CLoNe is a new method to target single progenitors and study their progeny in mouse and chick.

Cell lineage analysis enables us to address pivotal questions relating to: the embryonic origin of cells and sibling cell relationships in the adult body; the contribution of progenitors activated after trauma or disease; and the comparison across species in evolutionary biology. To address such fundamental questions, several techniques for clonal labelling have been developed, each with its shortcomings. Here, we report a novel method, CLoNe that is designed to work in all vertebrate species and tissues. CLoNe uses a cocktail of labelling, targeting and transposition vectors that enables targeting of specific subpopulations of progenitor types with a combination of fluorophores resulting in multifluorescence that describes multiple clones per specimen. Furthermore, transposition into the genome ensures the longevity of cell labelling. We demonstrate the robustness of this technique in mouse and chick forebrain development, and show evidence that CLoNe will be broadly applicable to study clonal relationships in different tissues and species.

Cranial neural crest cells form corridors prefiguring sensory neuroblast migration.

The majority of cranial sensory neurons originate in placodes in the surface ectoderm, migrating to form ganglia that connect to the central nervous system (CNS). Interactions between inward-migrating sensory neuroblasts and emigrant cranial neural crest cells (NCCs) play a role in coordinating this process, but how the relationship between these two cell populations is established is not clear. Here, we demonstrate that NCCs generate corridors delineating the path of migratory neuroblasts between the placode and CNS in both chick and mouse. In vitro analysis shows that NCCs are not essential for neuroblast migration, yet act as a superior substrate to mesoderm, suggesting provision of a corridor through a less-permissive mesodermal territory. Early organisation of NCC corridors occurs prior to sensory neurogenesis and can be recapitulated in vitro; however, NCC extension to the placode requires placodal neurogenesis, demonstrating reciprocal interactions. Together, our data indicate that NCC corridors impose physical organisation for precise ganglion formation and connection to the CNS, providing a local environment to enclose migrating neuroblasts and axonal processes as they migrate through a non-neural territory.

Changes in gene expression and cell shape characterise stages of epibranchial placode-derived neuron maturation in the chick.

Sensory neurons in the head are largely generated from neurogenic placodes. Previous studies have revealed early events in placode development; however, the process of maturation has not been studied. In this study, it has been shown that placodal neurogenesis follows a sequential progression with distinct stages defined by expression of specific markers. These markers highlight domains of maturation within the stream of migratory neuroblasts that extend between the placode and the neural tube. Commitment to neurogenesis occurs in the apical placode, with the newborn neuroblasts delaminating basally and entering a transition zone. The neuroblasts migrate through the transition zone, differentiating further and becoming post-mitotic as they approach the ganglionic anlage. It has further been demonstrated that this progression from the transition zone to the ganglionic anlage is accompanied by a switch from multipolar to bipolar cell morphology. This sequential progression parallels events observed elsewhere in the nervous system, but here the stages are distinct and anatomically segregated. It is proposed that placodal neurogenesis provides a tractable system to examine the transition between states in neurogenesis.

From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes.

The inner ear and the epibranchial ganglia constitute much of the sensory system in the caudal vertebrate head. The inner ear consists of mechanosensory hair cells, their neurons, and structures necessary for sound and balance sensation. The epibranchial ganglia are knots of neurons that innervate and relay sensory signals from several visceral organs and the taste buds. Their development was once thought to be independent, in line with their independent functions. However, recent studies indicate that both systems arise from a morphologically distinct common precursor domain: the posterior placodal area. This review summarises recent studies into the induction, morphogenesis and innervation of these systems and discusses lineage restriction and cell specification in the context of their common origin.

The formation of the cranial ganglia by placodally-derived sensory neuronal precursors.

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.

Professional nursing governance in a large Australian health service.

BACKGROUND: Professional nursing governance refers to the processes and structures that influence nursing practice within an organisation. This study measured the effect of structured meeting communication processes on nurses' perceptions of professional governance. METHOD: The intervention was implemented in eight hospital wards. After 3 months, nurses on the intervention wards and eight matched-control wards completed the Index of Professional Nursing Governance (N = 225). Data were compared with a sample of Magnet(®) (N = 3) and non-Magnet (N = 46) hospitals. RESULTS: There was substantial variation in nurses' perceptions of governance across the 16 wards, irrespective of the intervention. Compared to non-Magnet hospitals, the overall score and three of the six subscales scores were higher in this study. Magnet hospitals scores, however, were typically higher suggesting greater progress towards shared governance. CONCLUSIONS: Professional nursing governance can be highly variable across individual wards and tailored interventions should be considered.

The formation of the superior and jugular ganglia: insights into the generation of sensory neurons by the neural crest.

The superior and jugular ganglia (S/JG) are the proximal ganglia of the IXth and Xth cranial nerves and the sensory neurons of these ganglia are neural crest derived. However, it has been unclear the extent to which their differentiation resembles that of the Dorsal Root Ganglia (DRGs). In the DRGs, neural crest cells undergo neuronal differentiation just after the onset of migration and there is evidence suggesting that these cells are pre-specified towards a sensory fate. We have analysed sensory neuronal differentiation in the S/JG. We show, in keeping with previous studies, that neuronal differentiation initiates long after the cessation of neural crest migration. We also find no evidence for the existence of migratory neural crest cells pre-specified towards a sensory phenotype prior to ganglion formation. Rather our results suggest that sensory neuronal differentiation in the S/JG is the result of localised spatiotemporal cues.

Separating early sensory neuron and blood vessel patterning.

The anatomical association between sensory nerves and blood vessels is well recognised in the adult, and interactions between the two are important during development. Here we have examined the relationship between developing blood vessels and sensory neuronal cell bodies, which is less well understood. We show in the chick that the nascent dorsal root ganglia (DRG) lie dorsal to the longitudinal anastomosis, adjacent to the developing neural tube at the level of the sulcus limitans. Furthermore, the blood vessel is present prior to the neurons suggesting that it may play a role in positioning the DRG. We use the zebrafish cloche mutation to analyse DRG formation in the absence of blood vessels and show that the DRG are positioned normally. Thus, despite their close anatomical relationship, the patterning of the blood vessel and DRG alongside the neural tube is separable rather than interdependent.

Use of the viral 2A peptide for bicistronic expression in transgenic mice.

BACKGROUND: Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner. RESULTS: To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line. CONCLUSION: The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.

Migration of neuroblasts from neurogenic placodes.

Peripheral neurons involved in cephalic sensory systems are born in the ectoderm at a distance from the neural tube. The neuroblasts migrate internally, coalesce to form ganglia and extend axons to the central nervous system. This process has long been evident but little is known about the way it occurs. We have shown that coordination of the migration and integration with the hindbrain occurs through interaction with neural crest cells.

Absence of Tangentially Migrating Glutamatergic Neurons in the Developing Avian Brain.

Several neuronal populations orchestrate neocortical development during mammalian embryogenesis. These include the glutamatergic subplate-, Cajal-Retzius-, and ventral pallium-derived populations, which coordinate cortical wiring, migration, and proliferation, respectively. These transient populations are primarily derived from other non-cortical pallial sources that migrate to the dorsal pallium. Are these migrations to the dorsal pallium conserved in amniotes or are they specific to mammals? Using in ovo electroporation, we traced the entire lineage of defined chick telencephalic progenitors. We found that several pallial sources that produce tangential migratory neurons in mammals only produced radially migrating neurons in the avian brain. Moreover, ectopic expression of VP-specific mammalian Dbx1 in avian brains altered neurogenesis but did not convert the migration into a mammal-like tangential movement. Together, these data indicate that tangential cellular contributions of glutamatergic neurons originate from outside the dorsal pallium and that pallial Dbx1 expression may underlie the generation of the mammalian neocortex during evolution.

Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins.

Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.

Identification of molecular signatures specific for distinct cranial sensory ganglia in the developing chick.

BACKGROUND: The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process. RESULTS: Transcriptional profiles were generated for populations of differentiated neurons purified from distinct cranial sensory ganglia using microdissection in embryonic chicken followed by FAC-sorting and RNAseq. Whole transcriptome analysis confirmed the division into somato- versus viscerosensory neurons, with additional evidence for subdivision of the somatic class into general and special somatosensory neurons. Cross-comparison of distinct ganglia transcriptomes identified a total of 134 markers, 113 of which are novel, which can be used to distinguish trigeminal, vestibulo-acoustic and epibranchial neuronal populations. In situ hybridisation analysis provided validation for 20/26 tested markers, and showed related expression in the target region of the hindbrain in many cases. CONCLUSIONS: One hundred thirty-four high-confidence markers have been identified for placode-derived cranial sensory ganglia which can now be used to address the acquisition of specific cranial sensory modalities.

Delamination of cells from neurogenic placodes does not involve an epithelial-to-mesenchymal transition.

Neurogenic placodes are specialized regions of embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we examine in chick the mechanism underlying the delamination of cells from the epibranchial placodal ectoderm. We show that the placodal epithelium has a distinctive morphology, reflecting a change in cell shape, and is associated with a breach in the underlying basal lamina. Placodal cell delamination is distinct from neural crest cell delamination. In particular, exit of neuroblasts from the epithelium is not associated with the expression of Snail/Snail2 or of the Rho family GTPases required for the epithelial-to-mesenchymal transition seen in neural crest cell delamination. Indeed, cells leaving the placodes do not assume a mesenchymal morphology but migrate from the epithelium as neuronal cells. We further show that the placodal epithelium has a pseudostratified appearance. Examination of proliferation shows that the placodal epithelium is mitotically quiescent, with few phosphohistone H3-positive cells being identified. Where division does occur within the epithelium it is restricted to the apical surface. The neurogenic placodes thus represent specialized ectodermal niches that generate neuroblasts over a protracted period.

Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the chick.

Within the hindbrain region, neural crest cell migration is organized into three streams that follow the segmentation of the neuroepithelium into distinct rhombomeric compartments. Although the streaming of neural crest cells is known to involve signals derived from the neuroepithelium, the molecular properties underlying this process are poorly understood. Here, we have mapped the expression of the signaling component of two secreted class III Semaphorins, Semaphorin (Sema) 3A and Sema 3F, at time points that correspond to neural crest cell migration within the hindbrain region of the chick. Both Semaphorins are expressed within rhombomeres at levels adjacent to crest-free mesenchyme and expression of the receptor components essential for Semaphorin activity by neural crest cells suggests a function in restricting neural crest cell migration. By using bead implantation and electroporation in ovo, we define a role for both Semaphorins in the maintenance of neural crest cell streams in proximity to the neural tube. Attenuation of Semaphorin signaling by expression of soluble Neuropilin-Fc resulted in neural crest cells invading adjacent mesenchymal territories that are normally crest-free. The loss or misguidance of specific neural crest cell populations after changes in Semaphorin signaling also affects the integration of the cranial sensory ganglia. Thus, Sema 3A and 3F, expressed and secreted by the hindbrain neuroepithelium contributes to the appropriate positioning of neural crest cells in proximity to the neural tube, a process crucial for the subsequent establishment of neuronal connectivity within the hindbrain region.

Cannabinoid receptor, CB1, expression follows neuronal differentiation in the early chick embryo.

The role of the CB1 cannabinoid receptor and endocannabinoid signalling has been widely studied in the adult nervous system. However, an emerging body of evidence suggests that the CB1 receptor may also play a role during development. Here we have scrutinized the expression profile of the CB1 receptor from the onset of neurogenesis in the chick embryo. We find that this gene exhibits a dynamic expression pattern that spatially and temporally follows neuronal differentiation in the early embryo.

Significance of the cranial neural crest.

The cranial neural crest has long been viewed as being of particular significance. First, it has been held that the cranial neural crest has a morphogenetic role, acting to coordinate the development of the pharyngeal arches. By contrast, the trunk crest seems to play a more subservient role in terms of embryonic patterning. Second, the cranial crest not only generates neurons, glia, and melanocytes, but additionally forms skeletal derivatives (bones, cartilage, and teeth, as well as smooth muscle and connective tissue), and this potential was thought to be a unique feature of the cranial crest. Recently, however, several studies have suggested that the cranial neural crest may not be so influential in terms of patterning, nor so exceptional in the derivatives that it makes. It is now becoming clear that the morphogenesis of the pharyngeal arches is largely driven by the pharyngeal endoderm. Furthermore, it is now apparent that trunk neural crest cells have skeletal potential. However, it has now been demonstrated that a key role for the cranial neural crest streams is to organise the innervation of the hindbrain by the cranial sensory ganglia. Thus, in the past few years, our views of the significance of the cranial neural crest for head development have been altered. Developmental Dynamics 229:5-13, 2004.

Early steps in the production of sensory neurons by the neurogenic placodes.

Neurogenic placodes are specialized regions of the embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we have accurately determined the onset of neurogenesis in each of the placodes in the chick, and we have also analyzed the expression profiles of genes that are believed to be involved in determining the types of sensory neurons produced by each placode. Interestingly, we find that there is a major difference in the expression domains of neurogenin-1 and neurogenin-2 in the chick, when compared with those reported for the mouse. We do find, however, that Brn-3a and Phox-2a and Phox-2b which are also associated with the specification of neuronal type are expressed in the same domains in the chick as they are in the mouse. These results suggest that neurogenin-1 and neurogenin-2 are functionally interchangeable in neurogenic placodes. We have also found major differences between the ophthalmic and maxillomandibular trigeminal placodes, and while all of the other placodes generate mitotically active cells the ophthalmic trigeminal placode seems to throw off postmitotic neuronal cells.