BMP receptor 1b is required for axon guidance and cell survival in the developing retina

Previous work has documented the importance of BMPs in eye development. Loss-of-function studies in mice, with targeted deletions in either the Bmp7 or Bmp4 genes, have shown that these molecules are critical for early eye development. On the basis of the asymmetry in the dorsal-ventral expression patterns of several members of this family, it has been proposed that these molecules are critical for some aspect of dorsal-ventral patterning in the eye; however, it has been difficult to test this hypothesis because of the early requirement for BMPs in eye development. We have therefore examined the effects of loss of one of the BMP receptors, the BmprIb, on the development of the eye by using targeted deletion. We have found that BmprIb is expressed exclusively in the ventral retina during embryonic development and is required for normal ventral ganglion cell axon targeting to the optic nerve head. In mice with a targeted deletion of the BmprIb gene, many axons arising from the ventrally located ganglion cells fail to enter the optic nerve head, and instead, make abrupt turns in this region. A second phenotype in these mice is a significantly elevated inner retinal apoptosis during a distinct phase of postnatal development, at the end of neurogenesis. Our results therefore show two distinct requirements for BmprIb in mammalian retinal development.     

Robo1 and Robo2 have distinct roles in pioneer longitudinal axon guidance

Pioneer longitudinal axons grow long distances parallel to the floor plate and precisely maintain their positions using guidance molecules released from the floor plate. Two receptors, Robo1 and Robo2, are critical for longitudinal axon guidance by the Slit family of chemorepellents. Previous studies showed that Robo1^−/−;2^−/− double mutant mouse embryos have disruptions in both ventral and dorsal longitudinal tracts. However, the role of each Robo isoform remained unclear, because Robo1 or 2 single mutants have mild or no errors. Here we utilized a more sensitive genetic strategy to reduce Robo levels for determining any separate functions of the Robo1 and 2 isoforms. We found that Robo1 is the predominant receptor for guiding axons in ventral tracts and prevents midline crossing. In contrast, Robo2 is the main receptor for directing axons within dorsal tracts. Robo2 also has a distinct function in repelling neuron cell bodies from the floor plate. Therefore, while Robo1 and 2 have some genetic overlap to cooperate in guiding longitudinal axons, each isoform has distinct functions in specific longitudinal axon populations.     

Dual roles of the chemorepellent axon guidance molecule RGMa in establishing pioneering axon tracts and neural fate decisions in embryonic vertebrate forebrain

Repulsive guidance molecule A (RGMa) is a glycosylphosphatidylinositol‐anchored plasma membrane protein that was originally identified based on its chemorepulsive activity during axon navigation in the developing nervous system. Knock down of RGMa has previously shown to perturb axon navigation in the developing Xenopus forebrain (Wilson and Key, 2006). In order to further understand the in vivo role of RGMa in axon guidance, we have adopted an in vivo gain‐of‐function approach. RGMa was mosaically overexpressed in the developing Xenopus embryo by the injection of mRNA into single blastomeres. Ectopic expression of RGMa affected the morphology and the topography of developing axon tracts in vivo. Pioneer axons misrouted or aberrantly projected in response to ectopic RGMa in the developing Xenopus forebrain, confirming the in vivo chemorepulsive activity of this ligand. In addition, we show here for the first time that overexpression of RGMa acts cell‐autonomously to generate ectopic neurons in the developing embryonic brain. Taken together, the current study reveals a pleiotropic role of RGMa in early vertebrate embryonic brain in the spatial organization of axon tracts, pioneer axon guidance, and neural cell differentiation. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Signalling and crosstalk of Rho GTPases in mediating axon guidance

Axon extension during development of the nervous system is guided by many factors, but the signalling mechanisms responsible for triggering this extension remain mostly unknown. Here we have examined the role of Rho family small guanosine triphosphatases (GTPases) in mediating axon guidance by diffusible factors. Expression of either dominant-negative or constitutively active Cdc42 in cultured Xenopus laevis spinal neurons, at a concentration that does not substantially affect filopodial formation and neurite extension, abolishes the chemoattractive growth cone turning induced by a gradient of brain-derived neurotrophic factor that can activate Cdc42 and Rac in cultured neurons. Chemorepulsion induced by a gradient of lysophosphatidic acid is also abolished by the expression of dominant-negative RhoA. We also show that an asymmetry in Rho kinase or filopodial initiation across the growth cone is sufficient to trigger the turning response and that there is a crosstalk between the Cdc42 and RhoA pathways through their converging actions on the myosin activity essential for growth cone chemorepulsion.     

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. We demonstrate here that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies revealed that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also showed axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes were able to rescue some sdc visual-system phenotypes, but sdc+ transgenes were ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential.     

lin-28 controls the succession of cell fate choices via two distinct activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.     

The region-specific activities of lipid rafts during axon growth and guidance

The generation and control of cell polarity is a fundamental mechanism for directed migration of the cell. In developing neurons, the axonal growth cone recognizes environmental molecular cues and migrates toward its correct target, thereby forming neuronal networks. The spatial information provided by environmental cues directs axon growth and guidance through generating polarity of intracellular signals and cytoskeletal organization in the growth cone. This polarization process is dependent on lipid rafts, specialized microdomains in the cell membrane. Lipid rafts in specific regions of the growth cone are involved in axon growth and guidance. For example, forward migration of the growth cone requires raft membranes in its leading front. Recent experiments have suggested that lipid rafts function as a platform for localized signaling downstream of adhesion molecules and guidance receptors. The rafts assemble into an active membrane domain that captures and reorganizes the cytoskeletal machinery. In this way, the spatial control of signaling through raft membranes plays a critical role in translating extracellular information into polarized motility of the growth cone.     

The Arp2/3 activators WAVE and WASP have distinct genetic interactions with Rac GTPases in Caenorhabditis elegans axon guidance

In the developing nervous system, axons are guided to their targets by the growth cone. Lamellipodial and filopodial protrusions from the growth cone underlie motility and guidance. Many molecules that control lamellipodia and filopodia formation, actin organization, and axon guidance have been identified, but it remains unclear how these molecules act together to control these events. Experiments are described here that indicate that, in Caenorhabditis elegans, two WH2-domain-containing activators of the Arp2/3 complex, WVE-1/WAVE and WSP-1/WASP, act redundantly in axon guidance and that GEX-2/Sra-1 and GEX-3/Kette, molecules that control WAVE activity, might act in both pathways. WAVE activity is controlled by Rac GTPases, and data are presented here that suggest WVE-1/WAVE and CED-10/Rac act in parallel to a pathway containing WSP-1/WASP and MIG-2/RhoG. Furthermore, results here show that the CED-10/WVE-1 and MIG-2/WSP-1 pathways act in parallel to two other molecules known to control lamellipodia and filopodia and actin organization, UNC-115/abLIM and UNC-34/Enabled. These results indicate that at least three actin-modulating pathways act in parallel to control actin dynamics and lamellipodia and filopodia formation during axon guidance (WASP-WAVE, UNC-115/abLIM, and UNC-34/Enabled).     

Evolution and diversity of axon guidance Robo receptor family genes

The diversity of axon guidance (AG) receptors reflects gains in complexity of the animal nervous system during evolution. Members of the Roundabout (Robo) family of receptors interact with Slit proteins and play important roles in many developmental processes, including AG and neural crest cell migration. There are four members of the Robo gene family. However, the evolutionary history of Robo family genes remain obscure. We analyzed the distribution of Robo family members in metazoan species ranging in complexity from hydras to humans. We undertook a phylogenetic analysis in metazoans, synteny analysis, and ancestral chromosome mapping in vertebrates, and detected selection pressure and functional divergence among four mammalian Robo paralogs. Based on our analysis, we proposed that the ancestral Robo gene could have undergone a tandem duplication in the vertebrate ancestor; then one round of whole genome duplication events occurred before the divergence of ancestral lamprey and gnathostome, generating four paralogs in early vertebrates. Robo4 paralog underwent segmental loss in the following evolutionary process. Our results showed that Robo3 paralog is under more powerful purifying selection pressure compared with other three paralogs, which could correlate with its unique expression pattern and function. Furthermore, we found four sites under positive selection pressure on the Ig1‐2 domains of Robo4 that might interfere with its binding to Slits ligand. Diverge analysis at the amino acid level showed that Robo4 paralog have relatively greater functional diversifications than other Robo paralogs. This coincides with the fact that Robo4 predominantly functions in vascular endothelial cells but not the nervous system.     

Functional redundancy of type II BMP receptor and type IIB activin receptor in BMP2-induced osteoblast differentiation

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor β (TGF-β), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.     

FGFR3 induces degradation of BMP type I receptor to regulate skeletal development

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia.     

Isolation of two distinct type I angiotensin II receptor genes.

A rat genomic Southern blot, probed with a type I angiotensin II receptor probe, demonstrated that two highly homologous type I angiotensin II receptors were present. A rat genomic library was subsequently screened and four clones were isolated. From restriction mapping, differential hybridization, polymerase chain reaction amplification and sequence analyses we have determined that there are two unique type I angiotensin II receptor genes. The first of these genes corresponds to the published rat vascular complementary DNA sequence; the second, corresponds to a novel receptor not previously described.     

Axon guidance: receptor complexes and signaling mechanisms

The generation of a functional neuronal network requires that axons navigate precisely to their appropriate targets. Molecules that specify guidance decisions have been identified, and the signaling events that occur downstream of guidance receptors are beginning to be understood. New research shows that guidance receptor signaling can be hierarchical -- one receptor silencing the other -- thereby allowing navigating growth cones to interpret opposing guidance cues. Among the known intracellular signaling molecules shared by all guidance receptor families, Rho GTPases appear to be primary regulators of actin dynamics and growth cone guidance. Novel effector molecules complete the picture and suggest additional signaling mechanisms.     

The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb

Mice carrying a targeted disruption of BmprIB were generated by homologous recombination in embryonic stem cells. BmprIB(-/-) mice are viable and, in spite of the widespread expression of BMPRIB throughout the developing skeleton, exhibit defects that are largely restricted to the appendicular skeleton. Using molecular markers, we show that the initial formation of the digital rays occurs normally in null mutants, but proliferation of prechondrogenic cells and chondrocyte differentiation in the phalangeal region are markedly reduced. Our results suggest that BMPRIB-mediated signaling is required for cell proliferation after commitment to the chondrogenic lineage. Analyses of BmprIB and Gdf5 single mutants, as well as BmprIB; Gdf5 double mutants suggests that GDF5 is a ligand for BMPRIB in vivo. BmprIB; Bmp7 double mutants were constructed in order to examine whether BMPRIB has overlapping functions with other type I BMP receptors. BmprIB; Bmp7 double mutants exhibit severe appendicular skeletal defects, suggesting that BMPRIB and BMP7 act in distinct, but overlapping pathways. These results also demonstrate that in the absence of BMPRIB, BMP7 plays an essential role in appendicular skeletal development. Therefore, rather than having a unique role, BMPRIB has broadly overlapping functions with other BMP receptors during skeletal development.     

Lipid rafts as organizing platforms for cell chemotaxis and axon guidance

Lipid rafts are thought to serve as plasma membrane platforms for localized trafficking and signaling. Recent findings reported by Guirland et al. in this issue of Neuron and by Gómez-Moutón in a recent issue of JCB support a direct role of lipid microdomains in organizing spatial signaling during axon guidance and cell chemotaxis by concentrating the gradient-sensing machinery at the leading edge.     

Syndecan regulates cell migration and axon guidance in C. elegans

During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. As there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. Here, we show that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, our experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans.     

Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance

Extending axons in the developing nervous system are guided in part by repulsive cues. Genetic analysis in Drosophila, reported in a companion to this paper, identifies the Slit protein as a candidate ligand for the repulsive guidance receptor Roundabout (Robo). Here we describe the characterization of three mammalian Slit homologs and show that the Drosophila Slit protein and at least one of the mammalian Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins. Furthermore, recombinant Slit2 can repel embryonic spinal motor axons in cell culture. These results support the hypothesis that Slit proteins have an evolutionarily conserved role in axon guidance as repulsive ligands for Robo receptors.     

Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes

We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic "fingerprints" of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.