Roger Bradley

Cadherin Function Is Required for Axon Outgrowth in Retinal Ganglion Cells In Vivo

The cell-cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia.

Previous studies have suggested that the segmental pattern of neural-crest-derived sympathetic ganglia arises as a direct result of signals that restrict neural crest cell migratory streams through rostral somite halves. We recently showed that the spatiotemporal pattern of chick sympathetic ganglia formation is a two-phase process. Neural crest cells migrate laterally to the dorsal aorta, then surprisingly spread out in the longitudinal direction, before sorting into discrete ganglia. Here, we investigate the function of two families of molecules that are thought to regulate cell sorting and aggregation. By blocking Eph/ephrins or N-cadherin function, we measure changes in neural crest cell migratory behaviors that lead to alterations in sympathetic ganglia formation using a recently developed sagittal slice explant culture and 3D confocal time-lapse imaging. Our results demonstrate that local inhibitory interactions within inter-ganglionic regions, mediated by Eph/ephrins, and adhesive cell-cell contacts at ganglia sites, mediated by N-cadherin, coordinate to sculpt discrete sympathetic ganglia.

NF-protocadherin, a novel member of the cadherin superfamily, is required for Xenopus ectodermal differentiation

BACKGROUND The assembly of complex tissues during embryonic development is thought to depend on differential cell adhesion, mediated in part by the cadherin family of cell-adhesion molecules. The protocadherins are a new subfamily of cadherins; their extracellular domains comprise cadherin-like repeats but their intracellular domains differ significantly from those of classical cadherins. Little is known about the ability of protocadherins to mediate the adhesion of embryonic cells, or whether they play a role in the formation of embryonic tissues. RESULTS We report the isolation and characterization of a novel protocadherin, termed NF-protocadherin (NFPC), that is expressed in Xenopus embryos. NFPC showed a striking pattern of expression in early embryos, displaying predominant expression within the deep, sensorial layer of the embryonic ectoderm and in a restricted group of cells in the neural folds, but was largely absent from the neural plate and surrounding placodal regions. Ectopic expression in embryos demonstrated that NFPC could mediate cell adhesion within the embryonic ectoderm. In addition, expression of a dominant-negative form of NFPC disrupted the integrity of embryonic ectoderm, causing cells in the deep layer to dissociate, though leaving the outer layer relatively intact. CONCLUSIONS Our results indicate that NFPC is required as a cell-adhesion molecule during embryonic development, and its function is distinct from that of classical cadherins in governing the formation of a two-layer ectoderm. These results suggest that NFPC, and protocadherins in general, are involved in novel cell-cell adhesion mechanisms that play important roles in tissue histogenesis.

NF-Protocadherin and TAF1 Regulate Retinal Axon Initiation and Elongation In Vivo

NF-protocadherin (NFPC)-mediated cell–cell adhesion plays a critical role in vertebrate neural tube formation. NFPC is also expressed during the period of axon tract formation, but little is known about its function in axonogenesis. Here we have tested the role of NFPC and its cytosolic cofactor template-activating factor 1 (TAF1) in the emergence of the Xenopus retinotectal projection. NFPC is expressed in the developing retina and optic pathway and is abundant in growing retinal axons. Inhibition of NFPC function in developing retinal ganglion cells (RGCs) severely reduces axon initiation and elongation and suppresses dendrite genesis. Furthermore, an identical phenotype occurs when TAF1 function is blocked. These data provide evidence that NFPC regulates axon initiation and elongation and indicate a conserved role for TAF1, a transcriptional regulator, as a downstream cytosolic effector of NFPC in RGCs.

The Cytoplasmic Domain of Xenopus NF-Protocadherin Interacts with TAF1/Set

Protocadherins are members of the cadherin superfamily of cell adhesion molecules proposed to play important roles in early development, but whose mechanisms of action are largely unknown. We examined the function of NF-protocadherin (NFPC), a novel cell adhesion molecule essential for the histogenesis of the embryonic ectoderm in Xenopus, and demonstrate that the cellular protein TAF1, previously identified as a histone-associated protein, binds the NFPC cytoplasmic domain. NFPC and TAF1 coprecipitate from embryo extracts when ectopically expressed, and TAF1 can rescue the ectodermal disruptions caused by a dominant-negative NFPC construct lacking the extracellular domain. Furthermore, disruptions in either NFPC or TAF1 expression, using NFPC- or TAF1-specific antisense morpholinos, result in essentially identical ectodermal defects. These results indicate a role for TAF1 in the differentiation of the embryonic ectoderm, as a cytosolic cofactor of NFPC.

Chicken protocadherin-1 functions to localize neural crest cells to the dorsal root ganglia during PNS formation

In vertebrate embryos, neural crest cells emerge from the dorsal neural tube and migrate along well defined pathways to form a wide diversity of tissues, including the majority of the peripheral nervous system (PNS). Members of the cadherin family of cell adhesion molecules play key roles during the initiation of migration, mediating the delamination of cells from the neural tube. However, a role for cadherins in the sorting and re-aggregation of the neural crest to form the PNS has not been established. We report the requirement for a protocadherin, chicken protocadherin-1 (Pcdh1), in neural crest cell sorting during the formation of the dorsal root ganglia (DRG). In embryos, cPcdh1 is highly expressed in the developing DRG, where it co-localizes with the undifferentiated and mitotically active cells along the perimeter. Pcdh1 can promote cell adhesion in vivo and disrupting Pcdh1 function in embryos results in fewer neural crest cells localizing to the DRG, with a concomitant increase in cells that migrate to the sympathetic ganglia. Furthermore, those cells that still localize to the DRG, when Pcdh1 is inhibited, are no longer found at the perimeter, but are instead dispersed throughout the DRG and are now more likely to differentiate along the sensory neuron pathway. These results demonstrate that Pcdh1-mediated cell adhesion plays an important role as neural crest cells coalesce to form the DRG, where it serves to sort cells to the mitotically active perimeter.

Grasshopper haemagglutinin: immunochemical localization in haemocytes and investigation of opsonic properties

The haemagglutinin (lectin) present in the haemolymph of the grasshoppers Melanoplus differentialis and M. sanguinipes has been localized to the haemocytes, and the in vitro opsonic capacity of purified grasshopper haemagglutinin has been investigated. Immunocytochemical labelling of fixed haemocyte monolayers with monoclonal antibody revealed that agglutinin is present in approx. 25% of the granular cells. Plasmatocytes (phagocytes) do not contain agglutinin. Potential haemocytic membrane receptors for haemagglutinin were indicated by the binding of fluorescein-conjugated peanut agglutinin to granular haemocytes. However, comparable binding of purified grasshopper haemagglutinin could not be demonstrated by immunochemical labelling. Haemocytes, in vitro, interact with asialo human erythrocytes, Bacillus thuringiensis bacteria (vegetative cells) and spores of Nosema locustae. No increase in binding or phagocytic (opsonic) activity was observed when these three foreign particle types were incubated in either grasshopper serum or purified haemagglutinin prior to overlayering the haemocyte monolayers, or when monolayers were exposed to purified agglutinin prior to overlayering with foreign partices. The results indicate that although grasshopper haemolymphatic haemagglutinin is present in a subpopulation of grasshopper haemocytes, it does not contribute to the in vitro association of haemocytes with these foreign particles.

Site of synthesis of the haemolymph agglutinin of Melanoplus differentialis (Acrididae: Orthoptera)

Abstract Several species of Melanoplus grasshoppers contain a haemolymph agglutinin exhibiting both d -galactosidic and d -glucosidic binding specificities. With the exception of the flesh fly Sarcophaga peregrina , the site(s) of synthesis of insect agglutinins is uncertain. Primary cultures of M. differentialis fat body, haemocytes, ovary and testes were established. An ELISA assay demonstrated that the fat body, ovary and testes tissues released significant amounts of agglutinin into the culture medium over a 4-day period. Immunoprecipitated agglutinin from the culture medium was radiolabelled when the cultures were metabolically labelled with [ 35 S] methionine. Haemocyte culture medium and cell lysates of the 4 tissues did not contain detectable amounts of radiolabelled agglutinin. Attempts to alter the kinetics of agglutinin release from fat body cultures through addition of microbial cell wall components (lipopolysaccharide, peptidoglycan, zymosan, or laminarin) were unsuccessful.

From dinosaurs to birds: a tail of evolution.

A particularly critical event in avian evolution was the transition from long- to short-tailed birds. Primitive bird tails underwent significant alteration, most notably reduction of the number of caudal vertebrae and fusion of the distal caudal vertebrae into an ossified pygostyle. These changes, among others, occurred over a very short evolutionary interval, which brings into focus the underlying mechanisms behind those changes. Despite the wealth of studies delving into avian evolution, virtually nothing is understood about the genetic and developmental events responsible for the emergence of short, fused tails. In this review, we summarize the current understanding of the signaling pathways and morphological events that contribute to tail extension and termination and examine how mutations affecting the genes that control these pathways might influence the evolution of the avian tail. To generate a list of candidate genes that may have been modulated in the transition to short-tailed birds, we analyzed a comprehensive set of mouse mutants. Interestingly, a prevalent pleiotropic effect of mutations that cause fused caudal vertebral bodies (as in the pygostyles of birds) is tail truncation. We identified 23 mutations in this class, and these were primarily restricted to genes involved in axial extension. At least half of the mutations that cause short, fused tails lie in the Notch/Wnt pathway of somite boundary formation or differentiation, leading to changes in somite number or size. Several of the mutations also cause additional bone fusions in the trunk skeleton, reminiscent of those observed in primitive and modern birds. All of our findings were correlated to the fossil record. An open question is whether the relatively sudden appearance of short-tailed birds in the fossil record could be accounted for, at least in part, by the pleiotropic effects generated by a relatively small number of mutational events.

The Order and Place of Neuronal Differentiation Establish the Topography of Sensory Projections and the Entry Points within the Hindbrain

Establishing topographical maps of the external world is an important but still poorly understood feature of the vertebrate sensory system. To study the selective innervation of hindbrain regions by sensory afferents in the zebrafish embryo, we mapped the fine-grained topographical representation of sensory projections at the central level by specific photoconversion of sensory neurons. Sensory ganglia located anteriorly project more medially than do ganglia located posteriorly, and this relates to the order of sensory ganglion differentiation. By single-plane illumination microscopy (SPIM) in vivo imaging, we show that (1) the sequence of arrival of cranial ganglion inputs predicts the topography of central projections, and (2) delaminated neuroblasts differentiate in close contact with the neural tube, and they never loose contact with the neural ectoderm. Afferent entrance points are established by plasma membrane interactions between primary differentiated peripheral sensory neurons and neural tube border cells with the cooperation of neural crest cells. These first contacts remain during ensuing morphological growth to establish pioneer axons. Neural crest cells and repulsive slit1/robo2 signals then guide axons from later-differentiating neurons toward the neural tube. Thus, this study proposes a new model by which the topographical representation of cranial sensory ganglia is established by entrance order, with the entry points determined by cell contact between the sensory ganglion cell bodies and the hindbrain.