Carrie L. Hehr

Matrix metalloproteinases are required for retinal ganglion cell axon guidance at select decision points.

Axons receive guidance information from extrinsic cues in their environment in order to reach their targets. In the frog Xenopus laevis, retinal ganglion cell (RGC) axons make three key guidance decisions en route through the brain. First, they cross to the contralateral side of the brain at the optic chiasm. Second, they turn caudally in the mid-diencephalon. Finally, they must recognize the optic tectum as their target. The matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families are zinc (Zn)-dependent proteolytic enzymes. The latter functions in axon guidance, but a similar role has not yet been identified for the MMP family. Our previous work implicated metalloproteinases in the guidance decisions made by Xenopus RGC axons. To test specifically the importance of MMPs, we used two different in vivo exposed brain preparations in which RGC axons were exposed to an MMP-specific pharmacological inhibitor (SB-3CT), either as they reached the optic chiasm or as they extended through the diencephalon en route to the optic tectum. Interestingly, SB-3CT affected only two of the guidance decisions, with misrouting defects at the optic chiasm and tectum. Only at higher concentrations was RGC axon extension also impaired. These data implicate MMPs in the guidance of vertebrate axons, and suggest that different metalloproteinases function to regulate axon behaviour at distinct choice points: an MMP is important in guidance at the optic chiasm and the target, while either a different MMP or an ADAM is required for axons to make the turn in the mid-diencephalon.

Targeting of Retinal Axons Requires the Metalloproteinase ADAM10

The role of extrinsic cues in guiding developing axons is well established; however, the means by which the activity of these extrinsic cues is regulated is poorly understood. A disintegrin and metalloproteinase (ADAM) enzymes are Zn-dependent proteinases that can cleave guidance cues or their receptors in vitro. Here, we identify the first example of a metalloproteinase that functions in vertebrate axon guidance in vivo. Specifically, ADAM10 is required for formation of the optic projection by Xenopus retinal ganglion cell (RGC) axons. Xadam10 mRNA is expressed in the dorsal neuroepithelium through which RGC axons extend. Pharmacological or molecular inhibition of ADAM10 within the brain each resulted in a failure of RGC axons to recognize their target. In contrast, molecular inhibition of ADAM10 within the RGC axons themselves had no effect. These data argue strongly that in the dorsal brain ADAM10 acts cell non-autonomously to regulate the guidance of RGC axons.

Multiple signaling pathways regulate FGF-2-induced retinal ganglion cell neurite extension and growth cone guidance

Growth cones use cues in their environment in order to grow in a directed fashion to their targets. In Xenopus laevis, fibroblast growth factors (FGFs) participate in retinal ganglion cell (RGC) axon guidance in vivo and in vitro. The main intracellular signaling cascades known to act downstream of the FGF receptor include the mitogen-activated protein kinase (MAPK), phospholipase Cgamma (PLCgamma) and phosphotidylinositol 3-kinase (PI3K) pathways. We used pharmacological inhibitors to identify the signaling cascade(s) responsible for FGF-2-stimulated RGC axon extension and chemorepulsion. The MAPK, PI3K and PLCgamma pathways were blocked by U0126, LY249002 and U73122, respectively. D609 was used to test a role for the phosphotidylcholine-PLC (PC-PLC) pathway. We determined that the MAPK and two PLC pathways are required for FGF-2 to stimulate RGC neurite extension in vitro, but the response of axons to FGF-2 applied asymmetrically to the growth cone depended only on the PLC pathways.

Dynamic Expression of Axon Guidance Cues Required for Optic Tract Development Is Controlled by Fibroblast Growth Factor Signaling

Axons are guided to their targets by molecular cues expressed in their environment. How is the presence of these cues regulated? Although some evidence indicates that morphogens establish guidance cue expression as part of their role in patterning tissues, an important question is whether morphogens are then required to maintain guidance signals. We found that fibroblast growth factor (FGF) signaling sustains the expression of two guidance cues, semaphorin3A (xsema3A) and slit1 (xslit1), throughout the period of Xenopus optic tract development. With FGF receptor inhibition, xsema3A and xslit1 levels were rapidly diminished, and retinal ganglion cell axons arrested in the mid-diencephalon, before reaching their target. Importantly, direct downregulation of XSema3A and XSlit1 mostly phenocopied this axon guidance defect. Thus, FGFs promote continued presence of specific guidance cues critical for normal optic tract development, suggesting a second later role for morphogens, independent of tissue patterning, in maintaining select cues by acting to regulate their transcription.

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axons cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.

TGFβ ligands promote the initiation of retinal ganglion cell dendrites in vitro and in vivo

Each type of neuron develops a unique morphology critical to its function, but almost all start with the basic plan of one long axon and multiple short, branched dendrites. Though extrinsic signals are known to direct many steps in the development of neuronal structure, little is understood about the initiation of processes, particularly dendrites. We find that Xenopus retinal ganglion cells (RGCs) explanted early will extend axons and not dendrites in dissociated cultures. If RGCs develop longer in vivo prior to culturing, many now extend dendrite-like processes in vitro, suggesting that an extrinsic factor is required to stimulate dendrite initiation. Members of the transforming growth factor beta (TGFbeta) superfamily, bone morphogenetic protein 2 (BMP2), and growth and differentiation factor 11 (GDF11), can signal cultured RGCs to form dendrites. Furthermore, TGFbeta ligands have an endogenous role: blocking BMP/GDF signaling with a secreted antagonist or inhibitory receptors reduces the number of primary dendrites extended in vivo.

LIMK1 acts downstream of BMP signaling in developing retinal ganglion cell axons but not dendrites.

The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites.

Zac1 Promotes a Muller Glial Cell Fate and Interferes With Retinal Ganglion Cell Differentiation in Xenopus Retina

The timing of cell cycle exit is tightly linked to cell fate specification in the developing retina. Accordingly, several tumor suppressor genes, which are key regulators of cell cycle exit in cancer cells, play critical roles in retinogenesis. Here we investigated the role of Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, in retinal development. Strikingly, in gain‐of‐function assays in Xenopus, mouse Zac1 promotes proliferation and apoptosis at an intermediate stage of retinogenesis. Zac1 also influences cell fate decisions, preferentially promoting the differentiation of tumor‐like clusters of abnormal neuronal cells in the ganglion cell layer, as well as inducing the formation of supernumerary Müller glial cells at the expense of other cell types. Thus Zac1 has the capacity to influence cell cycle exit, and cell fate specification and differentiation decisions by retinal progenitors, suggesting that further functional studies will uncover new insights into how retinogenesis is regulated. Developmental Dynamics 236:192–202, 2007.

Neuropilin-1 biases dendrite polarization in the retina

The majority of neurons in the nervous system exhibit a polarized morphology, with multiple short dendrites and a single long axon. It is clear that multiple factors govern polarization in developing neurons, and the biased accumulation of intrinsic determinants to one side of the cell, coupled with responses to asymmetrically localized extrinsic factors, appears to be crucial. A number of intrinsic factors have been identified, but surprisingly little is known about the identity of the extrinsic signals. Here, we show in vivo that neuropilin-1 (Nrp1) and its co-receptor plexinA1 (Plxna1) are necessary to bias the extension of the dendrites of retinal ganglion cells to the apical side of the cell, and ectopically expressed class III semaphorins (Sema3s) disrupt this process. Importantly, the requirement for Nrp1 and Plxna1 in dendrite polarization occurs at a developmental time point after the cells have already extended their basally directed axon. Thus, we propose a novel mechanism whereby an extrinsic factor, probably a Sema3, acts through Nrp1 and Plxna1 to promote the asymmetric outgrowth of dendrites independently of axon polarization.

Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. We know little about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. Here, we show that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. We propose a novel, tissue-autonomous mechanism of organ cohesion, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain.