Deletion or inhibition of PTPRO prevents ectopic fat accumulation and induces healthy obesity with markedly reduced systemic inflammation.

AIMS: Chronic inflammation plays crucial roles in obesity-induced metabolic diseases. Protein tyrosine phosphatase receptor type O (PTPRO) is a member of the R3 subfamily of receptor-like protein tyrosine phosphatases. We previously suggested a role for PTPRO in the inactivation of the insulin receptor. The present study aimed to elucidate the involvement of PTPRO in the control of glucose and lipid metabolism as well as in obesity-induced systemic inflammation. MATERIALS AND METHODS: Lipid accumulation in adipose tissue and the liver, the expression of inflammatory cytokines, and insulin resistance associated with systemic inflammation were investigated in hyper-obese Ptpro-KO mice by feeding a high-fat/high-sucrose diet (HFHSD). The effects of the administration of AKB9778, a specific inhibitor of PTPRO, to ob/ob mice and cultured 3T3-L1 preadipocyte cells were also examined. KEY FINDINGS: Ptpro was highly expressed in visceral white adipose tissue and macrophages. Ptpro-KO mice fed HFHSD were hyper-obese, but did not have ectopic fat accumulation in the liver, dysfunctional lipid and glucose homeostasis, systemic inflammation, or insulin resistance. The administration of AKB9778 reproduced "the healthy obese phenotypes" of Ptpro-KO mice in highly obese ob/ob mice. Furthermore, the inhibition of PTPRO promoted the growth of lipid droplets in adipocytes through an increase in the phosphorylation of Tyr(117) in vimentin. SIGNIFICANCE: Healthy systemic conditions with the attenuation of inflammation in hyper-obese Ptpro-KO mice were associated with the expansion of adipose tissue and low activation of NF-κb. Therefore, PTPRO may be a promising target to ameliorate hepatic steatosis and metabolic dysfunction.

Protein Tyrosine Phosphatase Receptor Type J (PTPRJ) Regulates Retinal Axonal Projections by Inhibiting Eph and Abl Kinases in Mice.

Eph receptors play pivotal roles in the axon guidance of retinal ganglion cells (RGCs) at the optic chiasm and the establishment of the topographic retinocollicular map. We previously demonstrated that protein tyrosine phosphatase receptor type O (PTPRO) is specifically involved in the control of retinotectal projections in chicks through the dephosphorylation of EphA and EphB receptors. We subsequently revealed that all the mouse R3 subfamily members (PTPRB, PTPRH, PTPRJ, and PTPRO) of the receptor protein tyrosine phosphatase (RPTP) family inhibited Eph receptors as their substrates in cultured mammalian cells. We herein investigated the functional roles of R3 RPTPs in the projection of mouse retinal axon of both sexes. Ptpro and Ptprj were expressed in mouse RGCs; however, Ptprj expression levels were markedly higher than those of Ptpro Consistent with their expression levels, Eph receptor activity was significantly enhanced in Ptprj-knock-out (Ptprj-KO) retinas. In Ptprj-KO and Ptprj/Ptpro-double-KO (DKO) mice, the number of retinal axons that projected ipsilaterally or to the contralateral eye was significantly increased. Furthermore, retinal axons in Ptprj-KO and DKO mice formed anteriorly shifted ectopic terminal zones in the superior colliculus (SC). We found that c-Abl (Abelson tyrosine kinase) was downstream of ephrin-Eph signaling for the repulsion of retinal axons at the optic chiasm and in the SC. c-Abl was identified as a novel substrate for PTPRJ and PTPRO, and the phosphorylation of c-Abl was upregulated in Ptprj-KO and DKO retinas. Thus, PTPRJ regulates retinocollicular projections in mice by controlling the activity of Eph and c-Abl kinases.SIGNIFICANCE STATEMENT Correct retinocollicular projection is a prerequisite for proper vision. Eph receptors have been implicated in retinal axon guidance at the optic chiasm and the establishment of the topographic retinocollicular map. We herein demonstrated that protein tyrosine phosphatase receptor type J (PTPRJ) regulated retinal axonal projections by controlling Eph activities. The retinas of Ptprj-knock-out (KO) and Ptpro/Ptprj double-KO mice exhibited significantly enhanced Eph activities over those in wild-type mice, and their axons showed defects in pathfinding at the chiasm and retinocollicular topographic map formation. We also revealed that c-Abl (Abelson tyrosine kinase) downstream of Eph receptors was regulated by PTPRJ. These results indicate that the regulation of the ephrin-Eph-c-Abl axis by PTPRJ plays pivotal roles in the proper central projection of retinal axons during development.

PTPRJ Inhibits Leptin Signaling, and Induction of PTPRJ in the Hypothalamus Is a Cause of the Development of Leptin Resistance.

Leptin signaling in the hypothalamus plays a crucial role in the regulation of body weight. Leptin resistance, in which leptin signaling is disrupted, is a major obstacle to the improvement of obesity. We herein demonstrated that protein tyrosine phosphatase receptor type J (Ptprj) is expressed in hypothalamic neurons together with leptin receptors, and that PTPRJ negatively regulates leptin signaling by inhibiting the activation of JAK2, the primary tyrosine kinase in leptin signaling, through the dephosphorylation of Y813 and Y868 in JAK2 autophosphorylation sites. Leptin signaling is enhanced in Ptprj-deficient mice, and they exhibit lower weight gain than wild-type mice because of a reduced food intake. Diet-induced obesity and the leptin treatment up-regulated PTPRJ expression in the hypothalamus, while the overexpression of PTPRJ induced leptin resistance. Thus, the induction of PTPRJ is a factor contributing to the development of leptin resistance, and the inhibition of PTPRJ may be a potential strategy for improving obesity.

The R3 receptor-like protein tyrosine phosphatase subfamily inhibits insulin signalling by dephosphorylating the insulin receptor at specific sites.

The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6-17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj-deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo, and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent.

Loss-of-Function Mutation in APC2 Causes Sotos Syndrome Features.

Sotos syndrome, characterized by intellectual disability and characteristic facial features, is caused by haploinsufficiency in the NSD1 gene. We conducted an etiological study on two siblings with Sotos features without mutations in NSD1 and detected a homozygous frameshift mutation in the APC2 gene by whole-exome sequencing, which resulted in the loss of function of cytoskeletal regulation in neurons. Apc2-deficient (Apc2-/-) mice exhibited impaired learning and memory abilities along with an abnormal head shape. Endogenous Apc2 expression was downregulated by the knockdown of Nsd1, indicating that APC2 is a downstream effector of NSD1 in neurons. Nsd1 knockdown in embryonic mouse brains impaired the migration and laminar positioning of cortical neurons, as observed in Apc2-/- mice, and this defect was rescued by the forced expression of Apc2. Thus, APC2 is a crucial target of NSD1, which provides an explanation for the intellectual disability associated with Sotos syndrome.

SPIG1 negatively regulates BDNF maturation.

We previously identified SPARC-related protein-containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4) as one of the dorsal-retina-specific molecules expressed in the developing chick retina. We here demonstrated that the knockdown of SPIG1 in the retinal ganglion cells (RGCs) of developing chick embryos induced the robust ectopic branching of dorsal RGC axons and failed to form a tight terminal zone at the proper position on the tectum. The knockdown of SPIG1 in RGCs also led to enhanced axon branching in vitro. However, this was canceled by the addition of a neutralizing antibody against brain-derived neurotrophic factor (BDNF) to the culture medium. SPIG1 and BDNF were colocalized in vesicle-like structures in cells. SPIG1 bound with the proform of BDNF (proBDNF) but very weakly with mature BDNF in vitro. The expression and secretion of mature BDNF were significantly decreased when SPIG1 was exogenously expressed with BDNF in HEK293T or PC12 cells. The amount of mature BDNF proteins as well as the tyrosine phosphorylation level of the BDNF receptor, tropomyosin-related kinase B (TrkB), in the hippocampus were significantly higher in SPIG1-knockout mice than in wild-type mice. Here the spine density of CA1 pyramidal neurons was consistently increased. Together, these results suggest that SPIG1 negatively regulated BDNF maturation by binding to proBDNF, thereby suppressing axonal branching and spine formation.

Substrate specificity of R3 receptor-like protein-tyrosine phosphatase subfamily toward receptor protein-tyrosine kinases.

Receptor-like protein-tyrosine phosphatases (RPTPs) are involved in various aspects of cellular functions, such as proliferation, differentiation, survival, migration, and metabolism. A small number of RPTPs have been reported to regulate activities of some cellular proteins including receptor protein-tyrosine kinases (RPTKs). However, our understanding about the roles of individual RPTPs in the regulation of RPTKs is still limited. The R3 RPTP subfamily reportedly plays pivotal roles in the development of several tissues including the vascular and nervous systems. Here, we examined enzyme-substrate relationships between the four R3 RPTP subfamily members and 21 RPTK members selected from 14 RPTK subfamilies by using a mammalian two-hybrid system with substrate-trapping RPTP mutants. Among the 84 RPTP-RPTK combinations conceivable, we detected 30 positive interactions: 25 of the enzyme-substrate relationships were novel. We randomly chose several RPTKs assumed to be substrates for R3 RPTPs, and validated the results of this screen by in vitro dephosphorylation assays, and by cell-based assays involving overexpression and knock-down experiments. Because their functional relationships were verified without exception, it is probable that the RPTKs identified as potential substrates are actually physiological substrates for the R3 RPTPs. Interestingly, some RPTKs were recognized as substrates by all R3 members, but others were recognized by only one or a few members. The enzyme-substrate relationships identified in the present study will shed light on physiological roles of the R3 RPTP subfamily.

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration.

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.

Directional neuronal migration is impaired in mice lacking adenomatous polyposis coli 2.

Adenomatous polyposis coli 2 (APC2) is a family member of APC and mainly expressed in the nervous system. We previously reported that APC2 plays a critical role in axonal projection through the regulation of microtubule stability. Here, we show that a lack of Apc2 induces severe laminary defects in some regions of the mouse brain, including the cerebral cortex and cerebellum. In vivo BrdU labeling and immunohistochemical analyses with specific markers revealed that the laminary abnormalities are a result of dysregulated neuronal migration by a cell-autonomous mechanism. Using total internal reflection fluorescent microscopy, we found that APC2 is distributed along actin fibers as well as microtubules. Cerebellar granule cells in dissociated cultures and in vivo showed that BDNF-stimulated directional migration is impaired in Apc2-deficient neurons. We revealed that this impairment stems from the dysregulations of Rho family GTPase activation and TrkB localization, which disrupts the formation of BDNF-stimulated F-actin at the leading edge. Thus, APC2 is an essential mediator of the cytoskeletal regulation at leading edges in response to extracellular signals.

APC2 plays an essential role in axonal projections through the regulation of microtubule stability.

Growth cones at the tip of growing axons are key cellular structures that detect guidance cues and mediate axonal growth. An increasing number of studies have suggested that the dynamic regulation of microtubules in the growth cone plays an essential role in growth cone steering. The dynamic properties of microtubules are considered to be regulated by variegated cellular factors but, in particular, through microtubule-interacting proteins. Here, we examined the functional role of adenomatous polyposis coli-like molecule 2 (APC2) in the development of axonal projections by using the chick retinotectal topographic projection system. APC2 is preferentially expressed in the nervous system from early developmental stages through to adulthood. Immunohistochemical analysis revealed that APC2 is distributed along microtubules in growth cones as well as axon shafts of retinal axons. Overexpression of APC2 in cultured cells induced the stabilization of microtubules, whereas the knockdown of APC2 in chick retinas with specific short hairpin RNA reduced the stability of microtubules in retinal axons. APC2 knockdown retinal axons showed abnormal growth attributable to a reduced response to ephrin-A2 in vitro. Furthermore, they showed drastic alterations in retinotectal projections without making clear target zones in the tectum in vivo. These results suggest that APC2 plays a critical role in the development of the nervous system through the regulation of microtubule stability.

Activation of maxi-anion channel by protein tyrosine dephosphorylation.

The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5'-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)zeta, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPzeta gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPzeta in mouse fibroblasts, but not in C127 cells.

Functional mode of FoxD1/CBF2 for the establishment of temporal retinal specificity in the developing chick retina.

Two winged-helix transcription factors, FoxG1 (previously called chick brain factor1, CBF1) and FoxD1 (chick brain factor2, CBF2), are expressed specifically in the nasal and temporal regions of the developing chick retina, respectively. We previously demonstrated that FoxG1 controls the expression of topographic molecules including FoxD1, and determines the regional specificity of the nasal retina. FoxD1 is known to prescribe temporal specificity, however, molecular mechanisms and downstream targets have not been elucidated. Here we addressed the genetic mechanisms for establishing temporal specificity in the developing retina using an in ovo electroporation technique. Fibroblast growth factor (Fgf) and Wnt first play pivotal roles in inducing the region-specific expression of FoxG1 and FoxD1 in the optic vesicle. Misexpression of FoxD1 represses the expression of FoxG1, GH6, SOHo1, and ephrin-A5, and induces that of EphA3 in the retina. GH6 and SOHo1 repress the expression of FoxD1. In contrast to the inhibitory effect of FoxG1 on bone morphogenic protein (BMP) signaling, FoxD1 does not alter the expression of BMP4 or BMP2. Studies with chimeric mutants of FoxD1 showed that FoxD1 acts as a transcription repressor in controlling its downstream targets in the retina. Taken together with previous findings, our data suggest that FoxG1 and FoxD1 are located at the top of the gene cascade for regional specification along the nasotemporal (anteroposterior) axis in the retina, and FoxD1 determines temporal specificity.

Identification of retinal ganglion cells and their projections involved in central transmission of information about upward and downward image motion.

The direction of image motion is coded by direction-selective (DS) ganglion cells in the retina. Particularly, the ON DS ganglion cells project their axons specifically to terminal nuclei of the accessory optic system (AOS) responsible for optokinetic reflex (OKR). We recently generated a knock-in mouse in which SPIG1 (SPARC-related protein containing immunoglobulin domains 1)-expressing cells are visualized with GFP, and found that retinal ganglion cells projecting to the medial terminal nucleus (MTN), the principal nucleus of the AOS, are comprised of SPIG1+ and SPIG1(-) ganglion cells distributed in distinct mosaic patterns in the retina. Here we examined light responses of these two subtypes of MTN-projecting cells by targeted electrophysiological recordings. SPIG1+ and SPIG1(-) ganglion cells respond preferentially to upward motion and downward motion, respectively, in the visual field. The direction selectivity of SPIG1+ ganglion cells develops normally in dark-reared mice. The MTN neurons are activated by optokinetic stimuli only of the vertical motion as shown by Fos expression analysis. Combination of genetic labeling and conventional retrograde labeling revealed that axons of SPIG1+ and SPIG1(-) ganglion cells project to the MTN via different pathways. The axon terminals of the two subtypes are organized into discrete clusters in the MTN. These results suggest that information about upward and downward image motion transmitted by distinct ON DS cells is separately processed in the MTN, if not independently. Our findings provide insights into the neural mechanisms of OKR, how information about the direction of image motion is deciphered by the AOS.

Protein tyrosine phosphatase receptor type Z dephosphorylates TrkA receptors and attenuates NGF-dependent neurite outgrowth of PC12 cells.

Protein tyrosine phosphatase receptor type Z (Ptprz/Ptpzeta/RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.

[Molecular mechanisms for the formation of topographic retinotectal projection].

Topographic maps are a fundamental feature of neural networks in the nervous system. Understanding the molecular mechanisms by which topographically ordered neuronal connections are established during development has long been a major challenge in developmental neurobiology. The retinotectal projection of lower vertebrates including birds has been used as a readily accessible model system. In this projection, the temporal (posterior) retina is connected to the rostral (anterior) part of the contralateral optic tectum, the nasal (anterior) retina to the caudal (posterior) tectum, and likewise the dorsal and ventral retina to the ventral (lateral) and dorsal (medial) tectum, respectively. Thus, images received by the retina are precisely projected onto the tectum in a reversed manner. For the formation of topographic maps, molecular gradients in origin and targets are essential. To search for topographic molecules in the embryonic retina, we performed a large-scale screening and successfully identified a variety of molecules with various asymmetrical expression patterns along both axes in the developing retina. Included were many novel molecules with unknown functions, together with known molecules. Through analyses of these molecules, we can now present gene cascades for the retinal patterning and for the establishment of topographic retinotectal projection. In addition, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that regulates Eph receptors. We show that Ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays crucial roles in the topographic projection.

Expression of SPIG1 reveals development of a retinal ganglion cell subtype projecting to the medial terminal nucleus in the mouse.

Visual information is transmitted to the brain by roughly a dozen distinct types of retinal ganglion cells (RGCs) defined by a characteristic morphology, physiology, and central projections. However, our understanding about how these parallel pathways develop is still in its infancy, because few molecular markers corresponding to individual RGC types are available. Previously, we reported a secretory protein, SPIG1 (clone name; D/Bsp120I #1), preferentially expressed in the dorsal region in the developing chick retina. Here, we generated knock-in mice to visualize SPIG1-expressing cells with green fluorescent protein. We found that the mouse retina is subdivided into two distinct domains for SPIG1 expression and SPIG1 effectively marks a unique subtype of the retinal ganglion cells during the neonatal period. SPIG1-positive RGCs in the dorsotemporal domain project to the dorsal lateral geniculate nucleus (dLGN), superior colliculus, and accessory optic system (AOS). In contrast, in the remaining region, here named the pan-ventronasal domain, SPIG1-positive cells form a regular mosaic and project exclusively to the medial terminal nucleus (MTN) of the AOS that mediates the optokinetic nystagmus as early as P1. Their dendrites costratify with ON cholinergic amacrine strata in the inner plexiform layer as early as P3. These findings suggest that these SPIG1-positive cells are the ON direction selective ganglion cells (DSGCs). Moreover, the MTN-projecting cells in the pan-ventronasal domain are apparently composed of two distinct but interdependent regular mosaics depending on the presence or absence of SPIG1, indicating that they comprise two functionally distinct subtypes of the ON DSGCs. The formation of the regular mosaic appears to be commenced at the end of the prenatal stage and completed through the peak period of the cell death at P6. SPIG1 will thus serve as a useful molecular marker for future studies on the development and function of ON DSGCs.

Role of bone morphogenic protein 2 in retinal patterning and retinotectal projection.

It has been long believed that the anteroposterior (A-P) and dorsoventral (D-V) axes in the developing retina are determined independently and also that the retinotectal projection along the two axes is controlled independently. However, we recently demonstrated that misexpression of Ventroptin, a bone morphogenic protein (BMP) antagonist, in the developing chick retina alters the retinotectal projection not only along the D-V (or mediolateral) axis but also along the A-P axis. Moreover, the dorsal-high expression of BMP4 is relieved by the dorsotemporal-high expression of BMP2 at embryonic day 5 (E5) in the retina, during which Ventroptin continuously counteracts the two BMPs keeping on the countergradient expression pattern, respectively. Here, we show that the topographic molecules so far reported to have a gradient only along the D-V axis and ephrin-A2 so far only along the A-P axis are both controlled by the BMP signal, and that they are expressed in a gradient manner along the tilted axis from E6 on in the developing chick retina: the expression patterns of these oblique-gradient molecules are all changed, when BMP2 expression is manipulated in the developing retina. Furthermore, in both BMP2 knockdown embryos and ephrin-A2-misexpressed embryos, the retinotectal projection is altered along the two orthogonal axes. The expressional switching from BMP4 to BMP2 thus appears to play a key role in the retinal patterning and topographic retinotectal projection by tilting the D-V axis toward the posterior side during retinal development. Our results also indicate that BMP2 expression is essential for the maintenance of regional specificity along the revised D-V axis.

Eph receptors are negatively controlled by protein tyrosine phosphatase receptor type O.

Eph receptors are activated by the autophosphorylation of tyrosine residues upon the binding of their ligands, the ephrins; however, the protein tyrosine phosphatases (PTPs) responsible for the negative regulation of Eph receptors have not been elucidated. Here, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that efficiently dephosphorylates both EphA and EphB receptors as substrates. Biochemical analyses revealed that Ptpro dephosphorylates a phosphotyrosine residue conserved in the juxtamembrane region, which is required for the activation and signal transmission of Eph receptors. Ptpro thus seems to moderate the amount of maximal activation of Eph receptors. Using the chick retinotectal projection system, we show that Ptpro controls the sensitivity of retinal axons to ephrins and thereby has a crucial role in the establishment of topographic projections. Our findings explain the molecular mechanism that determines the threshold of the response of Eph receptors to ephrins in vivo.