Receptor tyrosine phosphatases regulate axon guidance across the midline of the Drosophila embryo

Neural receptor-linked protein tyrosine phosphatases (RPTPs) are required for guidance of motoneuron and photoreceptor growth cones in Drosophila. These phosphatases have not been implicated in growth cone responses to specific guidance cues, however, so it is unknown which aspects of axonal pathfinding are controlled by their activities. Three RPTPs, known as DLAR, DPTP69D, and DPTP99A, have been genetically characterized thus far. Here we report the isolation of mutations in the fourth neural RPTP, DPTP10D. The analysis of double mutant phenotypes shows that DPTP10D and DPTP69D are necessary for repulsion of growth cones from the midline of the embryonic central nervous system. Repulsion is thought to be triggered by binding of the secreted protein Slit, which is expressed by midline glia, to Roundabout (Robo) receptors on growth cones. Robo repulsion is downregulated by the Commissureless (Comm) protein, allowing axons to cross the midline. Here we show that the Rptp mutations genetically interact with robo, slit and comm. The nature of these interactions suggests that DPTP10D and DPTP69D are positive regulators of Slit/Roundabout repulsive signaling. We also show that elimination of all four neural RPTPs converts most noncrossing longitudinal pathways into commissures that cross the midline, indicating that tyrosine phosphorylation controls the manner in which growth cones respond to midline signals.     

Pathfinding and error correction by retinal axons: the role of astray/robo2.

To address how the highly stereotyped retinotectal pathway develops in zebrafish, we used fixed-tissue and time-lapse imaging to analyze morphology and behavior of wild-type and mutant retinal growth cones. Wild-type growth cones increase in complexity and pause at the midline. Intriguingly, they make occasional ipsilateral projections and other pathfinding errors, which are always eventually corrected. In the astray/robo2 mutant, growth cones are larger and more complex than wild-type. astray axons make midline errors not seen in wild-type, as well as errors both before and after the midline. astray errors are rarely corrected. The presumed Robo ligands Slit2 and Slit3 are expressed near the pathway in patterns consistent with their mediating pathfinding through Robo2. Thus, Robo2 does not control midline crossing of retinal axons, but rather shapes their pathway, by both preventing and correcting pathfinding errors.     

Ongoing sonic hedgehog signaling is required for dorsal midline formation in the developing forebrain

The division of the mammalian forebrain into distinct left and right hemispheres represents a critical step in neural development. Several signaling molecules including sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and bone morphogenetic proteins (BMPs) have been implicated in dorsal midline development, and prior work suggests that the organizing centers from which these proteins are secreted mutually regulate one another during development. To explore the role of the ventral organizing center in the formation of two hemispheres, we assessed dorsal midline development in Shh mutant embryos and in wildtype embryos treated with the SHH signaling inhibitor HhAntag. Collectively, our findings demonstrate that SHH signaling plays an important role in maintaining the normal expression patterns of Fgf8 and Bmp4 in the developing forebrain. We further show that FGF8 can induce the expression of Zic2, which is normally expressed at the midline and is required in vivo for hemispheric cleavage, suggesting that FGF signaling may stimulate dorsal midline development by inducing Zic2 expression.     

Short-range and long-range guidance by slit and its Robo receptors. Robo and Robo2 play distinct roles in midline guidance

Previous studies showed that Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline, even though they cross and recross it. Here we show that the Drosophila genome encodes three Robo receptors and that Robo and Robo2 have distinct functions, which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit.     

Spatially discrete FGF-mediated signalling directs glial morphogenesis.

Neurons provide critical signals that regulate both the number and differentiation of glia. In addition, glia are attracted to and enwrap neuronal axonal processes. FGF-like signalling is thought to be one of the many potential axon-derived morphogenetic signals, however, the multiple roles of FGFs have made experimental tests of these signals difficult in vivo. In the Drosophila FGF receptor mutant heartless, glia migrate to axons, but fail to elongate around them. This study shows that in the similar but larger grasshopper CNS, FGF signalling is likely to mediate one step in the close interaction between glia and axons. FGF2-coated beads attract glia in the CNS and compete with axons for their resident, enwrapped glia. In addition, bath applied FGF2 causes mature axonal glia, which normally enwrap axon tracts, to round up. FGF2 activates the product of the grasshopper heartless FGF receptor gene and probably interferes with the normal function of an endogenous axon-associated FGF-like molecule. It is proposed that insect axons provide a critical spatially restricted FGF-like signal that induces glia to enwrap them.     

Synchronous and symmetric migration of Drosophila caudal visceral mesoderm cells requires dual input by two FGF ligands

Caudal visceral mesoderm (CVM) cells migrate synchronously towards the anterior of the Drosophila embryo as two distinct groups located on each side of the body, in order to specify longitudinal muscles that ensheath the gut. Little is known about the molecular cues that guide cells along this path, the longest migration of embryogenesis, except that they closely associate with trunk visceral mesoderm (TVM). The expression of the fibroblast growth factor receptor (FGFR) heartless and its ligands, pyramus (pyr) and thisbe (ths), within CVM and TVM cells, respectively, suggested FGF signaling may influence CVM cell guidance. In FGF mutants, CVM cells die before reaching the anterior region of the TVM. However, an earlier phenotype observed was that the two cell clusters lose direction and converge at the midline. Live in vivo imaging and tracking analyses identified that the movements of CVM cells were slower and no longer synchronous. Moreover, CVM cells were found to cross over from one group to the other, disrupting bilateral symmetry, whereas such mixing was never observed in wild-type embryos. Ectopic expression of either Pyr or Ths was sufficient to redirect CVM cell movement, but only when the endogenous source of these ligands was absent. Collectively, our results show that FGF signaling regulates directional movement of CVM cells and that native presentation of both FGF ligands together is most effective at attracting cells. This study also has general implications, as it suggests that the activity supported by two FGF ligands in concert differs from their activities in isolation.     

DASB -in vitro binding characteristics on human recombinant monoamine transporters with regard to its potential as positron emission tomography (PET) tracer

The efficiency of serotonergic signal transduction is controlled by the density of serotonegic synapses and by the activity of the serotonin transporter (SERT), which selectively clears the synaptic cleft of the neurotransmitter. SERT is located in axons, where it is concentrated in varicosities and terminal boutons and thus is an exquisite marker for serotonergic synapses. This finding has been taken advantage of for neuroimaging serotonergic synaptic contact sites. Previous positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies were often carried out using radioligands that bind with high affinity to SERTs in the brainstem but also exhibit high affinity for dopamine and norepinephrine transporters and therefore did not allow quantification of serotonergic innervations in brain regions also containing dopaminergic or noradrenergic terminals. In order to visualize SERT availability more selectively, in recent years new tracers have been developed, one of which is [11C]DASB (N,N-dimethyl-2-2-amino-4-cyanophenylthiobenzylamine). Here, we have performed a detailed pharmacological characterization of unlabelled as well as radioactive DASB on recombinant human monoamine transporter proteins. Our results show that DASB selectively binds to SERT with high affinity (KD = 3.5 nm) to a site distinct from the serotonin (5-HT) recognition/translocation site. 5-HT inhibits DASB binding to SERT with more than one order of magnitude lower affinity than that of DASB binding (IC50 = 82.4 nm). These findings suggest DASB to be a highly selective PET tracer to visualize the density of serotonergic synapses in human brain.     

Slit is the midline repellent for the robo receptor in Drosophila

Previous studies suggested that Roundabout (Robo) is a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. Here we present genetic evidence that Slit is the midline Robo ligand; a companion paper presents biochemical evidence that Slit binds Robo. Slit is a large extracellular matrix protein expressed by midline glia. In slit mutants, growth cones enter the midline but never leave it; they abnormally continue to express high levels of Robo while at the midline. slit and robo display dosage-sensitive genetic interactions, indicating that they function in the same pathway. slit is also required for migration of muscle precursors away from the midline. Slit appears to function as a short-range repellent controlling axon crossing of the midline and as a long-range chemorepellent controlling mesoderm migration away from the midline.     

Flotillin‐1 interacts with the serotonin transporter and modulates chronic corticosterone response

Aberrant serotonergic neurotransmission in the brain is considered at the core of the pathophysiological mechanisms involved in neuropsychiatric disorders. Gene by environment interactions contribute to the development of depression and involve modulation of the availability and functional activity of the serotonin transporter (SERT). Using behavioral and in vivo electrophysiological approaches together with biochemical, molecular‐biological and molecular imaging tools we establish Flotillin‐1 (Flot1) as a novel protein interacting with SERT and demonstrate its involvement in the response to chronic corticosterone (CORT) treatment. We show that genetic Flot1 depletion augments chronic CORT‐induced behavioral despair and describe concomitant alterations in the expression of SERT, activity of serotonergic neurons and alterations of the glucocorticoid receptor transport machinery. Hence, we propose a role for Flot1 as modulatory factor for the depressogenic consequences of chronic CORT exposure and suggest Flotillin‐1‐dependent regulation of SERT expression and activity of serotonergic neurotransmission at the core of the molecular mechanisms involved.     

Semaphorin 3d guides laterality of retinal ganglion cell projections in zebrafish

The optic chiasm is an important choice point at which retinal ganglion cell (RGC) axons either cross the midline to innervate the contralateral brain or turn back to innervate the ipsilateral brain. Guidance cues that regulate this decision, particularly those directing the midline crossing of contralateral axons, are still not well understood. Here we show that Sema3d, a secreted semaphorin expressed at the midline, guides the crossing of RGC axons in zebrafish. Both Sema3d knockdown and ubiquitous overexpression induced aberrant ipsilateral projections, suggesting that Sema3d normally guides axons into the contralateral optic tract. Live imaging in vivo showed that RGC growth cones responded to ubiquitous Sema3d overexpression by pausing for extended periods and increasing their exploratory behavior at the midline, suggesting that Sema3d overexpression causes the midline environment to become less favorable for RGC axon extension. Interestingly, Sema3d overexpression did not affect growth cone behaviors before the midline, suggesting that RGC axons normally respond to Sema3d only upon reaching the midline. After Sema3d knockdown, growth cones grew across the midline but then paused or repeatedly retracted, impairing their ability to leave the midline region. Our results indicate that a proper balance of Sema3d is needed at the midline for the progression of RGC axons from the chiasm midline into the contralateral optic tract.     

Growth cone guidance by floor plate cells in the spinal cord of zebrafish embryos.

The spinal cord of early zebrafish embryos contains a small number of neuronal classes whose growth cones all follow stereotyped, cell-specific pathways to their targets. Two classes of spinal neurons make cell-specific turns at or near the ventral midline of the spinal cord, which is occupied by a single row of midline floor plate cells. We tested whether these cells guide the growth cones by examining embryos missing the midline floor plate cells due either to laser ablation of the cells or to a mutation (cyc-1). In these embryos the growth cones followed both normal and aberrant pathways once near the ventral midline. This suggests that normally the midline floor plate cells do provide guidance cues, but that these cues are not obligatory.     

Regulation of rho family GTPases is required to prevent axons from crossing the midline

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. Our data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho.     

Ligand Effects on Cross-linking Support a Conformational Mechanism for Serotonin Transport

Serotonin transporter (SERT) is responsible for the re-uptake of 5-hydroxytryptamine (5-HT) from the synaptic cleft after release from serotonergic neurons. We show here that cysteine residues at positions in transmembranes 1 and 3 of SERT, like the corresponding positions in the γ-aminobutyric acid transporter, can be cross-linked using copper(II)(1,10-phenanthroline)₃. The presence of a cross-link was detected by a novel methionine mutagenesis strategy. A change in mobility for an N-terminal cyanogen bromide fragment accompanied disulfide cross-linking of the two cysteine residues. Cross-linking also inhibited transport, and this process was blocked by cocaine, which is expected to stabilize SERT in conformations where the two positions are separated, but cocaine did not decrease accessibility of either of the two cysteines to modification by 2-aminoethyl methanethiosulfonate. Cysteine was required at both positions on the same molecule for efficient cross-linking, indicating that the reaction was intramolecular.     

Spatial restriction of spontaneous activity towards the rostral primary initiating zone during development of the embryonic mouse hindbrain

In the developing embryonic mouse hindbrain, we have previously shown that synchronized spontaneous activity is driven by midline serotonergic neurons at E11.5. This is mediated, at least in part, by the 5-HT2A receptor, which is expressed laterally in the hindbrain. Activity at E11.5 is widespread within the hindbrain tissue, propagating from the midline to more lateral regions. Using rapid acquisition of [Ca2+]i events along the midline, we now show that the rostral midline, primarily in the region of former rhombomere r2, is the primary initiating zone for this activity. We propose that at E11.5, the combined events along the rostral-caudal axis in combination with events propagating along the medial-lateral axis could assign positional information to developing neurons within the hindbrain. With further development, to E13.5, both the lateral and caudal dimensions of spontaneous activity retract to the rostral midline, occupying an area only 14% of that exhibited at E11.5. We also show that increased levels of [K+]o (to 8 mM) at E13.5 are able to increase the spread of spontaneous activity laterally and rostro-caudally. This suggests that spontaneous activity in the hindbrain depends in a dynamic way on the dominant initiating zone of the rostral midline, and that this relationship changes over development.     

The EGF and FGF receptors mediate neuroglian function to control growth cone decisions during sensory axon guidance in Drosophila

Cell adhesion molecules (CAMs) implement the process of axon guidance by promoting specific selection and attachment to substrates. We show that, in Drosophila, loss-of-function conditions of either the Neuroglian CAM, the FGF receptor coded by the gene heartless, or the EGF receptor coded by DER display a similar phenotype of abnormal substrate selection and axon guidance by peripheral sensory neurons. Moreover, neuroglian loss-of-function phenotype can be suppressed by the expression of gain-of-function conditions of heartless or DER. The results are consistent with a scenario where the activity of these receptor tyrosine kinases is controlled by Neuroglian at choice points where sensory axons select between alternative substrates for extension.     

Fibroblast growth factor 13 is a microtubule-stabilizing protein regulating neuronal polarization and migration

Secretory fibroblast growth factors (FGFs) and their receptors are known for their regulatory function in the early stages of neural development. FGF13, a nonsecretory protein of the FGF family, is expressed in cerebral cortical neurons during development and is a candidate gene for syndromal and nonspecific forms of X-chromosome-linked mental retardation (XLMR). However, its function during development remains unclear. We show that FGF13 acts intracellularly as a microtubule-stabilizing protein required for axon and leading process development and neuronal migration in the cerebral cortex. FGF13 is enriched in axonal growth cones and interacts directly with microtubules. Furthermore, FGF13 polymerizes tubulins and stabilizes microtubules. The loss of FGF13 impairs neuronal polarization and increases the branching of axons and leading processes. Genetic deletion of FGF13 in mice results in neuronal migration defects in both the neocortex and the hippocampus. FGF13-deficient mice also exhibit weakened learning and memory, which is correlated to XLMR patients' intellectual disability.     

The FGF receptor uses the endocannabinoid signaling system to couple to an axonal growth response

A key role for DAG lipase activity in the control of axonal growth and guidance in vitro and in vivo has been established. For example, DAG lipase activity is required for FGF-stimulated calcium influx into neuronal growth cones, and this response is both necessary and sufficient for an axonal growth response. The mechanism that couples the hydrolysis of DAG to the calcium response is not known. The initial hydrolysis of DAG at the sn-1 position (by DAG lipase) will generate 2-arachidonylglycerol, and this molecule is well established as an endogenous cannabinoid receptor agonist in the brain. In the present paper, we show that in rat cerebellar granule neurons, CB1 cannabinoid receptor antagonists inhibit axonal growth responses stimulated by N-cadherin and FGF2. Furthermore, three CB1 receptor agonists mimic the N-cadherin/FGF2 response at a step downstream from FGF receptor activation, but upstream from calcium influx into cells. In contrast, we could find no evidence for the CB1 receptor coupling the TrkB neurotrophin receptor to an axonal growth response in the same neurons. The observation that the CB1 receptor can couple the activated FGF receptor to an axonal growth response raises novel therapeutic opportunities.     

Calmodulin and profilin coregulate axon outgrowth in Drosophila

Coordinated regulation of actin cytoskeletal dynamics is critical to growth cone movement. The intracellular molecules calmodulin and profilin actively regulate actin-based motility and participate in the signaling pathways used to steer growth cones. Here we show that in the developing Drosophila embryo, calmodulin and profilin convey complimentary information that is necessary for appropriate growth cone advance. Reducing calmodulin activity by expression of a dominant inhibitor (KA) stalls axon extension of pioneer neurons within the CNS, while a partial loss of profilin function decreases extension of motor axons in the periphery. Yet, surprisingly, when calmodulin and profilin are simultaneously reduced, the ability of both CNS pioneer axons and motor axons to extend beyond the choice points is restored. In the CNS, at the time when growth cones must decide whether to cross or not to cross the midline, a reduction in calmodulin and/or roundabout signaling causes axons to cross the midline inappropriately. These inappropriate crossings are suppressed when profilin activity is simultaneously reduced. Interestingly, the mutual suppression of calmodulin and profilin activity requires a minimal level of profilin. In KA combinations with profilin null alleles, defects in axon extension and midline guidance are synergistically enhanced rather than suppressed. Together, our data indicate that the growth cone must coordinate the activity of both calmodulin and profilin in order to advance past selected choice points, including those dictating midline crossovers.     

Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy

In the central nervous system serotonergic neurotransmission is terminated by the rapid removal of serotonin (5-hydroxytryptamine, 5HT) out of the extra-cellular space back into the presynaptic neuron. This task is fulfilled by a specific serotonin transporter (SERT) protein which controls the concentration of extra-cellular 5HT. Consequently, one mechanism to regulate the efficacy of serotonergic neurotransmission is via modulation of the density of SERT molecules on the cell membrane.In this regard it has been shown, that chronic activation of the p38 mitogen-activated protein kinase (p38 MAPK) leads to enhanced SERT surface expression whereas activation of protein kinase C (PKC) reduces SERT surface expression. In addition, it has been reported that exposure to selective serotonin re-uptake inhibitors (SSRIs) leads to a down-regulation of SERT expression in vivo and in vitro in different cellular systems. Here, we have studied interactions between kinase- and SSRI-induced SERT internalization in mouse stem cell-derived serotonergic neurons expressing the native SERT allele in its natural surroundings. Therefore we established a method to quantify the amount of cell surface-expressed SERT molecules on individual cells by antibody detection combined with confocal laser scanning microscopy.Using this methodology we could show that activation of PKC, inhibition of the p38 MAPK as well as exposure to the SSRI citalopram each induced a significant reduction of cell surface-expressed SERT over time. Combinations of PKC activation, p38 MAPK inhibition and SSRI exposure led to a more pronounced down-regulation of SERT surface expression depending on the time of drug exposure.