FORSE-1: A Positionally Regulated Epitope in the Developing Rat Central Nervous System

We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1- positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non-CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5–10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high- molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development

Specific glial populations regulate hippocampal morphogenesis

The hippocampus plays an integral role in spatial navigation, learning and memory, and is a major site for adult neurogenesis. Critical to these functions is the proper organization of the hippocampus during development. Radial glia are known to regulate hippocampal formation, but their precise function in this process is yet to be defined. We find that in Nuclear Factor I b (Nfib)-deficient mice, a subpopulation of glia from the ammonic neuroepithelium of the hippocampus fail to develop. This results in severe morphological defects, including a failure of the hippocampal fissure, and subsequently the dentate gyrus, to form. As in wild-type mice, immature nestin-positive glia, which encompass all types of radial glia, populate the hippocampus in Nfib-deficient mice at embryonic day 15. However, these fail to mature into GLAST- and GFAP-positive glia, and the supragranular glial bundle is absent. In contrast, the fimbrial glial bundle forms, but alone is insufficient for proper hippocampal morphogenesis. Dentate granule neurons are present in the mutant hippocampus but their migration is aberrant, likely resulting from the lack of the complete radial glial scaffold usually provided by both glial bundles. These data demonstrate a role for Nfib in hippocampal fissure and dentate gyrus formation, and that distinct glial bundles are critical for correct hippocampal morphogenesis

Tbr1 Misexpression Alters Neuronal Development in the Cerebral Cortex

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

Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate

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

Lateral thalamic eminence – a novel origin for mGluR1/lot cells

A unique population of cells, called "lot cells," circumscribes the path of the lateral olfactory tract (LOT) in the rodent brain and acts to restrict its position at the lateral margin of the telencephalon. Lot cells were believed to originate in the dorsal pallium (DP). We show that Lhx2 null mice that lack a DP show a significant increase in the number of mGluR1/lot cells in the piriform cortex, indicating a non-DP origin of these cells. Since lot cells present common developmental features with Cajal-Retzius (CR) cells, we analyzed Wnt3a- and Dbx1-reporter mouse lines and found that mGluR1/lot cells are not generated in the cortical hem, ventral pallium, or septum, the best characterized sources of CR cells. Finally, we identified a novel origin for the lot cells by combining in utero electroporation assays and histochemical characterization. We show that mGluR1/lot cells are specifically generated in the lateral thalamic eminence and that they express mitral cell markers, although a minority of them express DeltaNp73 instead. We conclude that most mGluR1/lot cells are prospective mitral cells migrating to the accessory olfactory bulb (OB), whereas mGluR1+, DeltaNp73+ cells are CR cells that migrate through the LOT to the piriform cortex and the OB

Surface markers of regionalization in the vertebrate nervous system

In order to identify new molecules that might play a role in regional specification of the nervous system, we generated and characterized monoclonal antibodies (mAbs) that have positionally-restricted labeling patterns. The FORSE-1 mAb was generated using a strategy designed to produce mAbs against neuronal cell surface antigens that might be regulated by regionally-restricted transcription factors in the developing central nervous system (CNS). FORSE-1 staining is enriched in the forebrain as compared to the rest of the CNS until E18. Between E11.5-E13.5, only certain areas of the forebrain are labeled. There is also a dorsoventrally-restricted region of labeling in the hindbrain and spinal cord. The mAb labels a large proteoglycan-like cell-surface antigen (>200 kD). The labeling pattern of FORSE-1 is conserved in various mammals and in chick. To determine whether the FORSE-1 labeling pattern is similar to that of known transcription factors, the expression of BF-1 and Dlx-2 was compared with FORSE-1. There is a striking overlap between BF-1 and FORSE-1 in the telencephalon. In contrast, FORSE-1 and Dlx-2 have very different patterns of expression in the forebrain, suggesting that regulation by Dlx-2 alone cannot explain the distribution of FORSE-1. They do, however, share some sharp boundaries in the diencephalon. In addition, FORSE-1 identifies some previously unknown boundaries in the developing forebrain. Thus, FORSE-1 is a new cell surface marker that can be used to subdivide the embryonic forebrain into regions smaller than previously described, providing further complexity necessary for developmental patterning. I also studied the expression of the cell surface protein CD9 in the developing and adult rat nervous system. CD9 is implicated in intercellular signaling and cell adhesion in the hematopoetic system. In the nervous system, CD9 may perform similar functions in early sympathetic ganglia, chromaffin cells, and motor neurons, all of which express the protein. The presence of CD9 on the surfaces of Schwann cells and axons at the appropriate time may allow the protein to participate in the cellular interactions involved in myelination.</p

Distribution of CD9 in the Developing and Mature Rat Nervous System

CD9 is a cell surface protein implicated in intercellular signaling that has been identified in selected cell types of the hematopoietic system. To begin a study of the role of CD9 in the developing and adult nervous system, we used the anti-rat CD9 monoclonal antibody ROCA2 to determine the distribution of this protein. The identity of the antigen in these tissues was confirmed by immunoblotting and peptide sequencing. Early embryonic sympathetic and dorsal root ganglion sensory neurons and adrenal chromaffin cells all express CD9. ROCA2 also labels the somas, axons, and growth cones of cultured sympathetic and sensory neurons. In the central nervous system (CNS), CD9 is transiently and specifically expressed in embryonic spinal motorneurons. In the adult, central and peripheral glia intensely express CD9. Thus, CD9 is developmentlly regulated in a variety of peripheral and central neurons and glia, including proliferating progenitors as well as mature cells. These findings suggest that CD9 may have diverse roles in the nervous system

Mechanisms underlying the specification, positional regulation, and function of the cortical hem

The cortical hem was first described as a potential signaling center at the telencephalic midline because of an enriched expression of multiple members of the Wnt and Bmp families of morphogens, and its position at the border between the presumptive cortex and the choroid plexus. There is now definitive evidence that the cortical hem is an organizing center in the telencephalon, and that it instructs the formation of the hippocampus. In this review, we present an analysis of the molecular and cellular events that lead to the formation of the cortical hem, and define its position and extent in the telencephalon. This directly controls the positioning of the hippocampus within the telencephalon. We conclude with a summary of the current understanding of the role of the hem as the hippocampal organizer

Patterning events and specification signals in the developing hippocampus

The mouse hippocampus is an attractive model system in which to study patterning of a cortical structure. Ongoing studies indicate that hippocampal areas or fields are specified many days before birth - possibly involving signals from within the cortical mantle. Although the hippocampal CA fields are distinguished by cytoarchitecture only after birth, molecular differences between fields appear by late gestation. Moreover, these embryonic fields are already specified to develop additional features that characterize the mature fields. The basic division of the hippocampus into fields may be specified still earlier. Thus, if medial cortical neuroepithelium is isolated in vitro early in hippocampal neurogenesis, it can autonomously generate features of a patterned hippocampus. In vivo, the spatial progression of initial field differentiation suggests that signals regulating growth and patterning could arise from sources close to the hippocampal poles. Observations of mouse mutants indicate that the cortical hem, an embryonic structure close to one pole of the hippocampus, is a source of such regulatory signals