BMPs as mediators of roof plate repulsion of commissural neurons

During spinal cord development, commissural (C) neurons, located near the dorsal midline, send axons ventrally and across the floor plate (FP). The trajectory of these axons toward the FP is guided in part by netrins. The mechanisms that guide the early phase of C axon extension, however, have not been resolved. We show that the roof plate (RP) expresses a diffusible activity that repels C axons and orients their growth within the dorsal spinal cord. Bone morphogenetic proteins (BMPs) appear to act as RP-derived chemorepellents that guide the early trajectory of the axons of C neurons in the developing spinal cord: BMP7 mimics the RP repellent activity for C axons in vitro, can act directly to collapse C growth cones, and appears to serve an essential function in RP repulsion of C axons.     

Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.     

Crossing the floor plate triggers sharp turning of commissural axons.

During development of the vertebrate CNS, commissural axons initially grow circumferentially toward the ventral midline floor plate. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. Although recent studies have unraveled the mechanisms that control navigation of these axons along the circumferential axis, those that result in the transition from circumferential to longitudinal trajectory remain unknown. Here, we examined whether an interaction with the floor plate is a prerequisite for the initiation of trajectory transition of commissural axons, using in vitro preparations of the rat metencephalon. We found that commissural axons in the metencephalon, once having crossed the floor plate, turned sharply to grow longitudinally. In contrast, axons extending in floor plate-deleted preparations, continued to grow circumferentially, ignoring the hypothetical turning point. These results suggest that a prior interaction of commissural axons with floor plate cells is a key step for these axons to activate a navigation program required for their change in axonal trajectory from the circumferential to the longitudinal axis.     

Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

F-Spondin is required for accurate pathfinding of commissural axons at the floor plate.

The commissural axons project toward and across the floor plate. They then turn into the longitudinal axis, extending along the contralateral side of the floor plate. F-spondin, a protein produced and secreted by the floor plate, promotes adhesion and neurite extension of commissural neurons in vitro. Injection of purified F-spondin protein into the lumen of the spinal cord of chicken embryos in ovo resulted in longitudinal turning of commissural axons before reaching the floor plate, whereas neutralizing antibody (Ab) injections caused lateral turning at the contralateral floor plate boundary. These combined in vitro and in vivo results suggest that F-spondin is required to prevent the lateral drifting of the commissural axons after having crossed the floor plate.     

A role for BMP signalling in heart looping morphogenesis in Xenopus

The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.     

The midline glial cells are required for regionalization of commissural axons in the embryonic CNS of Drosophila

 The ventral nerve cord of arthropods is characterised by the organisation of major axon tracts in a ladder-like pattern. The individual neuromeres are connected by longitudinal connectives whereas the contra-lateral connections are brought about through segmental commissures. In each neuromere of the embryonic central nervous system (CNS) of Drosophila an anterior and a posterior commissure is found. The development of these commissures requires a set of neurone-glia interactions at the midline. Here we show that both the anterior as well as the posterior commissures are subdivided into three axon-containing regions. Electron microscopy of the ventral nerve cord of mutations affecting CNS midline cells indicates that the midline glial cells are required for this subdivision. In addition the midline glial cells appear required for a crossing of commissural growth cones perpendicular to the longitudinal tracts, since in mutants with defective midline glial cells commissural axons frequently cross the midline at aberrant angles. Received: 6 July 1997 / Accepted: 27 August 1997     

Shh signaling guides spatial pathfinding of raphespinal tract axons by multidirectional repulsion

Relatively little is known about the molecular mechanisms underlying spatial pathfinding in the descending serotonergic raphespinal tract (RST) in the developing spinal cord, one of the most important nerve pathways for pain, sensory and motor functions. We provide evidence that ventral floor plate-secreted Sonic hedgehog (Shh) is responsible for the establishment of decreasing gradients in both the anterior-to-posterior (A-P) and the medial-to-lateral (M-L) directions in the ventral spinal cord during serotonergic RST axon projection. Downstream components of the Shh pathway, Patched 1 (Ptch1) and Smoothened (Smo), were expressed in the serotonergic caudal raphe nuclei and enriched in the descending serotonergic RST axons. Diffusible Shh repulsion of serotonergic RST axons was shown to be mediated by Shh-Ptch1 interactions and derepression of Smo. Using a co-culture assay, we showed that A-P graded repulsion mediated by Shh signaling pushed the serotonergic axons caudally through the ventral spinal cord and M-L graded repulsion mediated by Shh signaling simultaneously restricted the serotonergic axons to the ventral and ventral-lateral funiculus. Prominent pathfinding errors of serotonergic RST axons were observed in various Shh, Ptch1 and Smo mutants. We conclude that Shh signaling-mediated multidirectional repulsion is required to push descending serotonergic RST axons in the A-P direction, and to restrict these axons to the ventral and ventral-lateral funiculus in the M-L direction. This is the first demonstration that Shh signaling-mediated multidirectional repulsion of serotonergic RST axons maintains spatial axon pathfinding in the developing spinal cord.     

GDNF acts as a chemoattractant to support ephrinA-induced repulsion of limb motor axons

Despite the abundance of guidance cues in vertebrate nervous systems, little is known about cooperation between them. Motor axons of the lateral motor column (LMC(L)) require two ligand/receptor systems, ephrinA/EphA4 and glial cell line-derived neurotrophic factor (GDNF)/Ret, to project to the dorsal limb. Deletion of either EphA4 or Ret in mice leads to rerouting of a portion of LMC(L) axons to the ventral limb, a phenotype enhanced in EphA4;Ret double mutants. The guidance errors in EphA4 knockouts were attributed to the lack of repulsion from ephrinAs in the ventral mesenchyme. However, it has remained unclear how GDNF, expressed dorsally next to the choice point, acts on motor axons and cooperates with ephrinAs. Here we show that GDNF induces attractive turning of LMC(L) axons. When presented in countergradients, GDNF and ephrinAs cooperate in axon turning, indicating that the receptors Ret and EphA4 invoke opposite effects within the same growth cone. GDNF also acts in a permissive manner by reducing ephrinA-induced collapse and keeping the axons in a growth-competent state. This is the first example of two opposing cues promoting the same trajectory choice at an intermediate target.     

A role for the EphA family in the topographic targeting of vomeronasal axons

We have investigated the role of the Eph family of receptor tyrosine kinases and their ligands in the establishment of the vomeronasal projection in the mouse. Our data show intriguing differential expression patterns of ephrin-A5 on vomeronasal axons and of EphA6 in the accessory olfactory bulb (AOB), such that axons with high ligand concentration project onto regions of the AOB with high receptor concentration and vice versa. These data suggest a mechanism for development of this projection that is the opposite of the repellent interaction between Eph receptors and ligands observed in other systems. In support of this idea, when given the choice of whether to grow on lanes containing EphA-F(c)/laminin or F(c)/laminin protein (in the stripe assay), vomeronasal axons prefer to grow on EphA-F(c)/laminin. Analysis of ephrin-A5 mutant mice revealed a disturbance of the topographic targeting of vomeronasal axons to the AOB. In summary, these data, which are derived from in vitro and in vivo experiments, indicate an important role of the EphA family in setting up the vomeronasal projection.     

Sek4 and Nuk receptors cooperate in guidance of commissural axons and in palate formation.

Sek4 and Nuk are members of the Eph-related family of receptor protein-tyrosine kinases. These receptors interact with a set of cell surface ligands that have recently been implicated in axon guidance and fasciculation. We now demonstrate that the formation of the corpus callosum and anterior commissure, two major commissural axon tracts that connect the two cerebral hemispheres, is critically dependent on Sek4 and Nuk. While mice deficient in Nuk exhibit defects in pathfinding of anterior commissure axons, sek4 mutants have defects in corpus callosum formation. The phenotype in both axon tracts is markedly more severe in sek4/nuk1 double mutants, indicating that the two receptors act in a partially redundant fashion. sek4/nuk1 double mutants also exhibit specific guidance and fasciculation defects of diencephalic axon tracts. Moreover, while mice singly deficient in either Sek4 or Nuk are viable, most sek4/nuk1 double mutants die immediately after birth primarily due to a cleft palate. These results demonstrate essential and cooperative functions for Sek4 and Nuk in establishing axon pathways in the developing brain, and during the development of facial structures.     

Axon fasciculation and differences in midline kinetics between pioneer and follower axons within commissural fascicles

Early neuronal scaffold development studies suggest that initial neurons and their axons serve as guides for later neurons and their processes. Although this arrangement might aid axon navigation, the specific consequence(s) of such interactions are unknown in vivo. We follow forebrain commissure formation in living zebrafish embryos using timelapse fluorescence microscopy to examine quantitatively commissural axon kinetics at the midline: a place where axon interactions might be important. Although it is commonly accepted that commissural axons slow down at the midline, our data show this is only true for leader axons. Follower axons do not show this behavior. However, when the leading axon is ablated, follower axons change their midline kinetics and behave as leaders. Similarly, contralateral leader axons change their midline kinetics when they grow along the opposite leading axon across the midline. These data suggest a simple model where the level of growth cone exposure to midline cues and presence of other axons as a substrate shape the midline kinetics of commissural axons.     

Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons.

In both invertebrate and lower vertebrate species, decussated commissural axons travel away from the midline and assume positions within distinct longitudinal tracts. We demonstrate that in the developing chick and mouse spinal cord, most dorsally situated commissural neuron populations extend axons across the ventral midline and through the ventral white matter along an arcuate trajectory on the contralateral side of the floor plate. Within the dorsal (chick) and intermediate (mouse) marginal zone, commissural axons turn at a conserved boundary of transmembrane ephrin expression, adjacent to which they form a discrete ascending fiber tract. In vitro perturbation of endogenous EphB-ephrinB interactions results in the failure of commissural axons to turn at the appropriate dorsoventral position on the contralateral side of the spinal cord; consequently, axons inappropriately invade more dorsal regions of B-class ephrin expression in the dorsal spinal cord. Taken together, these observations suggest that B-class ephrins act locally during a late phase of commissural axon pathfinding to specify the dorsoventral position at which decussated commissural axons turn into the longitudinal axis.     

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.     

A case of repulsion in wheat

Molecular Genetics and Genomics -     

A case of repulsion in wheat

Ohne Zusammenfassung

---

A case of repulsion in wheat

     

A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic (DPP) and Screw (SCW). short gastrulation (sog), which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Here we demonstrate that SOG has a second function, which is to promote BMP signaling on the dorsal side of the embryo. We show that a weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and that the lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of SOG is directed against SCW, we present data that suggests that the positive activity of SOG is directed towards DPP. We demonstrate that Chordin shares the same apparent ligand specificity as does SOG, preferentially inhibiting SCW but not DPP activity. However, in Drosophila assays Chordin does not have the same capacity to elevate BMP signaling as does SOG, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.