Comparative analysis of extra-ventricular mitoses at early stages of cortical development in rat and human

Embryonic germinal zones of the dorsal and ventral telencephalon generate cortical neurons during the final week of gestation in rodent and during several months in human. Whereas the vast majority of cortical interneurons originate from the ventral telencephalon, excitatory neurons are locally generated within the germinal zone of the dorsal telencephalon, the future cerebral cortex, itself. However, a number of studies have described proliferating cells external to the ventricular and subventricular germinal zones in the developing dorsal telencephalon. In this study, we performed a comprehensive cell density analysis of such ‘extra-ventricular proliferating cells’ (EVPCs) during corticogenesis in rat and human using a mitotic marker anti-phospho-histone H3. Subsequently, we performed double-labelling studies with other mitotic and cell type specific markers to undertake phenotypic characterisation of EVPCs. Our findings show: (1) the densities of extra-ventricular H3-positive (H3⁺) cells were surprisingly similar in preplate stage rat and human; (2) extra-ventricular proliferation continues during mid-and late corticogenesis in rat and in early fetal human cortex; and (3) extra-ventricular cells appear to be mitotic precursors as they are not immunoreactive for a panel of early post-mitotic and cell type-specific markers, although (4) a subset of EVPCs are proliferating microglia. These data suggest that some aspects of early corticogenesis are conserved between rodent and human despite marked differences in the duration of neurogenesis and the anatomical organisation of the developing cerebral cortex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Neuropilin-1 is required for endothelial tip cell guidance in the developing central nervous system.

Recent evidence indicates that sprouting angiogenesis in the central nervous system (CNS) is a guided process similar to the guidance of axons and insect tracheal tubes. Specialized tip cells of vessel sprouts navigate in response to local depots or gradients of vascular endothelial growth factor (VEGF-A). Neuropilin-1 (Nrp-1) is a transmembrane receptor with a repulsive function in axon guidance. Nrp-1 also binds the VEGF-A splice isoform VEGF165, stimulates angiogenesis, and is necessary for vascular development in the mouse. However, the morphogenetic events controlled by Nrp-1 in angiogenesis have not been defined. Here, we analyzed endothelial tip cell guidance in the CNS of Nrp-1-deficient mice. We focused our attention on the developing hindbrain, which is normally vascularized in a stereotyped manner. Initially, angiogenic sprouts extend along radial glia from the pial surface toward the ventricles, but in the subventricular zone (SVZ), they leave the radial path, turn laterally, and fuse to form a capillary plexus. Radial sprout elongation correlated with tip cell filopodia extensions along nestin-positive radial glial processes, but in the SVZ, the tip cell filopodia also extended perpendicular to the glial tracks and made contact with filopodia of the neighboring sprouts. In Nrp-1-deficient mice, the tip cell filopodia remained associated with the radial glia in the SVZ, which correlated with a failure of sprout turning and elongation across this region. As a result, the sprouts remained blind-ended forming glomeruloid tufts in the SVZ. These observations suggest that Nrp-1 plays an important role in allowing the endothelial tip cell filopodia to switch substrate and protrude in a new direction at a specific location in the developing brain.     

Glial Reaction of the Subventricular Zone of the Telencephalon of the Rat Brain on Modeling of Alzheimer’s Disease

The aim of the present work was to study reactive changes in the subventricular zone (SVZ) of the lateral ventricles in the brain in rats at long time points after intraventricular administration of a neurotoxic substance, aggregated beta-amyloid peptide (BAP). At three months, ependymal cells in the SVZ responded to intraventricular administration of BAP by forming a characteristic gliotic structure similar to amyloid plaques in terms of composition, cell structures, and, probably, mechanism of formation. The atypical morphogenesis of these structures is suggested to result from the relationship between degenerative and regenerative processes involving both ependymal cells and progenitor cells of the proliferative zone of the telencephalon.     

Early formation of the vascular system in quail embryos

The relation between vascular development and translocation of the splanchnic mesodermal layers was studied in presomite to 20-somite quail embryos by scanning electron microscopy. In addition, serially sectioned embryos were stained immunohistochemically with monoclonal antibodies (αQH1 or αMB1) specific for endothelial and hemopoietic cells. By the formation of the foregut the anterior borders of the two splanchnic mesodermal layers of a presomite embryo are translocated to the lateral and ventral sides of the foregut and fuse in the ventral midline of a 4-somite embryo. Meanwhile the splanchnic mesoderm differentiates into a splanchnic mesothelial layer and a plexus of endothelial cells, facing the endoderm. From 4 somites onward the foregut is covered by a single endothelial plexus. At first the endothelial precursors bordering the anterior intestinal portal and those in the area of the ventral mesocardium lumenize, subsequently giving rise to the endocardium of the heart tube. Hereafter, the pharyngeal arch arteries and the dorsal aortae develop from the remaining precursors. During formation of the pharyngeal arches, the pharyngeal arch arteries maintain their connections with the splanchnic plexus through the developing ventral pharyngeal veins. After disappearance of the dorsal mesocardium, the midpharyngeal endothelial strand, which is a longitudinal strand of proendocardial cells, remains connected to the foregut. This strand will contribute to the formation of the pulmonary venous drainage into the left atrium. A bilateral accumulation of cardiac jelly developing between the promyocardium and proendocardial plexus only suggests that the heart develops from two tubes. The proendocardial layer, however, is not divided by the ventral mesocardium but initially forms just one endocardial heart tube. © 1993 Wiley-Liss, Inc.     

Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis

Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases.     

A new model of vasculogenesis and angiogenesis in vitro as compared with vascular growth in the avian area vasculosa

In cultures of dissociated quail epiblast the basic constituents of the vascular system, blood cells and endothelial cells can be induced by basic fibroblast growth factor (Flamme and Risau, Development, 116:435–439, 1992). As we show here, in those cultures three types of vascular plexus differentiate spontaneously under different culture conditions: At the 3rd day a vascular plexus appears in situ closely resembling the vascular plexus of the quail area opaca vasculosa (vasculogenesis). Vascular sprouts are formed, extending long filopodia at their tips. Such filopodia are shown to build the first intervascular bridges in the growing vascular plexus of the area vasculosa at embryonic day 3. Connections of filopodia turn out to be precursors of new capillaries interconnecting pre-existing blood vessels (angiogenesis). Two further types of in vitro capillary plexus differentiate in long term endothelial cell cultures derived from induced angioblasts. Whereas one closely resembles so-called angiogenesis in vitro, the third type comprises mainly multinucleated giant endothelial cells lining loop like capillaries and represents a differentiation of aging endothelial cell culture. Thus, the present in vitro model is an approach to the sequence of angioblast induction, vasculogenesis, and angiogenesis. © 1993 Wiley-Liss, Inc.     

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone‐rostral migratory stream

Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone‐rostral migratory stream (SVZ‐RMS) germinal niche that gives rise to many OB neurons. Results: Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ‐RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ‐RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. Conclusions: Tbr2 is essential for proper morphogenesis of the OB and SVZ‐RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons. Developmental Dynamics 243:440–450, 2014. © 2013 Wiley Periodicals, Inc.     

Comparative aspects of cortical neurogenesis in vertebrates

The mammalian neocortex consists of six layers. By contrast, the reptilian and avian cortices have only three, which are believed to be equivalent to layers I, V and VI of mammals. In mammals, the majority of cortical cell proliferation occurs in the ventricular and subventricular zones, but there are a small number of scattered individual divisions throughout the cortex. Neurogenesis in the cortical subventricular zone is believed to contribute to the supragranular layers. To estimate the proportions of different forms of divisions in reptiles and birds, we examined the site of proliferation in embryonic turtle (stages 18-25) and chick (embryonic days 8-15) brains using phospho-histone H3 (a G2 and M phase marker) immunohistochemistry. In turtle, only few scattered abventricular H3-immunoreactive cells were found outside the ventricular zone; the majority of the H3-immunoreactive cells were located in the ventricular zone throughout the entire turtle brain. Ventricular zone cell proliferation peaks at stages 18 and 20, before an increase of abventricular proliferation at stages 23 and 25. In turtle cortex, however, abventricular proliferation at any given stage never exceeded 17.5+/-2.47% of the total division and the mitotic profiles did not align parallel to the ventricular zone. Phospho-histone H3 immunoreactivity in embryonic chick brains suggests the lack of subventricular zone in the dorsal cortex, but the presence of subventricular zone in the ventral telencephalon. We were able to demonstrate that the avian subventricular zone is present in both pallial and subpallial regions of the ventral telencephalon during embryonic development, and we characterize the spatial and temporal organization of the subventricular zone. Comparative studies suggest that the subventricular zone was involved in the laminar expansion of the cortex to six layers in mammals from the three-layered cortex found in reptiles and birds. Within mammals, the number of neurons in a cortical column appears to be largely constant; nevertheless, there are considerable differences between the germinal zones in mammalian species. It is yet to be determined whether these elaborations of the subventricular zone may have contributed to cell diversity, tangential expansion or gyrus formation of the neocortex and whether it might have been the major driving force behind the evolution of the six-layered neocortex in mammals.     

人胚胎脑室脉络丛上皮细胞表达神经干细胞分子标志

目的 探讨人12周龄胚胎脑脉络丛上皮细胞是否具有神经干细胞的生物学特性.方法制备室管膜/室管膜下区和纹状体脑切片和脉络丛组织铺片,采用免疫荧光染色,在激光扫描共焦显微镜下观察结果,采集图像数据.结果人12周龄胚胎脑室脉络丛上皮细胞表达神经干细胞分子标志CD133、Nestin和Sox2.神经干细胞的分子标志在脉络丛上皮...     

Coronary stem development in wild‐type and Tbx1 null mouse hearts

Background: Coronary artery (CA) stems connect the ventricular coronary tree with the aorta. Defects in proximal CA patterning are a cause of sudden cardiac death. In mice lacking Tbx1, common arterial trunk is associated with an abnormal trajectory of the proximal left CA. Here we investigate CA stem development in wild‐type and Tbx1 null embryos. Results: Genetic lineage tracing reveals that limited outgrowth of aortic endothelium contributes to proximal CA stems. Immunohistochemistry and fluorescent tracer injections identify a periarterial vascular plexus present at the onset of CA stem development. Transplantation experiments in avian embryos indicate that the periarterial plexus originates in mesenchyme distal to the outflow tract. Tbx1 is required for the patterning but not timing of CA stem development and a Tbx1 reporter allele is expressed in myocardium adjacent to the left but not right CA stem. This expression domain is maintained in Sema3c^?/? hearts with a common arterial trunk and leftward positioned CA. Ectopic myocardial differentiation is observed on the left side of the Tbx1^?/? common arterial trunk. Conclusions: A periarterial plexus bridges limited outgrowth of the aortic endothelium with the ventricular plexus during CA stem development. Molecular differences associated with left and right CA stems provide new insights into the etiology of CA patterning defects. Developmental Dynamics 245:445–459, 2016. © 2015 Wiley Periodicals, Inc.     

Establishment of tooth blood supply and innervation is developmentally regulated and takes place through differential patterning processes

Teeth are richly supported by blood vessels and peripheral nerves. The aim of this study was to describe in detail the developmental time‐course and localization of blood vessels during early tooth formation and to compare that to innervation, as well as to address the putative role of vascular endothelial growth factor (VEGF), which is an essential regulator of vasculature development, in this process. The localization of blood vessels and neurites was compared using double immunofluorescence staining on sections at consecutive stages of the embryonic (E) and postnatal (PN) mandibular first molar tooth germ (E11‐PN7). Cellular mRNA expression domains of VEGF and its signaling receptor VEGFR2 were studied using sectional radioactive in situ hybridization. Expression of VEGF mRNA and the encoded protein were studied by RT‐PCR and western blot analysis, respectively, in the cap and early bell stage tooth germs, respectively. VEGFR2 was immunolocalized on tooth tissue sections. Smooth muscle cells were investigated by anti‐alpha smooth muscle actin (αSMA) antibodies. VEGF showed developmentally regulated epithelial and mesenchymal mRNA expression domains including the enamel knot signaling centers that correlated with the growth and navigation of the blood vessels expressing Vegfr2 and VEGFR2 to the dental papilla and enamel organ. Developing blood vessels were present in the jaw mesenchyme including the presumptive dental mesenchyme before the appearance of the epithelial dental placode and dental neurites. Similarly, formation of a blood vessel plexus around the bud stage tooth germ and ingrowth of vessels into dental papilla at E14 preceded ingrowth of neurites. Subsequently, pioneer blood vessels in the dental papilla started to receive smooth muscle coverage at the early embryonic bell stage. Establishment and patterning of the blood vessels and nerves during tooth formation are developmentally regulated, stepwise processes that likely involve differential patterning mechanisms. Development of tooth vascular supply is proposed to be regulated by local, tooth‐specific regulation by epithelial–mesenchymal tissue interactions and involving tooth target expressed VEGF signaling. Further investigations on tooth vascular development by local VEGF signaling, as well as how tooth innervation and development of blood vessels are integrated with advancing tooth organ formation by local signaling mechanisms, are warranted.     

Emx3 is required for the differentiation of dorsal telencephalic neurons

emx3 is first expressed in prospective telencephalic cells at the anterior border of the zebrafish neural plate. Knockdown of Emx3 function by morpholino reduces the expression of markers specific to dorsal telencephalon, and impairs axon tract formation. Rescue of both early and late markers requires low‐level expression of emx3 at the one‐ or two‐somite stage. Higher emx3 expression levels cause dorsal telencephalic markers to expand ventrally, which points to a possible role of emx3 in specifying dorsal telencephalon and a potential new function for Wnt/beta‐catenin pathway activation. In contrast to mice, where Emx2 plays a major role in dorsal telencephalic development, knockdown of zebrafish Emx2 apparently does not affect telencephalic development. Similarly, Emx1 knockdown has little effect. Previously, emx3 was thought to be fish‐specific. However, we found all three emx orthologs in Xenopus tropicalis and opossum (Monodelphis domestica) genomes, indicating that emx3 was present in an ancestral tetrapod genome. Developmental Dynamics 238:1984–1998, 2009. © 2009 Wiley‐Liss, Inc.     

Impairment of endothelial‐mesenchymal transformation during atrioventricular cushion formation in Tmem100 null embryos

Background: Endothelial‐mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin‐dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up‐regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively‐active calcineurin A, suggesting dysregulation of myocardial calcineurin‐VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium‐derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial‐specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium‐related signaling events. Developmental Dynamics 244:31–42, 2015. © 2014 Wiley Periodicals, Inc.     

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Background: Endothelial‐mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin‐dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up‐regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively‐active calcineurin A, suggesting dysregulation of myocardial calcineurin‐VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium‐derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial‐specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium‐related signaling events. Developmental Dynamics 244:31–42, 2015. © 2014 Wiley Periodicals, Inc.     

Branching of lung epithelium in vitro occurs in the absence of endothelial cells

Background: Early lung morphogenesis is driven by tissue interactions. Signals from the lung mesenchyme drive epithelial morphogenesis, but which individual mesenchymal cell types are influencing early epithelial branching and differentiation remains unclear. It has been shown that endothelial cells are involved in epithelial repair and regeneration in the adult lung, and they may also play a role in driving early lung epithelial branching. These data, in combination with evidence that endothelial cells influence early morphogenetic events in the liver and pancreas, led us to hypothesize that endothelial cells are necessary for early lung epithelial branching. Results: We blocked vascular endothelial growth factor (VEGF) signaling in embryonic day (E) 12.5 lung explants with three different VEGF receptor inhibitors (SU5416, Ki8751, and KRN633) and found that in all cases the epithelium was able to branch despite the loss of endothelial cells. Furthermore, we found that distal lung mesenchyme depleted of endothelial cells retained its ability to induce terminal branching when recombined with isolated distal lung epithelium (LgE). Additionally, isolated E12.5 primary mouse lung endothelial cells, or human lung microvascular endothelial cells (HMVEC‐L), were not able to induce branching when recombined with LgE. Conclusions: Our observations support the conclusion that endothelial cells are not required for early lung branching. Developmental Dynamics 244:553–563, 2015. © 2015 Wiley Periodicals, Inc.     

Putrescine as an important source of GABA in the postnatal rat subventricular zone

The subventricular zone (SVZ) is a neurogenic region that continually gives rise to olfactory bulb (OB) GABAergic interneurons in mammals. The newly generated neuroblasts already express GABA while migrating to this structure along the rostral migratory stream (RMS). Here, we investigate in early postnatal rat if SVZ/RMS cells undertake the same synthetic pathway by which GABA is produced in differentiated neurons, i.e. the decarboxylation of glutamate by the glutamic acid decarboxylase (GAD), or, if an alternative pathway, the conversion of putrescine into GABA, also contributes to GABA synthesis. We show here that GAD immunoreactivity is not significantly detectable within the SVZ/RMS. However, strong immunolabeling is found within the OB. Nevertheless, low GAD enzymatic activity (as compared with OB) is detected in the SVZ/RMS. SVZ/RMS explants convert approximately 30% of all captured radiolabeled putrescine into GABA in vitro, showing that this pathway is important for GABA synthesis in the SVZ. We also show that SVZ/RMS, OB and choroid plexus explants are able to synthesize putrescine, as analyzed by ornithine decarboxylase (ODC) activity, providing neuroblasts with different sources of putrescine for GABA production. During early stages of neuroblast differentiation, in which neurotransmitter choice may still be undefined, an alternative pathway for GABA synthesis guarantees the production of GABA, necessary for neuroblast proliferation and migration in the SVZ/RMS.     

Biological Plasticity in Penguin Heat‐Retention Structures

Insulation and vascular heat‐retention mechanisms allow penguins to forage for a prolonged time in water that is much cooler than core body temperature. Wing‐based heat retention involves a plexus of humeral arteries and veins, which redirect heat to the body core rather than to the wing periphery. The humeral arterial plexus is described here for Eudyptes and Megadyptes, the only extant penguin genera for which wing vascular anatomy had not previously been reported. The erect‐crested (Eudyptes sclateri) and yellow‐eyed (Megadyptes antipodes) penguins both have a plexus of three humeral arteries on the ventral surface of the humerus. The wing vascular system shows little variation between erect‐crested and yellow‐eyed penguins, and is generally conserved across the six extant genera of penguins, with the exception of the humeral arterial plexus. The number of humeral arteries within the plexus demonstrates substantial variation and correlates well with wing surface area. Little penguins (Eudyptula minor) have two humeral arteries and a wing surface area of ～ 75 cm², whereas emperor penguins (Aptenodytes forsteri) have up to 15 humeral arteries and a wing surface area of ～ 203 cm². Further, the number of humeral arteries has a stronger correlation with wing surface area than with sea water temperature. We propose that thermoregulation has placed the humeral arterial plexus under a strong selection pressure, driving penguins with larger wing surface areas to compensate for heat loss by developing additional humeral arteries. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.