning from lateral olfactory region to caudomedial territories. Genetic ablation of Lhx5 results in decreased reelin+ and p73+ cells in the neocortical anlage, in the cortical hem and in the septal, olfactory, and caudomedial telencephalic regions. The overall reduction in number of C-R cells in Lhx5 mutants is accompanied by formation of ectopic reelin+ cell clusters at the caudal telencephalon. Based on differential expression of molecular markers and by fluorescent cell tracing in cultured embryos, we located the origin of reelin+ ectopic cell clusters at the caudomedial telencephalic region. We also confirmed the existence of a normal migration stream of reelin+ cells from the caudomedial area to telencephalic olfactory territories in wild-type embryos. These results reveal a complex role for Lhx5 in regulating the development and normal distribution of C-R cells in the developing forebrain.
PMCID: PMC2927820  PMID: 20685998
cortex; homeodomain; Lhx1; migration; mouse; telencephalon
11.  Novel GABAergic circuits mediating excitation/inhibition of Cajal-Retzius cells in the developing hippocampus 
Cajal-Retzius cells are a class of neurons believed to play critical roles during cortical development. However, their network computational functions remain poorly understood. Although work in the neocortex and hippocampus has shown that Cajal-Retzius cells receive predominantly, if not exclusively, spontaneous GABAA receptor-mediated input, the cellular sources originating these events remain unclear. Yet, a precise definition of the presynaptic GABAergic interneurons contacting Cajal-Retzius cells is important to understand the microcircuits and network patterns controlling their activation. Here, we have taken advantage of electrophysiological and anatomical techniques applied to mouse hippocampal slices in vitro to directly address this question. Our paired recording experiments indicate that Cajal-Retzius cells receive small-amplitude, kinetically-slow synaptic input from stratum lacunosum-moleculare interneurons, anatomically identified as neurogliaform cells. In addition, a convergence of optogenetic, electrophysiological and pharmacological experiments show that Cajal-Retzius cell receive GABAergic input from O-LM cells, and that this input has different physiological properties (i.e. larger amplitude and faster kinetics) from the one provided by neurogliaform cells. Lastly, we show that GABAergic evoked synaptic input onto Cajal-Retzius cells may either increase their excitability and trigger action potentials or inhibit spontaneous firing by depolarization block. We propose that the specific type of response depends both on the membrane potential of Cajal-Retzius cells and on the kinetics of the received GABAergic input. In conclusion, we have unraveled a novel hippocampal microcircuit with complex GABAergic synaptic signaling, which we suggest may play a role in the refinement of the hippocampal network and connections during development.
PMCID: PMC3655532  PMID: 23536064
12.  Optogenetic Activation of Cajal-Retzius Cells Reveals Their Glutamatergic Output and a Novel Feedforward Circuit in the Developing Mouse Hippocampus 
The Journal of Neuroscience  2014;34(39):13018-13032.
Cajal-Retzius cells orchestrate the development of cortical circuits by secreting the glycoprotein reelin. However, their computational functions are still unknown. In fact, the nature of their postsynaptic targets, major neurotransmitter released, as well as the class of postsynaptic receptors activated by their firing remain unclear. Here, we have addressed these questions by activating Cajal-Retzius cells optogenetically in mouse hippocampal slices. Light delivered to stratum lacunosum-moleculare triggered EPSCs both on local interneurons and on pyramidal cells. Responses recorded under voltage-clamp conditions had identical short latencies and similar amplitudes, but were kinetically different (i.e., faster in interneurons vs pyramidal cells). In both cases, responses were blocked by TTX, indicating that they were generated by action potential-dependent release. Responses in interneurons were rescued by the addition of 4-AP to TTX, and decreased when presynaptic firing in Cajal-Retzius cells was reduced by the chemokine CXCL12, indicating the existence of a direct Cajal-Retzius cell-interneuron monosynaptic connection. Although the combined application of 4-AP and TTX did not rescue responses in pyramidal cells, neither were they affected by the GABAA receptor blocker gabazine, which would be expected if they were polysynaptic. Both connections showed physiological and pharmacological properties indicating the involvement of AMPA- and NMDA-type glutamate receptors. The connectivity from presynaptic Cajal-Retzius cells to interneurons was strong enough to generate long-latency feedforward GABAergic input onto pyramidal cells. We propose that this newly defined Cajal-Retzius cell-dependent microcircuit may regulate synaptic plasticity and dendritic development in stratum lacunosum-moleculare, thus impacting the integrative properties of the developing hippocampus.
PMCID: PMC4172799  PMID: 25253849
AMPA receptor; chemokine; GABAA receptor; glutamate; interneuron; NMDA receptor
13.  The Role of Robo3 in the Development of Cortical Interneurons 
Cerebral Cortex (New York, NY)  2009;19(Suppl 1):i22-i31.
A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal–Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons.
PMCID: PMC2693537  PMID: 19366869
interneuron; morphology; Robo
14.  Characterization of hippocampal Cajal-Retzius cells during development in a mouse model of Alzheimer's disease (Tg2576) 
Neural Regeneration Research  2014;9(4):394-401.
Cajal-Retzius cells are reelin-secreting neurons in the marginal zone of the neocortex and hippocampus. The aim of this study was to investigate Cajal-Retzius cells in Alzheimer's disease pathology. Results revealed that the number of Cajal-Retzius cells markedly reduced with age in both wild type and in mice over-expressing the Swedish double mutant form of amyloid precursor protein 695 (transgenic (Tg) 2576 mice). Numerous reelin-positive neurons were positive for activated caspase 3 in Tg2576 mice, suggesting that Cajal-Retzius neuronal loss occurred via apoptosis in this Alzheimer's disease model. Compared with wild type, the number of Cajal-Retzius cells was significantly lower in Tg2576 mice. Western blot analysis confirmed that reelin levels were markedly lower in Tg2576 mice than in wild-type mice. The decline in Cajal-Retzius cells in Tg2576 mice was found to occur concomitantly with the onset of Alzheimer's disease amyloid pathology and related behavioral deficits. Overall, these data indicated that Cajal-Retzius cell loss occurred with the onset and development of Alzheimer's disease.
PMCID: PMC4146192  PMID: 25206826
nerve regeneration; neurodegeneration; Alzheimer's disease; Cajal-Retzius cells; hippocampus; development; neuronal apoptosis; reelin; Tg2576 mice; NSFC grant; neural regeneration
15.  Neuronal and microglial regulators of cortical wiring: usual and novel guideposts 
Neocortex functioning relies on the formation of complex networks that begin to be assembled during embryogenesis by highly stereotyped processes of cell migration and axonal navigation. The guidance of cells and axons is driven by extracellular cues, released along by final targets or intermediate targets located along specific pathways. In particular, guidepost cells, originally described in the grasshopper, are considered discrete, specialized cell populations located at crucial decision points along axonal trajectories that regulate tract formation. These cells are usually early-born, transient and act at short-range or via cell-cell contact. The vast majority of guidepost cells initially identified were glial cells, which play a role in the formation of important axonal tracts in the forebrain, such as the corpus callosum, anterior, and post-optic commissures as well as optic chiasm. In the last decades, tangential migrating neurons have also been found to participate in the guidance of principal axonal tracts in the forebrain. This is the case for several examples such as guideposts for the lateral olfactory tract (LOT), corridor cells, which open an internal path for thalamo-cortical axons and Cajal-Retzius cells that have been involved in the formation of the entorhino-hippocampal connections. More recently, microglia, the resident macrophages of the brain, were specifically observed at the crossroads of important neuronal migratory routes and axonal tract pathways during forebrain development. We furthermore found that microglia participate to the shaping of prenatal forebrain circuits, thereby opening novel perspectives on forebrain development and wiring. Here we will review the last findings on already known guidepost cell populations and will discuss the role of microglia as a potentially new class of atypical guidepost cells.
PMCID: PMC4505395
guidance molecules; glial cells; Cajal-Retzius cells; microglia; axon guidance
16.  Zcchc11 Uridylates Mature miRNAs to Enhance Neonatal IGF-1 Expression, Growth, and Survival 
PLoS Genetics  2012;8(11):e1003105.
The Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3′ terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression.
Author Summary
MicroRNAs (miRNAs) are molecules that regulate gene expression, usually serving silencing functions. Mechanisms regulating miRNAs are poorly understood. In test tube experiments, the enzyme Zcchc11 adds uridines to the ends of miRNAs and their precursors, with uridyation of miRNA precursors decreasing the quantities of mature miRNAs and uridylation of mature miRNAs decreasing their silencing activity. Whether, when, and to what effect Zcchc11 alters miRNA in living animals has never previously been reported. To understand functions of Zcchc11 in integrative biology, we generated mice deficient in Zcchc11. Mutant mice were born normally, but some died soon after birth and survivors grew poorly. No miRNA quantities were changed in tissues sampled from these mice, but mature miRNAs were less likely to have additional uridines on their ends. Some miRNAs that were uridylated by Zcchc11 targeted a critical growth factor known as insulin-like growth factor 1 (IGF-1), but they did so less effectively when uridylated. Zcchc11-deficient mice had decreased amounts of IGF-1 in the liver and blood. These data reveal that Zcchc11 is an important enzyme in living animals for uridylating mature miRNAs, enhancing IGF-1 expression, and promoting neonatal growth and survival, suggesting a novel mode of gene regulation that is biologically significant.
PMCID: PMC3510031  PMID: 23209448
17.  mGlu1α-dependent recruitment of excitatory GABAergic input to neocortical Cajal-Retzius cells 
Neuropharmacology  2012;63(3):486-493.
Cajal-Retzius cells are thought to play an important role for cortical development, and receive primarily spontaneous GABAergic input mediated by GABAA receptors. However, neither the effects of synaptically-released GABA on their excitability nor the cellular source(s) of spontaneous GABAergic currents have been yet determined. By directly recording electrophysiological responses from identified Cajal-Retzius cells of the CXCR4-EGFP mouse, we show that GABAergic input can trigger supra-threshold responses, and that the pharmacological activation of mGlu1α receptors with the group I agonist DHPG powerfully increases the frequency of spontaneous GABAergic currents. These effects appeared mediated by a network mechanism, because responses to DHPG were completely prevented both by surgical disconnection of layer I from lower layers and by exposure of slices to TTX.
We propose that the cellular source underlying the observed effect of DHPG are layer I-targeting Martinotti-like interneurons, which we show express functional group I mGluRs and respond to DHPG with supra-threshold depolarization already at early developmental stages.
In conclusion, our work suggests that conditions of enhanced glutamate release may be critical at early developmental stages for the recruitment of an mGlu1α-dependent micro-circuit, which then leads to the activation of Cajal-Retzius cells.
PMCID: PMC3372658  PMID: 22579657
18.  Perlecan controls neurogenesis in the developing telencephalon 
Perlecan is a proteoglycan expressed in the basal lamina of the neuroepithelium during development. Perlecan absence does not impair basal lamina assembly, although in the 55% of the mutants early disruptions of this lamina conducts to exencephaly, impairing brain development. The rest of perlecan-null brains complete its prenatal development, maintain basal lamina continuity interrupted by some isolated ectopias, and are microcephalic. Microcephaly consists of thinner cerebral walls and underdeveloped ganglionic eminences. We have studied the mechanisms that generate brain atrophy in telencephalic areas where basal lamina is intact.
Brain atrophy in the absence of perlecan started in the ventral forebrain and extended to lateral and dorsal parts of the cortex in the following stages. First, the subpallial forebrain developed poorly in early perlecan-null embryos, because of a reduced cell proliferation: the number of cells in mitosis decreased since the early stages of development. This reduction resulted in a decreased tangential migration of interneurons to the cerebral cortex. Concomitant with the early hypoplasia observed in the medial ganglionic eminences, Sonic Hedgehog signal decreased in the perlecan-null floor plate basal lamina at E12.5. Second, neurogenesis in the pallial neuroepithelium was affected in perlecan deficient embryos. We found reductions of nearly 50% in the number of cells exiting the cell cycle at E12–E13. The labeling index, which was normal at this age, significantly decreased with advancing corticogenesis. Moreover, nestin+ or PCNA+ progenitors increased since E14.5, reaching up to about 150% of the proportion of PCNA+ cells in the wild-type at E17.5. Thus, labeling index reduction together with increased progenitor population, suggests that atrophy is the result of altered cell cycle progression in the cortical progenitors. Accordingly, less neurons populated the cortical plate and subplate of perlecan-null neocortex, as seen with the neuronal markers β-tubulin and Tbr1.
As a component of the basal lamina, perlecan both maintains this structure and controls the response of the neuroepithelium to growth factors. Less mitotic cells in the early medial ganglionic eminences, and impaired cell cycle progression in the late neocortex, suggests insufficient recruitment and signaling by neurogenic morphogens, such as SHH or FGF2.
PMCID: PMC1852307  PMID: 17411441
19.  Stromal-Derived Factor-1 (CXCL12) Regulates Laminar Position of Cajal-Retzius Cells in Normal and Dysplastic Brains 
Normal brain development requires a series of highly complex and interrelated steps. This process presents many opportunities for errors to occur, which could result in developmental defects in the brain, clinically referred to as malformations of cortical development. The marginal zone and Cajal-Retzius cells are key players in cortical development and are established early, yet there is little understanding of the factors resulting in the disruption of the marginal zone in many types of cortical malformation syndromes. We showed previously that treatment with methylazoxymethanol in rats causes marginal zone dysplasia with displacement of Cajal-Retzius cells to deeper cortical layers. Here we establish that loss of activity of the chemokine stromal-derived factor-1 (SDF1) (CXCL12), which is expressed by the leptomeninges, is necessary and sufficient to cause marginal zone disorganization in this widely used teratogenic animal model. We also found that mice with mutations in the main receptor for SDF1 (CXCR4) have Cajal-Retzius cells displaced to deeper cortical layers. Furthermore, by inhibiting SDF1 signaling in utero by intraventricular injection of a receptor antagonist, we establish that SDF1 signaling is required for the maintenance of Cajal-Retzius cell position in the marginal zone during normal cortical development. Our data imply that cortical layering is not a static process, but rather requires input from locally produced molecular cues for maintenance, and that complex syndromes of cortical malformation as a result of environmental insults may still be amenable to explanation by interruption of specific molecular signaling pathways.
PMCID: PMC2133346  PMID: 16971524
lamination; cortex; cortical dysplasia; migration; marginal zone; teratogen
20.  Nuclear Fragile X Mental Retardation Protein Is localized to Cajal Bodies 
PLoS Genetics  2013;9(10):e1003890.
Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.
Author Summary
Fragile X syndrome is the most common form of inherited mental retardation affecting approximately 1/7000 females and 1/4000 males worldwide. The syndrome is due to the silencing of a single gene, the Fragile Mental Retardation 1 (FMR1), that codes for a protein called the Fragile X mental retardation protein (FMRP). This protein, highly expressed in the brain, controls local protein synthesis essential for neuronal development and maturation. While considerable efforts have been focused on understanding FMRP functions in mental retardation, the pathophysiology of the syndrome is not well understood. Here, we show that in addition to the well-studied roles of FMRP in regulating protein synthesis, a minor species of FMRP different from the major one, is specifically found in structures called Cajal bodies present in the cell nucleus. Our observations suggest that different FMRP species, also called isoforms, might have independent cellular functions. These findings might open new avenues in search for new insights in the pathophysiology of Fragile X Syndrome.
PMCID: PMC3814324  PMID: 24204304
21.  Activity-dependent endogenous taurine release facilitates excitatory neurotransmission in the neocortical marginal zone of neonatal rats 
In the developing cerebral cortex, the marginal zone (MZ), consisting of early-generated neurons such as Cajal-Retzius cells, plays an important role in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by γ-aminobutyric acid (GABA) in the MZ. Cajal-Retzius cells express not only GABAA receptors but also α2/β subunits of glycine receptors, and exhibit glycine receptor-mediated depolarization due to high [Cl−]i. However, the physiological roles of glycine receptors and their endogenous agonists during neurotransmission in the MZ are yet to be elucidated. To address this question, we performed optical imaging from the MZ using the voltage-sensitive dye JPW1114 on tangential neocortical slices of neonatal rats. A single electrical stimulus evoked an action-potential-dependent optical signal that spread radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but was suppressed by either GABAA or glycine receptor antagonists. Combined application of both antagonists nearly abolished the signal. Inhibition of Na+, K+-2Cl− cotransporter by 20 µM bumetanide reduced the signal, indicating that this transporter contributes to excitation. Analysis of the interstitial fluid obtained by microdialysis from tangential neocortical slices with high-performance liquid chromatography revealed that GABA and taurine, but not glycine or glutamate, were released in the MZ in response to the electrical stimulation. The ambient release of taurine was reduced by the addition of a voltage-sensitive Na+ channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in Cajal-Retzius and non-Cajal-Retzius cells in the MZ, but was not localized in presynaptic structures. Our results suggest that activity-dependent non-synaptic release of endogenous taurine facilitates excitatory neurotransmission through activation of glycine receptors in the MZ.
PMCID: PMC3918584  PMID: 24574969
taurine; GABAA receptor; glycine receptor; marginal zone; NKCC1; GABA; microdialysis
22.  Reelin Is Involved in Transforming Growth Factor-β1-Induced Cell Migration in Esophageal Carcinoma Cells 
PLoS ONE  2012;7(2):e31802.
Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice.
PMCID: PMC3290530  PMID: 22393371
23.  Characterization of the Frizzled10-CreER™ Transgenic Mouse: An Inducible Cre Line for the Study of Cajal-Retzius Cell Development 
Genesis (New York, N.Y. : 2000)  2009;47(3):210-216.
Cajal-Retzius cells are an enigmatic class of neurons located in the most superficial layer of the cerebral cortex, and they play an important role in cortical development. Although many studies have indicated that CR cells are involved in regulating cell migration and cortical maturation, the function of these cells is still not fully understood. Here we describe an inducible Cre mouse line in which CreER™ is driven by the promoter for the Wnt receptor Frizzled10. Consistent with our previous studies on Frizzled10 expression and transgenic mouse lines using the Frizzled10 promoter, we found that in the developing telencephalon, Cre was mainly detected at the cortical hem, the largest source of CR cells. By crossing the Cre line to R26R reporter mice and injecting tamoxifen at different time points, we were able to detect via X-gal staining CR cells produced from the cortical hem at distinct stages during development. Thus, this transgenic Cre mouse line is a valuable tool for studying the molecular and cellular mechanisms of CR cell development.
PMCID: PMC2698876  PMID: 19241378
Cajal-Retzius cell; reelin; CreER™; Frizzled10; Wnt; cortical hem; cortical development; neuron migration
24.  Timing of Cortical Interneuron Migration Is Influenced by the Cortical Hem 
Cerebral Cortex (New York, NY)  2010;21(4):748-755.
Cerebral cortical γ-aminobutyric acid (GABA)ergic interneurons originate from the basal forebrain and migrate into the cortex in 2 phases. First, interneurons cross the boundary between the developing striatum and the cortex to migrate tangentially through the cortical primordium. Second, interneurons migrate radially to their correct neocortical layer position. A previous study demonstrated that mice in which the cortical hem was genetically ablated displayed a massive reduction of Cajal-Retzius (C-R) cells in the neocortical marginal zone (MZ), thereby losing C-R cell-generated reelin in the MZ. Surprisingly, pyramidal cell migration and subsequent layering were almost normal. In contrast, we find that the timing of migration of cortical GABAergic interneurons is abnormal in hem-ablated mice. Migrating interneurons both advance precociously along their tangential path and switch prematurely from tangential to radial migration to invade the cortical plate (CP). We propose that the cortical hem is responsible for establishing cues that control the timing of interneuron migration. In particular, we suggest that loss of a repellant signal from the medial neocortex, which is greatly decreased in size in hem-ablated mice, allows the early advance of interneurons and that reduction of another secreted molecule from C-R cells, the chemokine SDF-1/CXCL12, permits early radial migration into the CP.
PMCID: PMC3059882  PMID: 20713502
Cajal–Retzius cells; Cxcl12; Cxcr4; guidance cues
25.  Developmental Expression Mapping of a Gene Implicated in Multiple Neurodevelopmental Disorders, A2bp1 (Fox1) 
Developmental Neuroscience  2011;33(1):64-74.
The neuronal transcription splicing factor, A2BP1, has been implicated in a variety of neurodevelopmental disorders; however, the role of A2BP1 in brain development and gene regulatory function remains to be explicated. Here, we map A2bp1 gene expression, focusing on the developing forebrain of the C57BL6J mouse. Early in forebrain development, A2bp1 expression is highly reminiscent of the expression of genes marking postmitotic GABAergic cells emanating from the ventral telencephalon during migration to the dorsal pallium. Ventral pallial expression remains low after the migratory period. Broader dorsal pallial expression becomes more evident late prenatally and early postnatally. This is paralleled by dense, restricted expression in the ventrobasal dorsal thalamic complex and mid-hypothalamic region. Outside of the forebrain, there is significant expression in motor pathways. These data indicate that A2BP1 mutations may clinically affect very selective forebrain neuron types from early periods of development.
PMCID: PMC3080782  PMID: 21346316
C57BL6J mouse; Embryonic forebrain; Neurodevelopment; mRNA splicing regulation; Gene expression; GABA; Interneurons; Thalamus; Neocortex

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