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Angiogenesis is defined as a new blood vessel sprouting from pre-existing vessels. This highly regulated process take place through two non-exclusive events, the so-called endothelial sprouting or non-sprouting (intussusceptive) microvascular growth. This review article will provide a brief overview of some relevant topics defining sprouting angiogenesis and including: (i) The concept of functional specialization of endothelial cells during different phases of this process, involving the specification of endothelial cells into tip cells, stalk cells, and phalanx cells bearing different morphologies and functional properties; (ii) The interplay between numerous signaling pathways, including Notch and Notch ligands, VEGF and VEGFRs, semaphorins, and netrins, in the regulation and modulation of the phenotypic characteristics of these cells; (iii) Some fundamental and consecutive morphological processes, including lumen formation and perfusion, network formation, remodeling, pruning, leading to the final vessel maturation and stabilization.

Plexins (Plexs) comprise a large family of cell surface receptors for semaphorins (Semas) that function as evolutionarily conserved guidance molecules. GTPase activating protein (GAP) activity for Ras family small GTPases has been implicated in plexin signaling cascades through its RasGAP domain. However, little is known about how Ras family GTPases are controlled in vivo by plexin signaling. Here, we found that Drosophila Rap1, a member of the Ras family of GTPases, plays an important role controlling intersegmental nerve b motor axon guidance during neural development. Gain-of-function studies using dominant-negative and constitutively active forms of Rap1 indicate that Rap1 contributes to axonal growth and guidance. Genetic interaction analyses demonstrate that the Sema-1a/PlexA-mediated repulsive guidance function is regulated positively by Rap1. Furthermore, neuronal expression of mutant PlexA robustly restored defasciculation defects in PlexA null mutants when the catalytic arginine fingers of the PlexA RasGAP domain critical for GAP activity were disrupted. However, deleting the RasGAP domain abolished the ability of PlexA to rescue the PlexA guidance phenotypes. These findings suggest that PlexA-mediated motor axon guidance is dependent on the presence of the PlexA RasGAP domain, but not on its GAP activity toward Ras family small GTPases.

The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A., Highlights ► NRP1 and NRP2 are co-expressed in sympathetic neurons and their precursors. ► SEMA3A signals through NRP1 to regulate sympathetic gangliogenesis and axon guidance. ► SEMA3F signals through NRP2 to regulate sympathetic axon guidance. ► SEMA3A/NRP1 and SEMA3F/NRP2 act synergistically in sympathetic nervous system patterning. ► Abnormal sympathetic innervation of the heart leads to bradycardia in NRP1 mutants.

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, consists of numerous types of neurons, and glial cells, that are distributed in two intramuscular plexuses that extend along the entire length of the gut and control co-ordinated smooth muscle contractile activity and other gut functions. All enteric neurons and glia are derived from neural crest cells (NCC). Vagal (hindbrain) level NCC provide the majority of enteric precursors along the entire length of the gut, while a lesser contribution, that is restricted to the hindgut, arises from the sacral region of the neuraxis. After leaving the dorsal neural tube NCC undergo extensive migration, proliferation, survival and differentiation in order to form a functional ENS. This article reviews the molecular mechanisms underlying these key developmental processes and highlights the major groups of molecules that affect enteric NCC proliferation and survival (Ret/Gdnf and EdnrB/Et-3 pathways, Sox10 and Phox2b transcription factors), cell migration (Ret and EdnrB signalling, semaphorin 3A, cell adhesion molecules, Rho GTPases), and the development of enteric neuronal subtypes and morphologies (Mash1, Gdnf/neurturin, BMPs, Hand2, retinoic acid). Finally, looking to the future, we discuss the need to translate the wealth of data gleaned from animal studies to the clinical area and thus better understand, and develop treatments for, congenital human diseases affecting the ENS.

Gap junctions are proteinaceous channels that reside at the plasma membrane and permit the exchange of ions, metabolites, and second messengers between neighboring cells. Connexin proteins are the subunits of gap junction channels. Mutations in zebrafish cx43 cause the short fin (sofb123) phenotype which is characterized by short fins due to defects in length of the bony fin rays. Previous findings from our lab demonstrate that Cx43 is required for both cell proliferation and joint formation during fin regeneration. Here we demonstrate that semaphorin3d (sema3d) functions downstream of Cx43. Semas are secreted signaling molecules that have been implicated in diverse cellular functions such as axon guidance, cell migration, cell proliferation, and gene expression. We suggest that Sema3d mediates the Cx43-dependent functions on cell proliferation and joint formation. Using both in situ hybridization and quantitative RT-PCR, we validated that sema3d expression depends on Cx43 activity. Next, we found that knockdown of Sema3d recapitulates all of the sofb123 and cx43-knockdown phenotypes, providing functional evidence that Sema3d acts downstream of Cx43. To identify the potential Sema3d receptor(s), we evaluated gene expression of neuropilins and plexins. Of these, nrp2a, plxna1, and plxna3 are expressed in the regenerating fin. Morpholino-mediated knockdown of plxna1 did not cause cx43-specific defects, suggesting that PlexinA1 does not function in this pathway. In contrast, morpholino-mediated knockdown of nrp2a caused fin overgrowth and increased cell proliferation, but did not influence joint formation. Moreover, morpholino-mediated knockdown of plxna3 caused short segments, influencing joint formation, but did not alter cell proliferation. Together, our findings reveal that Sema3d functions in a common molecular pathway with Cx43. Furthermore, functional evaluation of putative Sema3d receptors suggests that Cx43-dependent cell proliferation and joint formation utilize independent membrane-bound receptors to mediate downstream cellular phenotypes.

The angioblast is an embryonic endothelial cell precursor that migrates long distances to reach its final position, navigating by sensing attractive and repulsive cues from the environment. Members of the semaphorin family have been implicated in controlling the behaviour of angioblast tip cells through repulsive signalling in vitro, but their in vivo roles are less clear. Here we show that zebrafish semaphorin3e (sema3e) is expressed by endothelial cells of the dorsal aorta, primary motoneurons, and endodermal cells. Further, loss of Sema3e leads to delayed exit of angioblasts from the dorsal aorta in ISV formation. Through transplant analysis, we show that Sema3e acts autonomously and non-autonomously in angioblasts to modulate interactions among themselves. The semaphorin receptors, PlexinD1 and PlexinB2, are expressed by zebrafish angioblasts. Loss of plxnB2 results in delayed ISV sprouting identical to that seen in sema3e morphants, while loss of plexinD1 in out of bounds (obd) mutants results in precocious ISV sprouting. Loss of either sema3e or plxnB2 in obd mutants generates an intermediate phenotype, suggesting that PlxnD1 and Sema3e/PlxnB2 antagonize each other to control timing of ISV sprouting. Consistent with this observation, we show that PlxnB2 acts cell autonomously in endothelial cells. This suggests a model where multiple semaphorin–plexin interactions control angioblast sprouting behaviour.

Semaphorins are a large class of proteins that function throughout the nervous system to guide axons. It had previously been shown that Semaphorin 5A (Sema5A) was a bifunctional axon guidance cue for mammalian midbrain neurons. We found that zebrafish sema5A was expressed in myotomes during the period of motor axon outgrowth. To determine whether Sema5A functioned in motor axon guidance, we knocked down Sema5A, which resulted in two phenotypes: a delay in motor axon extension into the ventral myotome and aberrant branching of these motor axons. Both phenotypes were rescued by injection of full-length rat Sema5A mRNA. However, adding back RNA encoding the sema domain alone significantly rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. Together, these data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The TSR domain promotes growth of developing motor axons into the ventral myotome whereas the sema domain mediates repulsion and keeps these motor axons from branching into surrounding myotome regions.

In vertebrates, class 3 semaphorins (SEMA3) control axon behaviour by binding to neuronal cell surface receptors composed of a ligand binding subunit termed neuropilin (NRP) and a signal transduction subunit of the A-type plexin family (PLXNA). We have determined the requirement for SEMA3/NRP/PLXN signalling in the development of the facial nerve, which contains axons from two motor neuron populations, branchiomotor and visceromotor neurons. Loss of either SEMA3A/NRP1 or SEMA3F/NRP2 caused defasciculation and ectopic projection of facial branchiomotor axons. In contrast, facial visceromotor axons selectively required SEMA3A/NRP1. Thus, the greater superficial petrosal nerve was defasciculated, formed ectopic projections and failed to branch in its target area when either SEMA3A or NRP1 were lost. To examine which A-type plexin conveyed SEMA3/neuropilin signals during facial nerve development, we combined an expression analysis with loss of function studies. Even though all four A-type plexins were expressed in embryonic motor neurons, PLXNA1 and PLXNA2 were not essential for facial nerve development. In contrast, loss of PLXNA4 phenocopied the defects of SEMA3A and NRP1 mutants, and loss of PLXNA3 phenocopied the defects of SEMA3F and NRP2 mutants. The combined loss of PLXNA3 and PLXNA4 impaired facial branchiomotor axon guidance more severely than loss of either plexin alone, suggesting that both pathways normally cooperate; in contrast, loss of both plexins did not impair facial visceromotor defects any worse than loss of PLXNA4. We conclude that PLXNA3 and PLXNA4 synergise to pattern the facial nerve, whereby both are required in branchiomotor neurons, but only PLXNA4 is essential for visceromotor neurons.

The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in both foregut endoderm and cardiogenic mesoderm and is required for earliest stages of heart development. Here, we report that isl1 is also required upstream of Shh. We find that, in isl1 null mice, Sonic hedgehog (Shh) is downregulated in foregut endoderm. Shh signals through the unique activating receptor smoothened (Smo). To investigate the role of hedgehog signaling in the isl1 domain, we ablated smo utilizing isl1-cre. Isl1-cre;smo mutants exhibit cardiovascular defects similar to those observed in Shh null mice, defining a spatial requirement for hedgehog signaling within isl1 expression domains for aortic arch and outflow tract formation. Semaphorin signaling through neuropilin receptors npn1 and npn2 is required for aortic arch and outflow tract formation. We find that expression of npn2 is downregulated in isl1-cre;smo mutants, suggesting an isl1/Shh/npn pathway required to affect morphogenesis at the anterior pole of the heart.

Ephrins and semaphorins regulate a wide variety of developmental processes, including axon guidance and cell migration. We have studied the roles of the ephrin EFN-4 and the semaphorin MAB-20 in patterning cell–cell contacts among the cells that give rise to the ray sensory organs of Caenorhabditis elegans. In wild-type, contacts at adherens junctions form only between cells belonging to the same ray. In efn-4 and mab-20 mutants, ectopic contacts form between cells belonging to different rays. Ectopic contacts also occur in mutants in regulatory genes that specify ray morphological identity. We used efn-4 and mab-20 reporters to investigate whether these ray identity genes function through activating expression of efn-4 or mab-20 in ray cells. mab-20 reporter expression in ray cells was unaffected by mutants in the Pax6 homolog mab-18 and the Hox genes egl-5 and mab-5, suggesting that these genes do not regulate mab-20 expression. We find that mab-18 is necessary for activating efn-4 reporter expression, but this activity alone is not sufficient to account for mab-18 function in controlling cell–cell contact formation. In egl-5 mutants, efn-4 reporter expression in certain ray cells was increased, inconsistent with a simple repulsion model for efn-4 action. The evidence indicates that ray identity genes primarily regulate ray morphogenesis by pathways other than through regulation of expression of semaphorin and ephrin.

Initial trajectories of dorsal root ganglion (DRG) axons are shaped by chemorepulsive signals from surrounding tissues. Although we have previously shown that axonin-1/SC2 expression on DRG axons is required to mediate a notochord-derived chemorepulsive signal (T. Masuda et al., 2000 , Dev. Biol. 224, 112–121), other molecules involved in the non-target-derived repulsive signals are largely unknown. Using coculture assays composed of tissues derived from the chick embryo or mutant mice treated with function-blocking antibodies and phosphatidylinositol-specific phospholipase C, we report here that the chemorepellent semaphorin 3A (Sema3A) and its receptor neuropilin-1 are required for mediating the dermamyotome- and notochord-derived, but not the ventral spinal cord-derived, chemorepulsive signal for DRG axons. The dermamyotome-derived chemorepulsion is exclusively dependent on Sema3A/neuropilin-1, whereas other molecules are also involved in the notochord-derived chemorepulsion. Chemorepulsion from the ventral spinal cord does not depend on Sema3A/neuropilin-1 but requires axonin-1/SC2 to repel DRG axons. Thus, differential chemorepulsive signals help shape the initial trajectories of DRG axons and are critical for the proper wiring of the nervous system.

Semaphorin3A (previously known as Semaphorin III, Semaphorin D, or collapsin-1) is a member of the semaphorin gene family, many of which have been shown to guide axons during nervous system development. Semaphorin3A has been demonstrated to be a diffusible chemorepulsive molecule for axons of selected neuronal populations in vitro. Analysis of embryogenesis in two independent lines of Semaphorin3A knockout mice support the hypothesis that this molecule is an important guidance signal for neurons of the peripheral nervous system (M. Taniguchi et al., 1997, Neuron 19, 519–530; E. Ulupinar et al., 1999, Mol. Cell. Neurosci. 13, 281–292). Surprisingly, newborn Semaphorin3A null mutant mice exhibit no significant abnormalities (O. Behar et al., 1996, Nature 383, 525–528). In this study we have tested the hypothesis that guidance abnormalities that occurred during early stages of Semaphorin3A null mice development are corrected later in development. We have found that the extensive abnormalities formed during early developmental stages in the peripheral nervous system are largely eliminated by embryonic day 15.5. We demonstrate further that at least in one distinct anatomical location these abnormalities are mainly the result of aberrant projections. In conclusion, these findings suggest the existence of correction mechanisms that eliminate most sensory axon pathfinding errors early in development.

Sema3A (Sema III, SemD, collapsin-1) can induce neuronal growth cone collapse and axon repulsion of distinct neuronal populations. To study Sema3A function in patterning afferent projections into the developing spinal cord, we employed the recombinant adenoviral vector technique in embryonic rat spinal cord slices. Virus solution was injected in the dorsal aspect of organotypic spinal cord cultures with segmentally attached dorsal root ganglia (sc-DRG). In cultures grown in the presence of nerve growth factor (NGF), injected either with the control virus AdCMVLacZ or with vehicle only, afferent innervation patterns were similar to those of control. However, unilateral injection of AdCMVSema3A/AdCMVLacZ in sc-DRG slices revealed a strong inhibitory effect on NGF-dependent sensory afferent growth. Ectopic Sema3A in the dorsal spinal cord, the target area of NGF-responsive DRG fibers in vivo, created an exclusion zone for these fibers and as a result they failed to reach and innervate their appropriate target zones. Taken together, gain of Sema3A function in the dorsal aspect of sc-DRG cultures revealed a dominant inhibitory effect on NGF-dependent, nociceptive sensory DRG afferents, an observation in line with the model proposed by E. K. Messersmith et al. (1995, Neuron 14, 949–959), suggesting that Sema3A secreted by spinal cord cells can act to repel central sensory fibers during the formation of lamina-specific connections in the spinal cord.

article i nfo The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC "routes"). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC "target"). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.

Myc and Mondo proteins are key regulators of cell growth, proliferation, and energy metabolism, yet often overlooked is their vital role in cell migration. Complex networks of protein–protein and protein–DNA interactions control the transcriptional activity of Myc and MondoA confounding their functional analysis in higher eukaryotes. Here we report the identification of the transcriptional activation arm of a simplified Myc-like network in Caenorhabditis elegans. This network comprises an Mlx ortholog, named MXL-2 for Max-like 2, and a protein that has sequence features of both Myc and Mondo proteins, named MML-1 for Myc and Mondo-like 1. MML-1/MXL-2 complexes have a primary function in regulating migration of the ray 1 precursor cells in the male tail. MML-1/MXL-2 complexes control expression of ECM components in the non-migratory epidermis, which we propose contributes to the substratum required for migration of the neighboring ray 1 precursor cells. Furthermore, we show that pro-migratory Wnt/β-catenin and semaphorin signaling pathways interact genetically with MML-1/MXL-2 to determine ray 1 position. This first functional analysis of the Myc superfamily in C. elegans suggests that MondoA and Myc may have more predominant roles in cell migration than previously appreciated, and their cooperation with other pro-migratory pathways provides a more integrated view of their role in cell migration.

In order to form a functional nervous system, neurones extend axons, often over long distances, to reach their targets. This process is controlled by extracellular receptors and their ligands, several families of which have been identified. These proteins may act to either repel or attract growth cones and a given receptor may transduce either type of signal, depending on the cellular context. In addition to these archetypal axon guidance molecules, it is becoming apparent that molecules previously known for their role in patterning can also direct axonal outgrowth. The growth cone receptors do not act in isolation and combine with members of the same or other families to produce a graded response or even a complete reversal in its polarity. These signals can be further combined and/or modulated by processing of the molecule both directly at the cell surface and by the network of intracellular signalling pathways which are activated. The result is a sophisticated and dynamic set of cues that enable a growth cone to successfully navigate to its destination, modulating its response to changing environmental cues along its pathway.

PLX-1 is a PlexinA transmembrane protein in Caenorhabditis elegans, and the transmembrane-type semaphorin, SMP-1, is a ligand for PLX-1. The SMP-1/PLX-1 system has been shown to be necessary for proper epidermal morphogenesis in the male tail and seam cells. Here, we show that the SMP-1/PLX-1 system also regulates vulval morphogenesis. In plx-1 and smp-1 mutants, hermaphrodites sometimes exhibit a protruding vulva or multiple vulva-like protrusions. Throughout the vulval development of plx-1 and smp-1 mutants, the arrangement of vulval cells is often disrupted. In the initial step of vulval morphogenesis, vulval precursor cells (VPCs) are generated normally but are subsequently arranged abnormally in mutants. Continuous observation revealed that plx-1 VPC fails to terminate longitudinal extension after making contact with neighbor VPCs. The arrangement defects of VPCs in plx-1 and smp-1 mutants are rescued by expressing the respective cDNA in VPCs. plx-1::egfp and smp-1::egfp transgenes are both expressed in all vulval cells, including VPCs, throughout vulval development. We propose that the SMP-1/PLX-1 system is responsible for a cell contact-mediated stop signal for VPC extension. Analyses using cell fate-specific markers showed that the arrangement defects of VPCs also affect cell fate specification and cell lineages, but in a relatively small fraction of plx-1 mutants.

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.

Plexins are a family of single-pass transmembrane proteins that serve as cell surface receptors for Semaphorins during the embryonic development of animals. Semaphorin–Plexin signaling is critical for many cellular aspects of organogenesis, including cell migration, proliferation and survival. Until recently, little was known about the function of PlexinD1, the sole member of the vertebrate-specific PlexinD (PlxnD1) subfamily. Here we review novel findings about PlxnD1's roles in the development of the cardiovascular, nervous and immune systems and salivary gland branching morphogenesis and discuss new insights concerning the molecular mechanisms of PlxnD1 activity.

The correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1(cond) and Npn1(Sema-) to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre(+);Npn1(cond-/-) mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1(Sema-) mutants appeared anatomically normal. Furthermore, Olig2-Cre(+);Npn1(cond) mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1(Sema-) mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre(+);Npn1(cond-/-) mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1(Sema-) mutants to compensate for their developmental deficits.

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.

CHARGE syndrome is caused by spontaneous loss-of-function mutations to the ATP-dependant chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7). It is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations. Disruption to the neural crest lineage has previously been emphasised in the aetiology of this developmental disorder. We present evidence for an additional requirement for CHD7 activity in the Mesp1-expressing anterior mesoderm during heart development. Conditional ablation of Chd7 in this lineage results in major structural cardiovascular defects akin to those seen in CHARGE patients, as well as a striking loss of cardiac innervation and embryonic lethality. Genome-wide transcriptional analysis identified aberrant expression of key components of the Class 3 Semaphorin and Slit-Robo signalling pathways in Chd7(fl/fl);Mesp1-Cre mutant hearts. CHD7 localises at the Sema3c promoter in vivo, with alteration of the local chromatin structure seen following Chd7 ablation, suggestive of direct transcriptional regulation. Furthermore, we uncover a novel role for CHD7 activity upstream of critical calcium handling genes, and demonstrate an associated functional defect in the ability of cardiomyocytes to undergo excitation-contraction coupling. This work therefore reveals the importance of CHD7 in the cardiogenic mesoderm for multiple processes during cardiovascular development.