Development of the mammalian main olfactory bulb.

The mammalian main olfactory bulb is a crucial processing centre for the sense of smell. The olfactory bulb forms early during development and is functional from birth. However, the olfactory system continues to mature and change throughout life as a target of constitutive adult neurogenesis. Our Review synthesises current knowledge of prenatal, postnatal and adult olfactory bulb development, focusing on the maturation, morphology, functions and interactions of its diverse constituent glutamatergic and GABAergic cell types. We highlight not only the great advances in the understanding of olfactory bulb development made in recent years, but also the gaps in our present knowledge that most urgently require addressing.

Key separable events in the remodelling of the pharyngeal arches.

The pharyngeal arches are a series of bulges on the lateral surface of the embryonic head. They are a defining feature of the most conserved, the phylotypic, stage of vertebrate development. In many vertebrate clades, the segmental arrangement of the pharyngeal arches is translated into the iterative anatomy of the gill arches. However, in amniotes the pharyngeal arches undergo a rearrangement during development and the segmental organisation of the pharynx is lost. This remodelling involves the expansion of the second arch which comes to overlie the more posterior arches. A transient sinus forms between the expanded second arch and the posterior arches, that is then lost, and the posterior arches are internalised. The morphogenesis of the second arch has been viewed as being central to this remodelling. Yet little is known about this process. Therefore, in this study, we have characterised the development of the second arch. We show that as the second arch expands, its posterior margin forms a leading edge and that the mesenchymal cells subjacent to this are in an elevated proliferative state. We further show that the posterior marginal epithelium is the site of expression of three key developmental signalling molecules: BMP7, FGF8 and SHH, and that their expression continues throughout the period of expansion. Using a novel approach, we have been able to simultaneously inhibit these three pathways, and we find that when this is done the second arch fails to establish its caudal projection and that there is a loss of proliferation in the posterior mesenchymal cells of the second arch. We have further used this manipulation to ask if the internalisation of the posterior arches is dependent upon the expansion of the second arch. We find that it is not-the posterior arches are still internalised when the expansion of the second arch is curtailed. We further show that while the collapse of the sinus is dependent upon thyroid hormone signalling, that this is not the case for the internalisation of the posterior pouches. Thus, the internalisation of the posterior arches is not dependent on the expansion of the second arch or on the collapse of the sinus. Finally, we show that the termination of expansion of the second arch correlates with a burst of morphogenetic cell death suggesting a mechanism for ending this. Thus, while it has long been thought that it is the morphogenesis of the second arch that drives the remodelling of the pharyngeal arches, we show that this is not the case. Rather the remodelling of the pharyngeal arches is a composite process that can split into contemporaneous but separate events: the expansion of the second arch, the internalisation of the posterior arches and the collapse of the sinus.

Extracellular matrix protein anosmin-1 modulates olfactory ensheathing cell maturation in chick olfactory bulb development.

Olfactory ensheathing cells (OECs) are a specialized class of glia, wrapping around olfactory sensory axons that target the olfactory bulb (OB) and cross the peripheral nervous system/central nervous system boundary during development and continue to do so post-natally. OEC subpopulations perform distinct subtype-specific functions dependent on their maturity status. Disrupted OEC development is thought to be associated with abnormal OB morphogenesis, leading to anosmia, a defining characteristic of Kallmann syndrome. Hence, anosmin-1 encoded by Kallmann syndrome gene (KAL-1) might modulate OEC differentiation/maturation in the OB. We performed in ovo electroporation of shRNA in the olfactory placode to knock-down kal in chick embryos, resulting in abnormal OB morphogenesis and loss of olfactory sensory axonal innervation into OB. BLBP-expressing OECs appeared to form a thinner and poorly organized outmost OB layer where SOX10 expressing OECs were completely absent with emergence of GFAP-expressing OECs. Furthermore, in embryonic day 10 chick OB explant cultures, GFAP expression in OECs accumulating along the OB nerve layers was dramatically reduced by recombinant anosmin-1. We then purified immature OECs from embryonic day 10 chick OB. These cells express GFAP after 7 days in vitro, exhibiting a multipolar morphology. Overexpression of chick anosmin, exogenous anosmin-1 or FGF2 could inhibit GFAP expression with cells presenting elongated morphology, which was blocked by the FGF receptor inhibitor Su5402. These data demonstrate that anosmin-1 functions via FGF signalling in regulating OEC maturation, thereby providing a permissive glial environment for axonal innervation into the OB during development.

A reappraisal and revision of the numbering of the pharyngeal arches.

The pharyngeal arches are a prominent and significant feature of vertebrate embryos. These are visible as a series of bulges on the lateral surface of the embryonic head. In humans, and other amniotes, there are five pharyngeal arches numbered 1, 2, 3, 4 and 6; note the missing '5'. This is the standard scheme for the numbering of these structures, and it is a feature of modern anatomy textbooks. In this article, we discuss the rationale behind this odd numbering, and consider its origins. One reason given is that there is a transient 5th arch that is never fully realized, while another is that this numbering reflects considerations from comparative anatomy. We show here, however, that neither of these reasons has substance. There is no evidence from embryology for a '5th' arch, and the comparative argument does not hold as it does not apply across the vertebrates. We conclude that there is no justification for this strange numbering. We suggest that the pharyngeal arches should simply be numbered 1, 2, 3, 4 and 5 as this would be in keeping with the embryology and with the general numbering of the pharyngeal arches across the vertebrates.

EphrinA6 on chick retinal axons is a key component for p75(NTR)-dependent axon repulsion and TrkB-dependent axon branching.

A characteristic of the ephrin/Eph family is their capacity for bi-directional signalling. This means that an ephrin, for example, can function either as a ligand for an Eph 'receptor', or as a receptor for an Eph 'ligand'. A system in which this phenomenon is well studied is the retinotectal projection in which the guidance of retinal ganglion cell (RGC) axons to their target area in the tectum is controlled by both Ephs and ephrins expressed in gradients in both the retina and tectum. Here we have analysed the receptor function of ephrinAs on RGC axons in further detail by focussing on ephrinA6, which is the most strongly expressed ephrinA in the chick retina. EphrinAs are GPI-anchored proteins and therefore require the interaction with transmembrane proteins to exert this receptor function. Previous work has shown that ephrinAs interact on RGC axons in cis with the neurotrophin receptors p75(NTR) and TrkB. P75(NTR) then was shown to be necessary for the repulsion of ephrinA-expressing RGC axons from an EphA substrate and for the downregulation of axon branching. In turn, an interaction of ephrinAs with TrkB as well as an increase in axonal ephrinA expression augments the axon branch-promoting activity of TrkB. We now show that ephrinA6 is the necessary ephrinA component of the repulsive ephrinA/p75(NTR) receptor complex on chick RGC axons as axons lacking ephrinA6 no longer avoid an EphA matrix in stripe assay experiments. We also demonstrate that the branch-promoting activity of TrkB is dependent on ephrinA6 as a knockdown of ephrinA6 renders RGC axons insensitive to BDNF, the high affinity ligand for TrkB. In sum our data further strengthen the hypothesis that a fine-tuned interplay of ephrinAs with p75(NTR) and TrkB is important for the guidance and branching of RGC axons.

A TrkB/EphrinA interaction controls retinal axon branching and synaptogenesis.

Toward understanding topographically specific branching of retinal axons in their target area, we have studied the interaction between neurotrophin receptors and members of the Eph family. TrkB and its ligand BDNF are uniformly expressed in the retina and tectum, respectively, and exert a branch-promoting activity, whereas EphAs and ephrinAs are expressed in gradients in retina and tectum and can mediate a suppression of axonal branching. We have identified a novel cis interaction between ephrinA5 and TrkB on retinal ganglion cell axons. TrkB interacts with ephrinA5 via its second cysteine-rich domain (CC2), which is necessary and sufficient for binding to ephrinA5. Their functional interaction is twofold: ephrinA5 augments BDNF-promoted retinal axon branching in the absence of its activator EphA7-Fc, whereas EphA7-Fc application abolishes branching in a local and concentration-dependent manner. The importance of TrkB in this process is shown by the fact that overexpression of an isolated TrkB-CC2 domain interfering with the ephrinA/TrkB interaction abolishes this regulatory interplay, whereas knockdown of TrkB via RNA interference diminishes the ephrinA5-evoked increase in branching. The ephrinA/Trk interaction is neurotrophin induced and specifically augments the PI-3 kinase/Akt pathway generally known to be involved in the promotion of branching. In addition, ephrinAs/TrkB modulate axon branching and also synapse formation of hippocampal neurons. Our findings uncover molecular mechanisms of how spatially restricted axon branching can be achieved by linking globally expressed branch-promoting with differentially expressed branch-suppressing activities. In addition, our data suggest that growth factors and the EphA-ephrinA system interact in a way that affects axon branching and synapse development.

Diminution of pharyngeal segmentation and the evolution of the amniotes.

BACKGROUND: The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos, and it is within these segments that components of the later anatomy are laid down. In most vertebrates, the post-otic pharyngeal arches will form the branchial apparatus, while in amniotes these segments are believed to generate the larynx. It has been unclear how the development of these segments has been altered with the emergence of the amniotes. RESULTS: In this study, we examined the development of pharyngeal arches in amniotes and show that the post-otic pharyngeal arches in this clade are greatly diminished. We find that the post-otic segments do not undergo myogenesis or skeletogenesis, but are remodelled before these processes occur. We also find that nested DLX expression, which is a feature of all the pharyngeal arches in anamniotes, is associated with the anterior segments but less so with the posterior arches in amniotes. We further show that the posterior arches of the mouse embryo fail to properly delineate, which demonstrates the lack of function of these posterior segments in later development. CONCLUSION: In amniotes, there has been a loss of the ancestral "branchial" developmental programme that is a general feature of gnathostomes; myogenesis and skeletogenesis This is likely to have facilitated the emergence of the larynx as a new structure not constrained by the segmental organisation of the posterior pharyngeal region.

Serotonin Receptor 1A (HTR1A), a Novel Regulator of GnRH Neuronal Migration in Chick Embryo.

The hypothalamic GnRH neurons are a small group of cells that regulate the reproductive axis. These neurons are specified within the olfactory placode, delaminate from this structure, and then migrate to enter the forebrain before populating the hypothalamus. We have used microarray technology to analyze the transcriptome of the olfactory placode at a number of key time points for GnRH ontogeny using the chick embryo. This resulted in the identification of a large number of genes whose expression levels change significantly over this period. This repertoire includes those genes that are known to be important for GnRH neuronal development as well as many novel genes, such as the serotonin receptor 1A, HTR1A. We find that HTR1A is expressed in the region of the olfactory placode that generates GnRH neurons. We further show that when this receptor is inactivated using a selective HTR1A antagonist as well as a gene knockdown approach using RNAi, this resulted in delayed migration causing the GnRH neurons to stall just outside the forebrain. These findings implicate HTR1A as being important for GnRH neuronal migration from the olfactory placode to the forebrain. Our study thus extends the repertoire of genes involved in GnRH neuron biology and thus identifies new candidate genes that can be screened for in patients who do not show mutations in any of the previously identified hypogonadotrophic hypogonadism/Kallmann syndrome genes.

Novel CHST6 nonsense and missense mutations responsible for macular corneal dystrophy.

PURPOSE: To identify the underlying mutations in two unrelated British families with macular corneal dystrophy (MCD) by screening the carbohydrate sulfotransferase (CHST6) gene. DESIGN: Case reports and results of DNA analysis. METHODS: Two subjects from two British families with MCD were studied. The genetic status of CHST6 was determined for all members of these MCD families. In addition, sulfated keratan sulfate (KS) assay from the probands was also undertaken. CHST6 gene was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by sequencing and restriction digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess KS presence in serum. RESULTS: Four compound heterozygous mutations were identified, three of which are novel. The ELISA showed that the probands were of MCD type I. CONCLUSIONS: These novel mutations are expected to result in loss of CHST6 function, which would account for the MCD phenotype.

Distinct patterns of heparan sulphate in pancreatic islets suggest novel roles in paracrine islet regulation.

Heparan sulphate proteoglycans (HSPGs) exist in pancreatic beta cells, and HS seems to modulate important interactions in the islet microenvironment. However, the intra-islet structures of HS in health or altered glucose homeostasis are currently unknown. Here we show that distinct spatial distribution of HS motifs is present in islets in the adult, that intra-islet HS motifs are mostly conserved between rodents and humans, and that HS is abundant in glucagon producing islet alpha cells. In beta cells HS is characterised by 2-O, 6-O and N-sulphated moieties, whereas HS in alpha cells is N-acetylated, N-, and 2-O sulphated and low in 6-O groups. Differential expression of three HS modifying genes in alpha and beta cells was observed and may account for the different HS patterns. Furthermore, we found that FGF1 and FGF2 were present in alpha cells, whereas functional FGFRs exist in beta cells, but not in the alpha cell line aTC1-6, or in primary alpha cells in islets. FGF1 induced signalling was dependent on 2-O, and 6-O HS sulphation in beta cells, and HS desulphation reduced beta cell proliferation and potentiated oxidant induced apoptosis. In leptin resistant animals and in islets from streptozotocin treated rats there was a reduction in alpha cell HS expression. These data demonstrate the distinct HS expression patterns in alpha and beta islet cells and propose a novel role for alpha cells as a source of paracrine FGF ligands to neighbouring beta cells with specific cell-associated HS domains mediating the activation and diffusion of paracrine ligands.

Mutations in the CACNA1F and NYX genes in British CSNBX families.

X-linked congenital stationary night blindness (CSNBX) is a genetically and phenotypically heterogeneous non-progressive disorder, characterised by impaired night vision but grossly normal retinal appearance. Other more variable features include reduction in visual acuity, myopia, nystagmus and strabismus. Genetic mapping studies by other groups, and our own studies of British patients, identified key recombination events indicating the presence of at least 2 disease genes on Xp11. Two causative genes (CACNA1F and NYX) for CSNBX have now been identified through positional cloning strategies. In this report, we present the results of comprehensive mutation screening in 14 CSNBX families, three with mutations in the CACNA1F gene and 10 with mutations in the NYX gene. In one family we failed to identify the mutation after testing RP2, RPGR, NYX and CACNA1F. NYX gene mutations are a more frequent cause of CSNBX, although there is evidence for founder mutations. Our report of patient population mutation screening for both CSNBX genes, and our exclusion of RP2 and RGPR, indicates that mutations in CACNA1F and NYX are likely to account for all CSNBX.

Refinement of the X-linked cataract locus (CXN) and gene analysis for CXN and Nance-Horan syndrome (NHS).

The X-linked congenital cataract (CXN) locus has been mapped to a 3-cM (approximately 3.5 Mb) interval on chromosome Xp22.13, which is syntenic to the mouse cataract disease locus Xcat and encompasses the recently refined Nance-Horan syndrome (NHS) locus. A positional cloning strategy has been adopted to identify the causative gene. In an attempt to refine the CXN locus, seven microsatellites were analysed within 21 individuals of a CXN family. Haplotypes were reconstructed confirming disease segregation with markers on Xp22.13. In addition, a proximal cross-over was observed between markers S3 and S4, thereby refining the CXN disease interval by approximately 400 Kb to 3.2 Mb, flanked by markers DXS9902 and S4. Two known genes (RAI2 and RBBP7) and a novel gene (TL1) were screened for mutations within an affected male from the CXN family and an NHS family by direct sequencing of coding exons and intron- exon splice sites. No mutations or polymorphisms were identified, therefore excluding them as disease-causative in CXN and NHS. In conclusion, the CXN locus has been successfully refined and excludes PPEF1 as a candidate gene. A further three candidates were excluded based on sequence analysis. Future positional cloning efforts will focus on the region of overlap between CXN, Xcat, and NHS.

GnRH neuronal migration and olfactory bulb neurite outgrowth are dependent on FGF receptor 1 signaling, specifically via the PI3K p110α isoform in chick embryo.

Fibroblast growth factor (FGF) signaling is essential for both olfactory bulb (OB) morphogenesis and the specification, migration, and maturation of the GnRH-secreting neurons. Disruption of FGF signaling contributes to Kallmann syndrome characterized by both anosmia and sexual immaturity. However, several unanswered questions remain as to which specific FGF receptor (FGFR)-1 signaling pathways are necessary for OB and GnRH neuronal development. Here, using pharmacological phosphatidylinositol 3-kinase (PI3K) isoform-specific inhibitors, we demonstrate a central role for the PI3K p110α isoform as a downstream effector of FGFR1 signaling for both GnRH neuronal migration and OB development. We show that signaling via the PI3K p110α isoform is required for GnRH neuronal migration in explant cultures of embryonic day (E) 4 chick olfactory placodes. We also show that in ovo administration of LY294002, a global PI3K inhibitor as well as an inhibitor to the PI3K p110α isoform into the olfactory placode of E3 chick embryo impairs GnRH neuronal migration toward the forebrain. In contrast, in ovo PI3K inhibitor treatment produced no obvious defects on primary olfactory sensory neuron axonal targeting and bundle formation. We also demonstrate that anosmin-1 and FGF2 induced neuronal migration of immortalized human embryonic GnRH neuroblast cells (FNC-B4-hTERT) is mediated by modulating FGFR1 signaling via the PI3K p110α isoform, specifically through phosphorylation of the PI3K downstream effectors, Akt and glycogen synthase kinase-3ß. Finally, we show that neurite outgrowth and elongation of OB neurons in E10 chick OB explants are also dependent on the PI3K p110α isoform downstream of FGFR1. This study provides mechanistic insight into the etiology of Kallmann syndrome.

Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance.

BACKGROUND: Retinotectal map formation develops via topographically specific guidance and branching of retinal axons in their target area. This process is controlled, in part, by reverse signalling of ephrinAs expressed on retinal axons. As glycosylphosphatidylinositol-anchored molecules, ephrinAs require transmembrane co-receptors to exert this function, for which the two neurotrophin receptors, p75NTR and TrkB, were recently proposed. RESULTS: We show here that the ligands for these receptors, the brain-derived neurotrophic factor precursor (proBDNF) and its processed form, BDNF, respectively, control the branching of retinal axons antagonistically, which they mediate by inducing the corresponding neurotrophin receptor-ephrinA complexes. Moreover, scavenging proneurotrophins, by adding antibodies specific for the pro-domain of proBNDF or a soluble extracellular domain of p75NTR, abolish repellent ephrinA reverse signalling in the stripe assay. CONCLUSIONS: This indicates that retinal cells secrete proneurotrophins, inducing the ephrinA-p75NTR interaction and enabling repellent axon guidance. The antagonistic functions of proBDNF and BDNF raise the possibility that topographic branching is controlled by local control of processing of proneurotrophins.

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment.

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.

Spectral tuning and evolution of short wave-sensitive cone pigments in cottoid fish from Lake Baikal.

The cottoid fishes of Lake Baikal in eastern Siberia provide a unique opportunity to study the evolution of visual pigments in a group of closely related species exposed to different photic environments. Members of this species flock are adapted to different depth habitats down to >1000 m, and both the rod and cone visual pigments display short wave shifts as depth increases. The blue-sensitive cone pigments of the SWS2 class cluster into two species groups with lambda(max) values of 450 and 430 nm, with the pigment in Cottus gobio, a cottoid fish native to Britain, forming a third group with a lambda(max) of 467 nm. The sequences of the SWS2 opsin gene from C. gobio and from two representatives of the 450 and 430 nm Baikal groups are presented. Approximately 6 nm of the spectral difference between C. gobio and the 450 nm Baikal group can be ascribed to the presence of a porphyropsin/rhodopin mixture in C. gobio. Subsequent analysis of amino acid substitutions by site-directed mutagenesis demonstrates that the remainder of the shift from 461 to 450 nm arises from a Thr269Ala substitution and the shift from 450 to 430 nm at least partly from Thr118Ala and Thr118Gly substitutions. The underlying adaptive significance of these substitutions in terms of spectral tuning and signal-to-noise ratio is discussed.

Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows the shortest lambda(max) values with peaks in different species in either the violet (390-435 nm) or ultraviolet (around 365 nm) regions of the spectrum. Phylogenetic evidence indicates that the ancestral pigment was probably UV-sensitive (UVS) and that the shifts between violet and UV have occurred many times during evolution. This is supported by the different mechanisms for these shifts in different species. All visual pigments possess a chromophore linked via a Schiff base to a Lys residue in opsin protein. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UVS pigments, it is almost certainly unprotonated. The generation of VS from ancestral UVS pigments most likely involved amino acid substitutions in the opsin protein that serve to stabilise protonation. The key residues in the opsin protein for this are at sites 86 and 90 that are adjacent to the Schiff base and the counterion at Glu113. In this review, the different molecular mechanisms for the UV or violet shifts are presented and discussed in the context of the structural model of bovine rhodopsin.

The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments.

The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with lambda(max) (the peak of maximal absorbance) values generally between 400 and 430 nm, and ultraviolet-sensitive (UVS), with a lambda(max)<380 nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86-->Tyr substitution is sufficient by itself to shift the lambda(max) of the goldfish pigment from a wild-type value of 360 nm to around 420 nm, and the reverse substitution of Tyr-86-Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.