Factors of Anti-Vascular Endothelial Growth Factor Therapy Withdrawal in Patients with Neovascular Age-Related Macular Degeneration: Implications for Improving Patient Adherence.

We investigated the factors associated with the discontinuation of anti-vascular endothelial growth factor (VEGF) therapies in patients with neovascular age-related macular degeneration (AMD). Japanese patients with AMD aged ≥50 years, reporting at least one prior injection of an anti-VEGF drug, completed an online survey covering reasons for discontinuation or dissatisfaction with therapy, quality of life (EQ-5D-5L) and patient activation (PAM-13). The respondents were divided into two cohorts: Cohort 1-patients who discontinued anti-VEGF therapy (n = 207); Cohort 2-patients continuing anti-VEGF therapy (n = 65). The most common reason for discontinuing therapy was the "doctor's decision" in 89.4% (Cohort 1-1). In the other 22 (10.6%) patients in Cohort 1 (Cohort 1-2), reasons included "no deterioration in vision", "financial burden" and "ineffective treatment". Patients in Cohort 2 were dissatisfied with "long waiting times" (77%), "financial burden" and "ineffective treatment". Pain/discomfort posed the greatest impact on quality of life. Only 5% of patients in Cohorts 1-1 and 2 and none in Cohort 1-2 were considered advocates for their own health. In conclusion, most patients who discontinued anti-VEGF therapy did so at their doctor's decision. Addressing the reasons associated with discontinuation or dissatisfaction with anti-VEGF therapies might help improve their continuation.

FGF8 acts as a classic diffusible morphogen to pattern the neocortex.

Gain- and loss-of-function experiments have demonstrated that a source of fibroblast growth factor (FGF) 8 regulates anterior to posterior (A/P) patterning in the neocortical area map. Whether FGF8 controls patterning as a classic diffusible morphogen has not been directly tested. We report evidence that FGF8 diffuses through the mouse neocortical primordium from a discrete source in the anterior telencephalon, forms a protein gradient across the entire A/P extent of the primordium, and acts directly at a distance from its source to determine area identity. FGF8 immunofluorescence revealed FGF8 protein distributed in an A/P gradient. Fate-mapping experiments showed that outside the most anterior telencephalon, neocortical progenitor cells did not express Fgf8, nor were they derived from Fgf8-expressing cells, suggesting that graded distribution of FGF8 results from protein diffusion from the anterior source. Supporting this conclusion, a dominant-negative high-affinity FGF8 receptor captured endogenous FGF8 at a distance from the FGF8 source. New FGF8 sources introduced by electroporation showed haloes of FGF8 immunofluorescence indicative of FGF8 diffusion, and surrounding cells reacted to a new source of FGF8 by upregulating different FGF8-responsive genes in concentric domains around the source. Reducing endogenous FGF8 with the dominant-negative receptor in the central neocortical primordium induced cells to adopt a more posterior area identity, demonstrating long-range area patterning by FGF8. These observations support FGF8 as a classic diffusible morphogen in neocortex, thereby guiding future studies of neocortical pattern formation.

Identification of protogenin, a novel immunoglobulin superfamily gene expressed during early chick embryogenesis.

We have identified a chick cDNA encoding the novel membrane protein, Protogenin. It has structural features of four immunoglobulin domains and five fibronectin type III domains and is closely related to the DCC/neogenin subclass of the Ig superfamily. protogenin gene is conserved among human, mouse and chick genome, and mapped on the same chromosome as other related members of the gene family such as neogenin, punc and nope. Expression of protogenin mRNA is most prominent in E1-E3 chick embryos and subsequently its expression level declines. The transcripts are found in early mesodermal cells, the neuroepithelial cell layer of the brain and the trunk neural tube, the neural layer of the retina and in the epithelial somite and dermomyotome.

Pigment cell lineage-specific expression activity of the ascidian tyrosinase-related gene.

Solitary ascidian tadpole larvae develop two types of black pigment cells in the major sensory organs of the brain. Such pigment cells have been demonstrated to express the melanogenic genes, tyrosinase and Tyrp/TRP (tyrosinase-related protein). To understand the genetic and developmental mechanisms underlying the differentiation of chordate pigment cells, we examined the function of the promoter region of Tyrp/TRP gene, an ascidian (Halocynthia roretzi) tyrosinase family gene. The expression of the gene in pigment cell lineage starts at the early-mid gastrula stages. To identify the transcriptional regulatory region of the gene allowing cell-type-specific expression, a deletion series of the HrTyrp 5' flanking region fused to a lacZ reporter gene was constructed and microinjected into ascidian fertilized eggs. The region of 73 bp in HrTyrp was identified as sufficient for expression in pigment cell-precursors of tailbud stage embryos. It is noteworthy that there is no M-box element highly conserved in the promoters for vertebrate tyrosinase family genes such as tyrosinase, Tyrp1/TRP-1 and Tyrp2/TRP-2 (Dct). Although the regulatory system of ascidian pigment-cell development is likely to contain most factors critical to vertebrate pigment-cell development, there might be critical differences in the mode of regulation, such as the developmental timing of interactions of factors, proteins and genes, involved in pigment cell differentiation and pigmentation.

Navigation of trochlear motor axons along the midbrain-hindbrain boundary by neuropilin 2.

Trochlear motor axons project dorsally along the midbrain-hindbrain boundary (MHB) to decussate at the dorsal midline. We report on the roles of neuropilin 2 and its ligands in the molecular mechanisms controlling this trajectory. In chick embryos, neuropilin 2 was expressed in the neuroepithelium of the dorsal isthmus in addition to the trochlear neurons, and Sema3F transcripts were localized along the caudal margin of the midbrain. Misexpression of Sema3F demonstrated that Sema3F displays repulsive activity in vivo that guides the trochlear motor axons along the MHB. An unexpected result was that misexpression of neuropilin 2 canceled the midbrain-evoked repulsion, allowing trochlear motor axons to cross the MHB and invade the tectum. A binding assay with neuropilin 2 ectodomain revealed the existence of neuropilin 2 ligands in the midbrain, which were masked by ectopic neuropilin 2. We therefore propose that neuropilin 2 neutralizes the repulsive activity in order to steer trochlear motor axons towards the dorsal decussation point. Taken together, our results suggest that the interaction of neuropilin 2 with its ligands has crucial roles for establishing trochlear trajectory along the MHB.

Cloning and functional analysis of ascidian Mitf in vivo: insights into the origin of vertebrate pigment cells.

The microphthalmia-associated transcription factor (Mitf) is a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor essential for the development and function of all melanin-producing pigment cells in vertebrates. To elucidate the evolutionary history of Mitf and the antiquity of its association with pigment cells, we have isolated and characterized HrMitf, a sole member of the Mitf-TFE bHLH-ZIP subfamily in the ascidian Halocynthia roretzi. Maternal HrMitf mRNA is detected in the fertilized egg and in the animal hemisphere from 4-cell stage through the gastrula stage. From the neurula through the early tailbud stage, HrMitf is preferentially expressed in the pigment-lineage cells that express the lineage-specific melanogenesis genes tyrosinase (HrTyr) and Tyrp. Overexpression of HrMitf induced ectopic expression of HrTyr enzyme activity in mesenchymal cells where the same enzyme activity was induced by overexpression of HrPax3/7, suggesting that a part(s) of the Pax3-Mitf-tyrosinase gene regulatory cascade seen in vertebrate melanocytes is operative during ascidian embryogenesis. We also show HrMitf and mouse Mitf-A, a Mitf isoform abundantly expressed in pigmented epithelial cells, share similar functional characteristics. These results suggest antiquity of the association of the Mitf-TFE subfamily with pigment cells and may support the idea that acquisition of multiple promoters (isoforms) by an ancestral Mitf gene has allowed the evolution of multiple pigment cell types.

Ascidian otx gene Hroth activates transcription of the brain-specific gene HrTRP.

The brain (sensory vesicle) of the ascidian larvae is thought to be homologous to the vertebrate forebrain and midbrain and, thus, is proposed as a simplified model to investigate mechanisms of brain formation in vertebrates. However, the genetic circuitry that governs formation of the sensory vesicle is largely unknown. To address this issue, we investigated the transcriptional regulation of the sensory vesicle-specific gene HrTRP by Hroth, the otx gene of the ascidian Halocynthia roretzi. A 133-bp 5'-flanking region of HrTRP, identified as a promoter that can drive expression of the reporter gene in the sensory vesicle, contains two otx binding consensus sites. When the two otx sites were deleted or mutated, the promoter activity of this region was decreased. Hroth overexpression can transactivate this promoter in an otx site-dependent manner. Transactivation of HrTRP promoter by Hroth overexpression was mimicked by overexpression of Hroth/VP16, which encodes a fusion protein of Hroth and the activator domain of VP16, and is suppressed by coexpression with Hroth/En, which encodes a fusion protein of Hroth and the Engrailed repressor domain. Finally, translational interference of Hroth by a morpholino oligonucleotide resulted in the reduction of HrTRP expression in the ascidian embryos. These results suggest that Hroth acts as a direct activator of HrTRP transcription during sensory vesicle development.