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Interneurons contribute to the complexity of neural circuits and maintenance of normal brain function. Rodent interneurons originate in embryonic ganglionic eminences, but developmental origins in other species are less understood. Here, we show that transcription factor expression patterns in porcine embryonic subpallium are similar to rodents, delineating a distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of Nkx2.1, Lhx6, and Dlx2 expression, in vitro differentiation into neurons expressing GABA, and robust migratory capacity in explant assays, we propose that cortical and hippocampal interneurons originate from a porcine MGE region. Following xenotransplantation into adult male and female rat hippocampus, we further demonstrate that porcine MGE progenitors, like those from rodents, migrate and differentiate into morphologically distinct interneurons expressing GABA. Our findings reveal that basic rules for interneuron development are conserved across species, and that porcine embryonic MGE progenitors could serve as a valuable source for interneuron-based xenotransplantation therapies. SIGNIFICANCE STATEMENT Here we demonstrate that porcine medial ganglionic eminence, like rodents, exhibit a distinct transcriptional and interneuron-specific antibody profile, in vitro migratory capacity and are amenable to xenotransplantation. This is the first comprehensive examination of embryonic interneuron origins in the pig; and because a rich neurodevelopmental literature on embryonic mouse medial ganglionic eminence exists (with some additional characterizations in other species, e.g., monkey and human), our work allows direct neurodevelopmental comparisons with this literature., (Copyright © 2021 the authors.)

The Gsx2 homeodomain transcription factor promotes neural progenitor identity in the lateral ganglionic eminence (LGE), despite upregulating the neurogenic factor Ascl1. How this balance in maturation is maintained is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in subapical progenitors that have unique transcriptional signatures in LGE ventricular zone (VZ) cells. Moreover, whereas Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors, the co-expression of Gsx2 with Ascl1 inhibits neurogenesis. Using luciferase assays, we found that Gsx2 reduces the ability of Ascl1 to activate gene expression in a dose-dependent and DNA binding-independent manner. Furthermore, Gsx2 physically interacts with the basic helix-loop-helix (bHLH) domain of Ascl1, and DNA-binding assays demonstrated that this interaction interferes with the ability of Ascl1 to bind DNA. Finally, we modified a proximity ligation assay for tissue sections and found that Ascl1-Gsx2 interactions are enriched within LGE VZ progenitors, whereas Ascl1-Tcf3 (E-protein) interactions predominate in the subventricular zone. Thus, Gsx2 contributes to the balance between progenitor maintenance and neurogenesis by physically interacting with Ascl1, interfering with its DNA binding and limiting neurogenesis within LGE progenitors., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Although the basic schema of the body plan is similar among different species of amniotes (mammals, birds, and reptiles), the lung is an exception. Here, anatomy and physiology are considerably different, particularly between mammals and birds. In mammals, inhaled and exhaled airs mix in the airways, whereas in birds the inspired air flows unidirectionally without mixing with the expired air. This bird-specific respiration system is enabled by the complex tubular structures called parabronchi where gas exchange takes place, and also by the bellow-like air sacs appended to the main part of the lung. That the lung is predominantly governed by the parasympathetic nervous system has been shown mostly by physiological studies in mammals. However, how the parasympathetic nervous system in the lung is established during late development has largely been unexplored both in mammals and birds. In this study, by combining immunocytochemistry, the tissue-clearing CUBIC method, and ink-injection to airways, we have visualized the 3-D distribution patterns of parasympathetic nerves and ganglia in the lung at late developmental stages of mice and chickens. These patterns were further compared between these species, and three prominent similarities emerged: (1) parasympathetic postganglionic fibers and ganglia are widely distributed in the lung covering the proximal and distal portions, (2) the gas exchange units, alveoli in mice and parabronchi in chickens, are devoid of parasympathetic nerves, (3) parasympathetic nerves are in close association with smooth muscle cells, particularly at the base of the gas exchange units. These observations suggest that despite gross differences in anatomy, the basic mechanisms underlying parasympathetic control of smooth muscles and gas exchange might be conserved between mammals and birds., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E 13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E 13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

There is growing evidence of a direct influence of vasculature on the development of neurons in the brain. The development of the cranial vasculature has been well described in zebrafish but its anatomical relationship with the adjacent developing sensory ganglia has not been addressed. Here, by 3D imaging of fluorescently labelled blood vessels and sensory ganglia, we describe for the first time the spatial organization of the cranial vasculature in relation to the cranial ganglia during zebrafish development. We show that from 24 h post-fertilization (hpf) onwards, the statoacoustic ganglion (SAG) develops in direct contact with two main blood vessels, the primordial hindbrain channel and the lateral dorsal aortae (LDA). At 48 hpf, the LDA is displaced medially, losing direct contact with the SAG. The relationship of the other cranial ganglia with the vasculature is evident for the medial lateral line ganglion and for the vagal ganglia that grow along the primary head sinus (PHS). We also observed that the innervation of the anterior macula runs over the PHS vessel. Our spatiotemporal anatomical map of the cranial ganglia and the head vasculature indicates physical interactions between both systems and suggests a possible functional interaction during development., (© 2017 Anatomical Society.)

Several concepts developed in the nineteenth century have formed the basis of much of our neuroanatomical teaching today. Not all of these were based on solid evidence nor have withstood the test of time. Recent evidence on the evolution and development of the autonomic nervous system, combined with molecular insights into the development and diversification of motor neurons, challenges some of the ideas held for over 100 years about the organization of autonomic motor outflow. This review provides an overview of the original ideas and quality of supporting data and contrasts this with a more accurate and in depth insight provided by studies using modern techniques. Several lines of data demonstrate that branchial motor neurons are a distinct motor neuron population within the vertebrate brainstem, from which parasympathetic visceral motor neurons of the brainstem evolved. The lack of an autonomic nervous system in jawless vertebrates implies that spinal visceral motor neurons evolved out of spinal somatic motor neurons. Consistent with the evolutionary origin of brainstem parasympathetic motor neurons out of branchial motor neurons and spinal sympathetic motor neurons out of spinal motor neurons is the recent revision of the organization of the autonomic nervous system into a cranial parasympathetic and a spinal sympathetic division (e.g., there is no sacral parasympathetic division). We propose a new nomenclature that takes all of these new insights into account and avoids the conceptual misunderstandings and incorrect interpretation of limited and technically inferior data inherent in the old nomenclature.

The enteric nervous system of jawed vertebrates arises primarily from vagal neural crest cells that migrate to the foregut and subsequently colonize and innervate the entire gastrointestinal tract. Here we examine development of the enteric nervous system in the basal jawless vertebrate the sea lamprey (Petromyzon marinus) to gain insight into its evolutionary origin. Surprisingly, we find no evidence for the existence of a vagally derived enteric neural crest population in the lamprey. Rather, labelling with the lipophilic dye DiI shows that late-migrating cells, originating from the trunk neural tube and associated with nerve fibres, differentiate into neurons within the gut wall and typhlosole. We propose that these trunk-derived neural crest cells may be homologous to Schwann cell precursors, recently shown in mammalian embryos to populate post-embryonic parasympathetic ganglia, including enteric ganglia. Our results suggest that neural-crest-derived Schwann cell precursors made an important contribution to the ancient enteric nervous system of early jawless vertebrates, a role that was largely subsumed by vagal neural crest cells in early gnathostomes.

Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2-3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 h in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 h confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Essential cellular components of the paired sensory organs of the vertebrate head are derived from transient thickenings of embryonic ectoderm known as cranial placodes. The epibranchial (EB) placodes give rise to sensory neurons of the EB ganglia that are responsible for relaying visceral sensations form the periphery to the central nervous system. Development of EB placodes and subsequent formation of EB ganglia is a multistep process regulated by various extrinsic factors, including fibroblast growth factors (Fgfs). We discovered that two Fgf ligands, Fgf3 and Fgf10a, cooperate to promote EB placode development. Whereas EB placodes are induced in the absence of Fgf3 and Fgf10a, they fail to express placode specific markers Pax2a and Sox3. Expression analysis and mosaic rescue experiments demonstrate that Fgf3 signal is derived from the endoderm, whereas Fgf10a is emitted from the lateral line system and the otic placode. Further analyses revealed that Fgf3 and Fgf10a activities are not required for cell proliferation or survival, but are required for placodal cells to undergo neurogenesis. Based on these data, we conclude that a combined loss of these Fgf factors results in a failure of the EB placode precursors to initiate a transcriptional program needed for maturation and subsequent neurogenesis. These findings highlight the importance and complexity of reiterated Fgf signaling during cranial placode formation and subsequent sensory organ development.

We review morphogenesis of the enteric nervous system from migratory neural crest cells, and defects of this process such as Hirschsprung disease, centering on cell motility and assembly, and cell adhesion and extracellular matrix molecules, along with cell proliferation and growth factors. We then review continuum and agent-based (cellular automata) models with rules of cell movement and logistical proliferation. Both movement and proliferation at the individual cell level are modeled with stochastic components from which stereotyped outcomes emerge at the population level. These models reproduced the wave-like colonization of the intestine by enteric neural crest cells, and several new properties emerged, such as colonization by frontal expansion, which were later confirmed biologically. These models predict a surprising level of clonal heterogeneity both in terms of number and distribution of daughter cells. Biologically, migrating cells form stable chains made up of unstable cells, but this is not seen in the initial model. We outline additional rules for cell differentiation into neurons, axon extension, cell-axon and cell-cell adhesions, chemotaxis and repulsion which can reproduce chain migration. After the migration stage, the cells re-arrange as a network of ganglia. Changes in cell adhesion molecules parallel this, and we describe additional rules based on Steinberg's Differential Adhesion Hypothesis, reflecting changing levels of adhesion in neural crest cells and neurons. This was able to reproduce enteric ganglionation in a model. Mouse mutants with disturbances of enteric nervous system morphogenesis are discussed, and these suggest future refinement of the models. The modeling suggests a relatively simple set of cell behavioral rules could account for complex patterns of morphogenesis. The model has allowed the proposal that Hirschsprung disease is mostly an enteric neural crest cell proliferation defect, not a defect of cell migration. In addition, the model suggests an explanations for zonal and skip segment variants of Hirschsprung disease, and also gives a novel stochastic explanation for the observed discordancy of Hirschsprung disease in identical twins., (© 2013 Elsevier Inc. All rights reserved.)

Vagal neural crest cells (VNCCs) arise in the hindbrain, and at (avian) embryonic day (E) 1.5 commence migration through paraxial tissues to reach the foregut as chains of cells 1-2 days later. They then colonise the rest of the gut in a rostrocaudal wave. The chains of migrating cells later resolve into the ganglia of the enteric nervous system. In organ culture, E4.5 VNCCs resident in the gut (termed enteric or ENCC) which have previously encountered vagal paraxial tissues, rapidly colonised aneural gut tissue in large numbers as chains of cells. Within the same timeframe, E1.5 VNCCs not previously exposed to paraxial tissues provided very few cells that entered the gut mesenchyme, and these never formed chains, despite their ability to migrate in paraxial tissue and in conventional cell culture. Exposing VNCCs in vitro to paraxial tissue normally encountered en route to the foregut conferred enteric migratory ability. VNCC after passage through paraxial tissue developed elements of retinoic acid signalling such as Retinoic Acid Binding Protein 1 expression. The paraxial tissue's ability to promote gut colonisation was reproduced by the addition of retinoic acid, or the synthetic retinoid Am80, to VNCCs (but not to trunk NCCs) in organ culture. The retinoic acid receptor antagonist CD 2665 strongly reduced enteric colonisation by E1.5 VNCC and E4.5 ENCCs, at a concentration suggesting RARα signalling. By FACS analysis, retinoic acid application to vagal neural tube and NCCs in vitro upregulated Ret; a Glial-derived-neurotrophic-factor receptor expressed by ENCCs which is necessary for normal enteric colonisation. This shows that early VNCC, although migratory, are incapable of migrating in appropriate chains in gut mesenchyme, but can be primed for this by retinoic acid. This is the first instance of the characteristic form of NCC migration, chain migration, being attributed to the application of a morphogen.

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve (1). Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components (2,3). Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

A defining characteristic of the normal development of the enteric nervous system (ENS) is the existence of mesoscale patterned entities called ganglia. Ganglia are clusters of neurons with associated enteric neural crest (ENC) cells, which form in the simultaneously growing gut wall. At first the precursor ENC cells proliferate and gradually differentiate to produce the enteric neurons; these neurons form clusters with ENC scattered around and later lying on the periphery of neuronal clusters. By immunolabelling neural cell-cell adhesion molecules, we infer that the adhesive capacity of neurons is greater than that of ENC cells. Using a discrete mathematical model, we test the hypothesis that local rules governing differential adhesion of neuronal agents and ENC agents will produce clusters which emulate ganglia. The clusters are relatively stable, relatively uniform and small in size, of fairly uniform spacing, with a balance between the number of neuronal and ENC agents. These features are attained in both fixed and growing domains, reproducing respectively organotypic in vitro and in vivo observations. Various threshold criteria governing ENC agent proliferation and differentiation and neuronal agent inhibition of differentiation are important for sustaining these characteristics. This investigation suggests possible explanations for observations in normal and abnormal ENS development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)(j1775) which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background & Aims: The majority of the enteric nervous system is derived from the vagal neural crest, with a second contribution, which is restricted to the post-umbilical gut, originating from the sacral neural crest. In mammals, although sacral neural crest cells (NCCs) have been shown to enter the hindgut, information on their development and role remains scant. Our aim was to determine the migratory routes of sacral NCCs to the hindgut, their timing and site of entry into the gut, and their migratory behaviors and differentiation within the hindgut., Methods: We used in situ cell labeling, whole embryo culture, immunofluorescence, organotypic culture, and time-lapse live-cell imaging in mouse embryos., Results: Sacral NCCs emigrated from the neural tube at embryonic day 9.5, accumulated bilateral to the hindgut to form prospective pelvic ganglia at embryonic day 11.5, and from there entered the distal hindgut through its ventrolateral side at embryonic day 13.5. They then migrated along nerve fibers extending from the pelvic ganglia toward the proximal hindgut, intermingling with rostrocaudally migrating vagal NCCs to differentiate into neurons and glia. In organotypic culture, genetically labeled sacral and vagal NCCs displayed different capabilities of entering the hindgut, implying differences in their intrinsic migratory properties. Time-lapse live-cell imaging on explants ex vivo showed that sacral NCCs migrated along nerve fibers and exhibited different migratory behaviors from vagal NCCs., Conclusions: Murine sacral NCCs are a distinct group of cells that migrate along defined pathways from neural tube to hindgut. They exhibit discrete migratory behaviors within the gut mesenchyme and contribute neurons and glial cells to the hindgut enteric nervous system., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

During rodent corticogenesis, a sizeable subpopulation of γ-aminobutyric acid (GABA)ergic interneurons arises extracortically from the medial ganglionic eminence (MGE). These neurons progressively acquire responsiveness to GABA in the course of corticopetal tangential migration, a process regulated by ambient GABA and mediated by GABA(A) receptors. Here, we combined patch clamp electrophysiology and single-cell reverse transcription-polymerase chain reaction to examine GABA(A) receptor expression in green fluorescent MGE-derived (eGFP+) cells in telencephalic slices from gestational day 14.5 BAC-Lhx6 embryos. GABA concentration-response curves revealed lower apparent affinity and efficacy in eGFP+ cells in and around the MGE than their counterparts in the cortex. Pharmacological tests revealed subunit-selective response profiles in the MGE and cortex consistent with differential expression of GABA(A) receptor isoforms. Profiling of GABA(A) receptor subunit transcripts (α1-5, β1-3, and γ1-3, δ) uncovered increased expression of the α1-, α2-, α5-, γ2-, and γ3-subunit messenger RNAs in the cortex. We propose that the dynamic expression of certain GABA(A) receptor subunits contributes to assembling receptor isoforms that confer functional attributes important in regulating the migration and maturation of primordial GABAergic cortical interneurons.

The peripheral growth cones of statoacoustic ganglion (SAG) neurons are presumed to sense molecular cues to navigate to their sensory targets during development. Based on previously reported expression data for Frizzled receptors, Wnt ligands, and Wnt inhibitors, we hypothesized that some members of the Wnt morphogen family may function as repulsive cues for SAG neurites. The responses of SAG neurons to mammalian Wnts -1, -4, -5a, -6, and -7b, and the Wnt inhibitors sFRP -1, -2, and -3, were tested in vitro by growing SAG explants from embryonic day 4 (E4) chicken embryos for two days in 3D collagen gels. Average neurite length and density were quantified to determine effects on neurite outgrowth. SAG neurites were strongly repelled by human Sema3E, demonstrating SAG neurons are responsive under these assay conditions. In contrast, SAG neurons showed no changes in neurite outgrowth when cultured in the presence of Wnts and Wnt inhibitors. As an alternative approach, Wnt4 and Wnt5a were also tested in vivo by injecting retroviruses encoding these genes into the chicken otocyst on E3. On E6, no differences were evident in the peripheral projections of SAG axons terminating in infected sensory organs as compared to uninfected organs on the contralateral side of the same embryo. For all Wnt sources, bioactivity was confirmed on E6 chick spinal cord explants by observing enhanced axon outgrowth, as reported previously in the mouse. These results suggest that the tested Wnts do not play a role in guiding peripheral axons in the chicken inner ear., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cadherins regulate the vertebrate nervous system development. We previously showed that cadherin-6 message (cdh6) was strongly expressed in the majority of the embryonic zebrafish cranial and lateral line ganglia during their development. Here, we present evidence that cdh6 has specific functions during cranial and lateral line ganglia and nerve development. We analyzed the consequences of cdh6 loss-of-function on cranial ganglion and nerve differentiation in zebrafish embryos. Embryos injected with zebrafish cdh6 specific antisense morpholino oligonucleotides (MOs, which suppress gene expression during development; cdh6 morphant embryos) displayed a specific phenotype, including (i) altered shape and reduced development of a subset of the cranial and lateral line ganglia (e.g., the statoacoustic ganglion and vagal ganglion) and (ii) cranial nerves were abnormally formed. These data illustrate an important role for cdh6 in the formation of cranial ganglia and their nerves., (Copyright © 2011 Wiley-Liss, Inc.)

Aims: To study the migration and developmental pattern of ganglion cells in fetuses aged 9-21 weeks, and to document whether the migration was occurring circumferentially equally in the entire axis or if there were discrepancies in different portions at the same level., Settings and Design: The hypothesis regarding the pathogenesis of Hirschsprung's disease mainly revolves around two schools. One is the single gradient migration of ganglia and the other is a dual gradient migration theory. Understanding the embryological development of enteric ganglia is necessary to study the pathogenesis of intestinal innervation disorders., Materials and Methods: We studied the development of intestinal ganglia in fetuses aged 9-21 weeks. Serial longitudinal sections from the colon were studied, the first one including the squamo-columnar junction, for the presence and the nature of ganglion cells with Hematoxylin and Eosin, and neurone-specific enolase immunostaining. Transverse sections from proximal gut were studied in a similar fashion. Thus, we evaluated the migration pattern as well as the nature of ganglia in the fetuses. We also measured the length of distal aganglionic segment in these growing fetuses., Results: We noted that ganglion cells appear first in the myenteric plexus followed by deep and superficial submucous plexus. We also found evidences in favor of dual migration theory, and the distal aganglionic segment varies around the circumference of the rectal wall., Conclusions: We got evidences in support of a dual migration pattern of intestinal ganglion cells. The level of distal aganglionic segments when measured from squamo-columnar junction varied with the age of gestation and the length was incongruous. The description of distal aganglionic segment may help surgeons while taking biopsies or during operative procedures.

Vertebrate cranial placodes contribute vitally to development of sensory structures of the head. Amongst posterior placodes, the otic placode forms the inner ear whereas nearby epibranchial placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common otic/epibranchial field, which later subdivides in response to other signals. However, we find that otic and epibranchial placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate otic expression of foxi1, a necessary step for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the otic placode forms first and induces epibranchial placodes through an Fgf-relay., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The generation of the sensory ganglia involves the migration of a precursor population to the site of ganglion formation and the differentiation of sensory neurons. There is, however, a significant difference between the ganglia of the head and trunk in that while all of the sensory neurons of the trunk are derived from the neural crest, the majority of cranial sensory neurons are generated by the neurogenic placodes. In this study, we have detailed the route through which the placodally-derived sensory neurons are generated, and we find a number of important differences between the head and trunk. Although, the neurogenic placodes release neuroblasts that migrate internally to the site of ganglion formation, we find that there are no placodally-derived progenitor cells within the forming ganglia. The cells released by the placodes differentiate during migration and contribute to the cranial ganglia as post-mitotic neurons. In the trunk, it has been shown that progenitor cells persist in the forming Dorsal Root Ganglia and that much of the process of sensory neuronal differentiation occurs within the ganglion. We also find that the period over which neuronal cells delaminate from the placodes is significantly longer than the time frame over which neural crest cells populate the DRGs. We further show that placodal sensory neuronal differentiation can occur in the absence of local cues. Finally, we find that, in contrast to neural crest cells, the different mature neurogenic placodes seem to lack plasticity. Nodose neuroblasts cannot be diverted to form trigeminal neurons and vice versa., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum-free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

Abnormalities in the development of enteric neural crest-derived progenitors (ENPs) that generate the enteric nervous system (ENS) can lead to aganglionosis in a variable portion of the distal gastrointestinal tract. Cumulative evidence suggests that variation of aganglionosis is due to gene interactions that modulate the ability of ENPs to populate the intestine; however, the developmental processes underlying this effect are unknown. We hypothesized that differences in enteric ganglion deficits could be attributable to the effects of genetic background on early developmental processes, including migration, proliferation, or lineage divergence. Developmental processes were investigated in congenic Sox10(Dom) mice, an established Hirschsprung disease (HSCR) model, on distinct inbred backgrounds, C57BL/6J (B6) and C3HeB/FeJ (C3Fe). Immuno-staining on whole-mount fetal gut tissue and dissociated cell suspensions was used to assess migration and proliferation. Flow cytometry utilizing the cell surface markers p75 and HNK-1 was used to isolate live ENPs for analysis of developmental potential. Frequency of ENPs was reduced in Sox10(Dom) embryos relative to wild-type embryos, but was unaffected by genetic background. Both migration and developmental potential of ENPs in Sox10(Dom) embryos were altered by inbred strain background with the most highly significant differences seen for developmental potential between strains and genotypes. In vivo imaging of fetal ENPs and postnatal ganglia demonstrates that altered lineage divergence impacts ganglia in the proximal intestine. Our analysis demonstrates that genetic background alters early ENS development and suggests that abnormalities in lineage diversification can shift the proportions of ENP populations and thus may contribute to ENS deficiencies in vivo.

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

The wiring of the nervous system arises from extensive directional migration of neuronal cell bodies and growth of processes that, somehow, end up forming functional circuits. Thus far, this feat of biological engineering appears to rely on sequences of pathfinding decisions upon local cues, each with little relationship to the anatomical and physiological outcome. Here, we uncover a straightforward cellular mechanism for circuit building whereby a neuronal type directs the development of its future partners. We show that visceral afferents of the head (that innervate taste buds) provide a scaffold for the establishment of visceral efferents (that innervate salivatory glands and blood vessels). In embryological terms, sensory neurons derived from an epibranchial placode--that we show to develop largely independently from the neural crest--guide the directional outgrowth of hindbrain visceral motoneurons and control the formation of neural crest-derived parasympathetic ganglia.

A cell's form and function is determined to a great extent by its cellular membrane and the underlying cytoskeleton. Understanding changes in the cellular membrane and cytoskeleton can provide insight into aging and disease of the cell. The atomic force microscope (AFM) allows unparalled resolution for the imaging of these cellular components and the ability to probe their mechanical properties. This report describes our progress toward the use of AFM as a tool in neuroscience applications. Elasticity measurements are reported on living chick embryo dorsal root ganglion and sympathetic neurons in vitro. The neuronal cellular body and growth cones regions are examined for variations in cellular maturity. In addition, cellular changes due to exposure to various environmental conditions and neurotoxins are investigated. This report includes data obtained on different AFM systems, using various AFM techniques and thus also provides knowledge of AFM instruments and methodology., ((c) 2009 Elsevier B.V. All rights reserved.)

Cranial placodes (which include the adenohypophyseal, olfactory, lens, otic, lateral line, profundal/trigeminal, and epibranchial placodes) give rise to many sense organs and ganglia of the vertebrate head. Recent evidence suggests that all cranial placodes may be developmentally related structures, which originate from a common panplacodal primordium at neural plate stages and use similar regulatory mechanisms to control developmental processes shared between different placodes such as neurogenesis and morphogenetic movements. After providing a brief overview of placodal diversity, the present review summarizes current evidence for the existence of a panplacodal primordium and discusses the central role of transcription factors Six1 and Eya1 in the regulation of processes shared between different placodes. Upstream signaling events and transcription factors involved in early embryonic induction and specification of the panplacodal primordium are discussed next. I then review how individual placodes arise from the panplacodal primordium and present a model of multistep placode induction. Finally, I briefly summarize recent advances concerning how placodal neurons and sensory cells are specified, and how morphogenesis of placodes (including delamination and migration of placode-derived cells and invagination) is controlled., (Copyright 2010 Elsevier Inc. All rights reserved.)

The amelogenins comprise 90% of the developing extracellular enamel matrix proteins and play a major role in the biomineralization and structural organization of enamel. Amelogenins were also detected, in smaller amounts, in postnatal calcifying mesenchymal tissues, and in several nonmineralizing tissues including brain. Low molecular mass amelogenin isoforms were suggested to have signaling activity; to produce ectopically chondrogenic and osteogenic-like tissue and to affect mouse tooth germ differentiation in vitro. Recently, some amelogenin isoforms were found to bind to the cell surface receptors; LAMP-1, LAMP-2 and CD63, and subsequently localize to the perinuclear region of the cell. The recombinant amelogenin protein (rHAM(+)) alone brought about regeneration of the tooth supporting tissues: cementum, periodontal ligament and alveolar bone, in the dog model, through recruitment of progenitor cells and mesenchymal stem cells. We show that amelogenin is expressed in various tissues of the developing mouse embryonic cranio-facial complex such as brain, eye, ganglia, peripheral nerve trunks, cartilage and bone, and is already expressed at E10.5 in the brain and eye, long before the initiation of tooth formation. Amelogenin protein expression was detected in the tooth germ (dental lamina) already at E13.5, much earlier than previously reported (E19). Application of amelogenin (rHAM(+)) beads together with DiI, on E13.5 and E14.5 embryonic mandibular mesenchyme and on embryonic tooth germ, revealed recruitment of mesenchymal cells. The present results indicate that amelogenin has an important role in many tissues of the cranio-facial complex during mouse embryonic development and differentiation, and might be a multifunctional protein., ((c) 2008 Wiley-Liss, Inc.)

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.

Background: The ability of a neuron to regenerate functional connections after injury is influenced by both its intrinsic state and also by extrinsic cues in its surroundings. Investigations of the transcriptional changes undergone by neurons during in vivo models of injury and regeneration have revealed many transcripts associated with these processes. Because of the complex milieu of interactions in vivo, these results include not only expression changes directly related to regenerative outgrowth and but also unrelated responses to surrounding cells and signals. In vitro models of neurite outgrowth provide a means to study the intrinsic transcriptional patterns of neurite outgrowth in the absence of extensive extrinsic cues from nearby cells and tissues., Results: We have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro. Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection. Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth. These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription. We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo., Conclusion: Many gene expression changes undergone by SCG and DRG during in vitro outgrowth are shared between these two tissue types and in common with in vivo regeneration models. This suggests that the genes identified in this in vitro study may represent new candidates worthy of further study for potential roles in the therapeutic regrowth of neuronal connections.

Incorporation of the thymidine analog bromodeoxyuridine (BrdU) was used to assess cytogenesis in the central nervous system (CNS) of the appendicularian Oikopleura dioica. A series of timed cumulative labelings carried out from 45 minutes (min) to 8 hours (h) after fertilization provided labeling patterns that showed when neurons and support cells residing at specific sites within the 9 h CNS became postmitotic. Throughout the CNS, which includes the cerebral ganglion, caudal ganglion and caudal nerve cord, neurogenesis occurs during an earlier time window than the genesis of support cells. Neurons are first generated at about 45 min to 1 h after fertilization in all 3 CNS regions, starting in the cerebral ganglion. Support cells are generated starting at about 2 h after fertilization. In both the cerebral ganglion and the caudal ganglion, neurons born during different time epochs settle in a specific spatial pattern, following a caudal to rostral gradient in the caudal ganglion and a more complex pattern in the cerebral ganglion. No such regional pattern was seen in the caudal nerve cord, where neurons born during different epochs were evenly distributed along the length of the cord. In the cerebral ganglion a small subpopulation of cells continued to incorporate BrdU from 8 h to at least 15 h and may represent a reserve of stem cells or progenitor cells that generate additional cells seen in the adult. The results show that this simple urochordate exhibits several vertebrate features of CNS cytogenesis, including a different timing of neurogenesis and gliogenesis (support cells being the likely candidates for glial cells in Oikopleura), gradients of neuron position according to birthdate, and a maintenance of neural cell precursors beyond embryonic and larval stages.

Sox5 is a member of the SoxD group of HMG-box transcription factors that, during the early stages of development, promotes neural crest generation. However, little is known about Sox5 function in neural crest derivatives such as the peripheral sensory nervous system. We have analysed the embryonic expression of Sox5 during chick cranial ganglia development, from the stages of ganglia condensation to those of differentiation. During this period, Sox5 expression is maintained in the crest-derived satellite glial cells in all the cranial ganglia. In contrast, Sox5 is only transiently expressed in a subpopulation of differentiating neurons of both neural crest and placode origin. This detailed analysis provides a good base to dissect the possible role of Sox5 in neural cell fate determination by future functional approaches., ((c) 2007 Wiley-Liss, Inc.)

The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/-), or null (-/-) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant., ((c) 2007 Wiley-Liss, Inc.)

The development of the caudal nerve cord and muscle innervation in the appendicularian Oikopleura dioica was assessed using differential interference contrast and confocal microscopy, phalloidin staining of actin, and in situ hybridization for the neuronal markers tubulin and choline acetyltransferase (ChAT). The caudal nerve cord first appears as a stream of tubulin mRNA-positive neurons that extends into the tail from the caudal ganglion. By this stage a few actin-rich nerve fibers course longitudinally along the cord. As the tail lengthens, the caudal nerve cord extends and becomes more fasciculated and the neurons cluster at stereotyped longitudinal positions. The number of neurons in the nerve cord reaches a relatively stable maximum of about 29. A subset of neurons in the caudal ganglion and caudal nerve cord expresses ChAT mRNA. These putative motoneurons are distributed along nearly the full extent of the tail in numbers consistent with an independent innervation of each tail muscle cell. The longitudinal series of putative motoneurons is not aligned with the muscle cells, but peripheral nerve fibers extending to the muscle cells are, indicating that motor axons grow along the cord before exiting adjacent to their peripheral target. Muscle innervation occurs roughly coincident with the onset of ChAT mRNA expression. Our results provide the first molecular identification of motoneurons and the first developmental characterization of the motor system in an appendicularian and help set the stage for gene expression studies aimed at understanding the evolution of developmental patterning in this part of the chordate central nervous system., ((c) 2007 Wiley-Liss, Inc.)

We previously reported that cadherin-4 (also called R-cadherin) was expressed by the majority of the developing zebrafish cranial and lateral line ganglia. Cadherin-4 (Cdh4) function in the formation of these structures in zebrafish was studied using morpholino antisense technology. Differentiation of the cranial and lateral line ganglia and lateral line nerve and neuromasts of the cdh4 morphants was analyzed using multiple neural markers. We found that a subset of the morphant cranial and lateral line ganglia were disorganized, smaller, with reduced staining, and/or with altered shape compared to control embryos. Increased cell death in the morphant ganglia likely contributed to these defects. Moreover, cdh4 morphants had shorter lateral line nerves and a reduced number of neuromasts, which was likely caused by disrupted migration of the lateral line primordia. These results indicate that Cdh4 plays a role in the normal formation of the zebrafish lateral line system and a subset of the cranial ganglia.

Notch activation inhibits neuronal differentiation during development of the nervous system; however, the dynamic role of Notch signaling in individual cell lineages remains poorly understood. We have characterized 3.4 kb 5'-regulatory sequence of a Notch target gene, her4, and used it to drive fluorescent gene expression in transgenic lines where the spatiotemporal pattern of Notch activation can be examined in vivo. The 3.4 kb her4 promoter contains five predicted Su(H) binding sites of which two proximal sites were confirmed to be required for Notch-mediated transcriptional activation. Without Notch, Su(H) effectively represses transcription regulated by the promoter. Analyses of transgenic zebrafish showed that while the expression of proneural genes and Notch activation were both critical for endogenous her4 expression, reporter gene expression was primarily regulated by Notch activity. This study also showed that her4 may be differently regulated in sensory cranial ganglia, where Notch activity is not essential for her4 expression and where Su(H) may repress her4 expression. The establishment of a reporter line with Notch-Su(H)-dependent fluorescent gene expression provides a tool to explore the complex role of Notch signaling in the development of vertebrate nervous system.

Cortical GABAergic interneurons in mice are largely derived from the subpallium. Work from our laboratory and others over the past five years has demonstrated that a developmental logic in space and time underlies the emergence of specific cortical interneuronal subtypes. Following on from the seminal work of the Rubenstein laboratory, we set out to fate map the output of the subpallial ganglionic eminences. Our initial approach utilized ultrasound backscatter microscopy to perform homotopic and heterotopic transplants of genetically marked progenitors from the lateral, medial and caudal ganglionic eminences (LGE, MGE and CGE, respectively) to unmarked host brains. The LGE, at least in the context of our transplant studies, did not appear to generate cortical interneurons. By contrast, we found that that approximately eighty percent of cortical interneurons arise from the MGE, while the remaining twenty percent were generated by the CGE. Hence, the majority of interneuron subtypes, including all fast spiking parvalbumin-positive basket cells and somatostatin-positive Martinotti cells appear to arise from the MGE. A more restricted set of cortical interneurons seems to be generated in the CGE, the majority of which are bipolar calretinin/VIP-positive interneurons. Complementing these results, we have recently demonstrated using inducible genetic fate mapping that the MGE produces specific cortical interneuron subtypes at discrete timepoints during development. These studies demonstrate that cortical interneurons arise from a precise developmental programme that acts in both space and time. Beyond this however, it seems likely that postmitotic events influence the specific function of subclasses of cortical interneurons. A primary challenge in the future will be to investigate what aspects of interneuron diversity are determined by intrinsic genetic programmes within each lineage versus those properties imposed by the local environment in the cortex.

BCS1L is a chaperone necessary for the incorporation of Rieske FeS and Qcr10p into complex III (CIII) of the respiratory chain. Mutations in the BCS1L gene cause early fetal growth restriction and a lethal neonatal disease in humans, however, the pathogenesis remains unclear. Here, we analysed the expression of BCS1L during mouse embryonic development and compared its expression with that of the mitochondrial markers Porin, GRIM19, Core I, and Rieske FeS. BCS1L was strongly expressed in embryonic tissues already at embryonic days 7 (E7) and 9 whereas the expression of Porin and Rieske FeS was not as evident at this time point. At E11, BCS1L, Porin, and Rieske FeS had overlapping expression patterns in organs known to contain high numbers of mitochondria such as heart, liver and somites. In contrast, BCS1L was differently distributed compared to the mitochondrial proteins Porin, Rieske FeS, Core I and Grim 19 in the floor plate of the E11, E12 and E13 neural tube. These results show that the expression pattern of BCS1L only partially overlaps with the expression of Porin and Rieske FeS. Thus, BCS1L alone or in cooperation with Rieske FES may during development have previously unknown functions beside its role in assembly of complex III. The floor plate of the neural tube is essential for dorsal ventral patterning and the guidance of the developing neurons to their targets. The predominant expression of BCS1L in this region, together with its presence in peripheral ganglia from E13 onwards, indicates a role for BCS1L in the development of neural structures.

The aim of the study was to determine the morphology of epicardiac ganglia in human fetuses at different stages of their development as these ganglia are considered to be of a pivotal clinical importance. Twenty-one fetal hearts were investigated applying a technique of histochemistry for acetylcholinesterase to visualize the epicardiac neural ganglionated plexus with its subsequent stereoscopic examination on total organs. In all of the examined fetuses, epicardiac neural plexus with numerous ganglia was well recognizable and could be clearly differentiated into seven ganglionated subplexuses, topography and structural organization of which were typical for hearts of adult human. The largest ganglion number comprising 77% of all counted ganglia was identified on the dorsal atrial surface. Fetal epicardiac plexus in gestation period of 15-40 weeks contained 929 +/- 62 ganglia, but ganglion amount did vary substantially from heart to heart. In conclusion, this study implies that the human fetal epicardiac ganglia occupy their definitive location already at gestation period from 15 weeks and their number as well as distribution on heart surface presumably is not age dependent.

Mutations in the Neurofibromatosis 2 (NF2) gene are associated with predisposition to vestibular schwannomas, spinal schwannomas, meningiomas, and ependymomas. Presently, how NF2 is expressed during embryonic development and in the tissues affected by neurofibromatosis type 2 (NF2) has not been well defined. To examine NF2 expression in vivo, we generated transgenic mice carrying a 2.4-kb NF2 promoter driving beta-galactosidase (beta-gal) with a nuclear localization signal. Whole-mount embryo staining revealed that the NF2 promoter directed beta-gal expression as early as embryonic day E5.5. Strong expression was detected at E6.5 in the embryonic ectoderm containing many mitotic cells. beta-gal staining was also found in parts of embryonic endoderm and mesoderm. The beta-gal staining pattern in the embryonic tissues was corroborated by in situ hybridization analysis of endogenous Nf2 RNA expression. Importantly, we observed strong NF2 promoter activity in the developing brain and in sites containing migrating cells including the neural tube closure, branchial arches, dorsal aorta, and paraaortic splanchnopleura. Furthermore, we noted a transient change of NF2 promoter activity during neural crest cell migration. While little beta-gal activity was detected in premigratory neural crest cells at the dorsal ridge region of the neural fold, significant activity was seen in the neural crest cells already migrating away from the dorsal neural tube. In addition, we detected considerable NF2 promoter activity in various NF2-affected tissues such as acoustic ganglion, trigeminal ganglion, spinal ganglia, optic chiasma, the ependymal cell-containing tela choroidea, and the pigmented epithelium of the retina. The NF2 promoter expression pattern during embryogenesis suggests a specific regulation of the NF2 gene during neural crest cell migration and further supports the role of merlin in cell adhesion, motility, and proliferation during development., ((c) 2006 Wiley-Liss, Inc.)

The RET gene is tightly regulated at the transcriptional level during embryo development, however in vitro experiments in cultured cells have failed to clarify the molecular mechanism of cell-type specificity of RET promoter activity. Therefore, we have generated transgenic mice in which the LacZ reporter gene is controlled by murine Ret promoter sequences to clarify in an in vivo model how this transcriptional regulation is achieved. We describe here the results of reporter gene expression in mice in which the transgene contained 380- and 1962-bp sequence upstream of the ATG start codon, derived from the mouse Ret promoter region, fused to the beta-galactosidase coding sequence. Transgenic mice showed well-defined patterns of beta-galactosidase staining obtained with both transgenes, suggesting that they were able per se to direct the reporter gene expression in specific districts, such as cranial ganglia, dorsal root ganglia, the heart and the kidney, partially recapitulating the profile of the endogenous Ret gene. In particular, proper expression in the developing excretory system seemed quite significant when considering that the appropriate regulation was obtained with a very short, 380 bp, fragment of Ret 5' flanking sequence.

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin alpha5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin alpha5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin alpha5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations.

Melanocytes are derived from the neural crest. We questioned whether the migratory mechanism during the invasive growth of melanoma cells is the same as that in neural crest cell migration. We transplanted human SK-Mel 28 melanoma cells into the neural tube of the chick embryo stage 11-13 and, after up to 6 days of total incubation, traced the cells by immunohistochemistry in serial paraffin sections. SK-Mel 28 cells were integrated into the host neural crest and were found in the roof plate of the neural tube, along the medial neural crest cell pathway, in the sclerotome and, finally, in developing sympathetic ganglia. At stage 21, massive segmental emigration between myotome and disintegrating dermatome was observed at the level of the upper limb bud. The melanoma cells, in contrast with the chick neural crest cells, were HNK-1-negative. They retained the premelanosome epitope HMB-45. For definite identification and exclusion of fusion with chick embryo cells, in situ hybridization with the human-specific Alu sequence was performed. The results showed that human SK-Mel 28 melanoma cells were capable of resuming neural crest cell migration in the embryo.

Nitric oxide (NO) is involved in normal bladder physiology by regulating local arteriolar tone and smooth muscle relaxation and modulating the production of extracellular matrix proteins in vitro. Little information is available regarding the nitrergic innervation of the bladder during development. In this study we investigated the changes in density and morphology of the intramural nitrergic neurons of the porcine urinary bladder during development using whole-mount preparation. Bladder specimens were obtained from porcine foetuses of gestational age 60 days (n=5) and 90 days (n=5) and from newborn piglets (n=5) after perfusion fixation. Bladders were divided into base, body, and dome. Whole-mount preparation using NADPH-diaphorase (NADPH-d) histochemistry was used to visualize nitrergic innervation of the urinary bladders and to measure density of NADPH-positive ganglia (including single neurons), number of NADPH-d positive neurons per ganglion, and size of individual neurons. One-way ANOVA and chi-square tests were used for statistical analysis with a p-value <0.05 considered statistically significant. NADPH-d positive ganglia were numerous in the muscular layer of all three age groups. At E60, ganglion density was significantly higher in the body (mean 880/cm(2)) than in the dome (397/cm(2)) or the base (676/cm(2)). The ganglion density significantly decreased with age. The number of NADPH-d positive neurons per ganglion increased significantly between E90 and birth (p<0.01). A marked increase in the size of individual neurons over time was also seen (p<0.001), predominantly due to an increase in cytoplasm. Our data on whole-mount preparations demonstrate that significant maturation in nitrergic neuronal density and morphology occurs in the porcine urinary bladder, at least until birth.

In this article we report the isolation of a novel zebrafish gene, pitx3, which plays an important role in the formation of several placode-derived structures. In wildtype embryos, pitx3 is first expressed in a crescent-shaped area in the anterior end of the embryo. At later stages, the primordia of the anterior pituitary, the lens, the olfactory sensory epithelium, and cranial ganglia express this gene. Pitx3 is not expressed in the more posterior preplacodal region that gives rise to the epibranchial, otic, and lateral line placodes. The dynamics of pitx3 in the anterior region of wildtype embryos suggests that pitx3 expression marks a common step in the formation of the pituitary, lens, olfactory placode as well as the trigeminal placode. Analysis of pitx3 expression in mutants lacking the hedgehog or nodal function demonstrates the differential dependence of pitx3 expression in these structures on nodal and hedgehog signaling. While the lens and trigeminal placodes express pitx3 in the absence of hedgehog and nodal signaling, there is no expression of pitx3 in the anteriormost ectoderm adjacent to the neural plate from which the anterior pituitary would derive. In mutants with impaired hedgehog signaling, the lens placode frequently extends into more anterior ventral regions of the embryo.

To identify a novel type II classical cadherin, we searched the genome database and found rat cadherin19 (cad19) with high similarity to human cadherin19. We also found nucleotide sequences corresponding to rat cad19 in mouse and chicken genomes. In situ hybridization of rat cad19 revealed that rat cad19 mRNA was initially expressed in cephalic neural crest cells, and then in the cranial ganglia, migrating trunk neural crest cells, the nascent dorsal root ganglia, and the sympathetic ganglia. Expression of cad19 overlapped with that of neural crest markers, including Sox10 and AP-2, but cad19 expression was confined to subpopulations of the neural crest-derived cells, those typically observed in the satellite glia at the periphery of the ganglia and Schwann cell precursors along the peripheral nerves. cad19 mRNA was not detected in cells expressing Phox2b, an epibranchial placode-derived neurons, nor in those expressing neuronal markers such as Hu protein. These observations suggest that cad19 is expressed in neural crest-derived, non-neuronal cells. Although the expression of cad19 mRNA persisted in Schwann cell precursors at E14.5, it was no longer detected in maturing Schwann cells at later stages. These results suggest that cad19 is an evolutionarily conserved cadherin and may be involved in the early development of Schwann cells in the peripheral nervous system.

The development of intrinsic ganglia, comprised of neurons and glia cells that innervate airway smooth muscle, is a recognized component of the growing lung. However, the embryological origin of these neurons and glia is unclear. The lung buds develop as an outgrowth of the foregut, which contains migrating neural crest cells (NCC) that ultimately give rise to the enteric nervous system (ENS) along the entire length of the gut. It has therefore been proposed that the intrinsic ganglia of the lung arise from a subset of NCC that leave the gut and migrate into the lung buds during early development. We have tested this hypothesis using quail-chick interspecies grafting to selectively label the hindbrain-derived neural crest cell population that colonizes the gut. In conjunction with antibody labeling and in situ hybridization, we demonstrate that: (i) lung ganglia arise from vagal NCC that migrate from the foregut into the lung buds; (ii) like ENS precursors, these NCC express the transcription factor Sox10, and the receptors EDNRB and RET; (iii) the co-receptor for RET, GFRalpha1, is expressed in the lung mesenchyme and in ganglia; (iv) ganglia persist within the lung throughout development and contain cells immunopositive for the pan-neuronal markers ANNA-1 and PGP9.5, the inhibitory neurotransmitter NO, as shown by NADPH-diaphorase staining, and the glial marker GFAP.

Sensory placodes are unique columnar epithelia with neurogenic potential that develop in the vertebrate head ectoderm next to the neural tube. They contribute to the paired sensory organs and the cranial sensory ganglia generating a wide variety of cell types ranging from lens fibres to sensory receptor cells and neurons. Although progress has been made in recent years to identify the molecular players that mediate placode specification, induction and patterning, the processes that initiate placode development are not well understood. One hypothesis suggests that all placode precursors arise from a common territory, the pre-placodal region, which is then subdivided to generate placodes of specific character. This model implies that their induction begins through molecular and cellular mechanisms common to all placodes. Embryological and molecular evidence suggests that placode induction is a multi-step process and that the molecular networks establishing the pre-placodal domain as well as the acquisition of placodal identity are surprisingly similar to those used in Drosophila to specify sensory structures.