Real-time monitoring of an endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation.

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential.

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Cell-fate plasticity, adhesion and cell sorting complementarily establish a sharp midbrain-hindbrain boundary.

The formation and maintenance of sharp boundaries between groups of cells play a vital role during embryonic development as they serve to compartmentalize cells with similar fates. Some of these boundaries also act as organizers, with the ability to induce specific cell fates and morphogenesis in the surrounding cells. The midbrain-hindbrain boundary (MHB) is such an organizer: it acts as a lineage restriction boundary to prevent the intermingling of cells with different developmental fates. However, the mechanisms underlying the lineage restriction process remain unclear. Here, using novel fluorescent knock-in reporters, live imaging, Cre/lox-mediated lineage tracing, atomic force microscopy-based cell adhesion assays and mutant analysis, we analyze the process of lineage restriction at the MHB and provide mechanistic details. Specifically, we show that lineage restriction occurs by the end of gastrulation, and that the subsequent formation of sharp gene expression boundaries in the developing MHB occur through complementary mechanisms, i.e. cell-fate plasticity and cell sorting. Furthermore, we show that cell sorting at the MHB involves differential adhesion among midbrain and hindbrain cells that is mediated by N-cadherin and Eph-ephrin signaling.

Single cell sequencing of radial glia progeny reveals the diversity of newborn neurons in the adult zebrafish brain.

Zebrafish display widespread and pronounced adult neurogenesis, which is fundamental for their regeneration capability after central nervous system injury. However, the cellular identity and the biological properties of adult newborn neurons are elusive for most brain areas. Here, we have used short-term lineage tracing of radial glia progeny to prospectively isolate newborn neurons from the her4.1+ radial glia lineage in the homeostatic adult forebrain. Transcriptome analysis of radial glia, newborn neurons and mature neurons using single cell sequencing identified distinct transcriptional profiles, including novel markers for each population. Specifically, we detected two separate newborn neuron types, which showed diversity of cell fate commitment and location. Further analyses showed that these cell types are homologous to neurogenic cells in the mammalian brain, identified neurogenic commitment in proliferating radial glia and indicated that glutamatergic projection neurons are generated in the adult zebrafish telencephalon. Thus, we prospectively isolated adult newborn neurons from the adult zebrafish forebrain, identified markers for newborn and mature neurons in the adult brain, and revealed intrinsic heterogeneity among adult newborn neurons and their homology with mammalian adult neurogenic cell types.

Isthmin1, a secreted signaling protein, acts downstream of diverse embryonic patterning centers in development.

Extracellular signals play essential roles during embryonic patterning by providing positional information in a concentration-dependent manner, and many such signals, like Wnt, fibroblast growth factor (FGF), Hedgehog (Hh), and retinoic acid, act by being secreted into the extracellular space, thereby triggering receptor-mediated responses in other cells. Isthmin1 (ism1) is a secreted protein whose gene expression pattern coincides with that of early dorsal determinants, nodal ligand genes like sqt and cyc, and with fgf8 during various phases of zebrafish development. Ism1 functions in early embryonic patterning and development are poorly understood; however, it has recently been shown to interact with nodal pathway genes to control organ asymmetry in chicken. Here, we show that misexpression of ism1 deletion constructs disrupts embryonic patterning in zebrafish and exhibits genetic interactions with both Fgf and nodal signaling. Unlike Fgf and nodal pathway mutants, CRISPR/Cas9-engineered ism1 mutants did not show obvious developmental defects. Further, in vivo single molecule fluorescence correlation spectroscopy (FCCS) showed that Ism1 diffuses freely in the extra-cellular space, with a diffusion coefficient similar to that of Fgf8a; however, our measurements do not support direct molecular interactions between Ism1 and either nodal ligands or Fgf8a in the developing zebrafish embryo. Together, data from gain- and loss-of-function experiments suggest that zebrafish Ism1 plays a complex role in regulating extracellular signals during early embryonic development.

Asymmetric inheritance of the apical domain and self-renewal of retinal ganglion cell progenitors depend on Anillin function.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.

Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology.

Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx-based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene-trap mutagenesis. The SAGFLEx gene-trap cassette comprises the rabbit ß-globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site-specific recombinases Cre and Flp. Insertion of the gene-trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene-trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial- and temporal-controlled manner.

Single-cell RNA sequencing unravels the transcriptional network underlying zebrafish retina regeneration.

In the lesioned zebrafish retina, Müller glia produce multipotent retinal progenitors that generate all retinal neurons, replacing lost cell types. To study the molecular mechanisms linking Müller glia reactivity to progenitor production and neuronal differentiation, we used single-cell RNA sequencing of Müller glia, progenitors and regenerated progeny from uninjured and light-lesioned retinae. We discover an injury-induced Müller glia differentiation trajectory that leads into a cell population with a hybrid identity expressing marker genes of Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. We further observe that neurogenic progenitors progressively differentiate to generate retinal ganglion cells first and bipolar cells last, similar to the events observed during retinal development. Our work provides a comprehensive description of Müller glia and progenitor transcriptional changes and fate decisions in the regenerating retina, which are key to tailor cell differentiation and replacement therapies for retinal dystrophies in humans.

Transcriptome analysis reveals an Atoh1b-dependent gene set downstream of Dlx3b/4b during early inner ear development in zebrafish.

The vertebrate inner ear is the sensory organ mediating hearing and balance. The entire organ develops from the otic placode, which itself originates from the otic-epibranchial progenitor domain (OEPD). Multiple studies in various species have shown the importance of the forkhead-box and distal-less homeodomain transcription factor families for OEPD and subsequent otic placode formation. However, the transcriptional networks downstream of these factors are only beginning to be understood. Using transcriptome analysis, we here reveal numerous genes regulated by the distal-less homeodomain transcription factors Dlx3b and Dlx4b (Dlx3b/4b). We identify known and novel transcripts displaying widespread OEPD expression in a Dlx3b/4b-dependent manner. Some genes, with a known OEPD expression in other vertebrate species, might be members of a presumptive vertebrate core module required for proper otic development. Moreover, we identify genes controlling early-born sensory hair cell formation as well as regulating biomineral tissue development, both consistent with defective sensory hair cell and otolith formation observed in dlx3b/4b mutants. Finally, we show that ectopic Atoh1b expression can rescue early sensorigenesis even in the absence of Dlx3b/4b. Taken together, our data will help to unravel the gene regulatory network underlying early inner ear development and provide insights into the molecular control of vertebrate inner ear formation to restore hearing loss in humans ultimately.

Dynamic coupling of pattern formation and morphogenesis in the developing vertebrate retina.

During embryonic development, pattern formation must be tightly synchronized with tissue morphogenesis to coordinate the establishment of the spatial identities of cells with their movements. In the vertebrate retina, patterning along the dorsal-ventral and nasal-temporal (anterior-posterior) axes is required for correct spatial representation in the retinotectal map. However, it is unknown how specification of axial cell positions in the retina occurs during the complex process of early eye morphogenesis. Studying zebrafish embryos, we show that morphogenetic tissue rearrangements during eye evagination result in progenitor cells in the nasal half of the retina primordium being brought into proximity to the sources of three fibroblast growth factors, Fgf8/3/24, outside the eye. Triple-mutant analysis shows that this combined Fgf signal fully controls nasal retina identity by regulating the nasal transcription factor Foxg1. Surprisingly, nasal-temporal axis specification occurs very early along the dorsal-ventral axis of the evaginating eye. By in vivo imaging GFP-tagged retinal progenitor cells, we find that subsequent eye morphogenesis requires gradual tissue compaction in the nasal half and directed cell movements into the temporal half of the retina. Balancing these processes drives the progressive alignment of the nasal-temporal retina axis with the anterior-posterior body axis and is controlled by a feed-forward effect of Fgf signaling on Foxg1-mediated cell cohesion. Thus, the mechanistic coupling and dynamic synchronization of tissue patterning with morphogenetic cell behavior through Fgf signaling leads to the graded allocation of cell positional identity in the eye, underlying retinotectal map formation.

Generation of a non-leaky heat shock-inducible Cre line for conditional Cre/lox strategies in zebrafish.

Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of the mammalian genome. Recently, we showed that Cre is also highly efficient in zebrafish and temporal control of recombination can be achieved by using the ligand-inducible CreER(T2). Previous attempts have been made to control recombination by using the temperature inducible hsp70l promoter to conditionally drive the expression of Cre or EGFP-Cre, respectively. However, in this study we demonstrate that the hsp70l promoter possesses a basal leakiness resulting in Cre-mediated recombination even at permissive temperatures. In order to prevent non-conditional recombination, we combined the hsp70l promoter with a mCherry-tagged ligand-inducible CreER(T2). At permissive temperatures and in the absence of the ligand tamoxifen (TAM), no non-conditional recombination is observed indicating tight regulation of CreER(T2). Instead, comprehensive site-specific recombination is mediated following heat induction and administration of TAM.

Reactivation of the Neurogenic Niche in the Adult Zebrafish Statoacoustic Ganglion Following a Mechanical Lesion.

Sensorineural hearing loss is caused by the loss of sensory hair cells and/or their innervating neurons within the inner ear and affects millions of people worldwide. In mammals, including humans, the underlying cell types are only produced during fetal stages making loss of these cells and the resulting consequences irreversible. In contrast, zebrafish produce sensory hair cells throughout life and additionally possess the remarkable capacity to regenerate them upon lesion. Recently, we showed that also inner ear neurogenesis continues to take place in the zebrafish statoacoustic ganglion (SAG) well into adulthood. The neurogenic niche displays presumptive stem cells, proliferating Neurod-positive progenitors and a high level of neurogenesis at juvenile stages. It turns dormant at adult stages with only a few proliferating presumptive stem cells, no proliferating Neurod-positive progenitors, and very low levels of newborn neurons. Whether the neurogenic niche can be reactivated and whether SAG neurons can regenerate upon damage is unknown. To study the regenerative capacity of the SAG, we established a lesion paradigm using injections into the otic capsule of the right ear. Upon lesion, the number of apoptotic cells increased, and immune cells infiltrated the SAG of the lesioned side. Importantly, the Neurod-positive progenitor cells re-entered the cell cycle displaying a peak in proliferation at 8 days post lesion before they returned to homeostatic levels at 57 days post lesion. In parallel to reactive proliferation, we observed increased neurogenesis from the Neurod-positive progenitor pool. Reactive neurogenesis started at around 4 days post lesion peaking at 8 days post lesion before the neurogenesis rate decreased again to low homeostatic levels at 57 days post lesion. Additionally, administration of the thymidine analog BrdU and, thereby, labeling proliferating cells and their progeny revealed the generation of new sensory neurons within 19 days post lesion. Taken together, we show that the neurogenic niche of the adult zebrafish SAG can indeed be reactivated to re-enter the cell cycle and to increase neurogenesis upon lesion. Studying the underlying genes and pathways in zebrafish will allow comparative studies with mammalian species and might provide valuable insights into developing cures for auditory and vestibular neuropathies.

Vision-related convergent gene losses reveal SERPINE3's unknown role in the eye.

Despite decades of research, knowledge about the genes that are important for development and function of the mammalian eye and are involved in human eye disorders remains incomplete. During mammalian evolution, mammals that naturally exhibit poor vision or regressive eye phenotypes have independently lost many eye-related genes. This provides an opportunity to predict novel eye-related genes based on specific evolutionary gene loss signatures. Building on these observations, we performed a genome-wide screen across 49 mammals for functionally uncharacterized genes that are preferentially lost in species exhibiting lower visual acuity values. The screen uncovered several genes, including SERPINE3, a putative serine proteinase inhibitor. A detailed investigation of 381 additional mammals revealed that SERPINE3 is independently lost in 18 lineages that typically do not primarily rely on vision, predicting a vision-related function for this gene. To test this, we show that SERPINE3 has the highest expression in eyes of zebrafish and mouse. In the zebrafish retina, serpine3 is expressed in Müller glia cells, a cell type essential for survival and maintenance of the retina. A CRISPR-mediated knockout of serpine3 in zebrafish resulted in alterations in eye shape and defects in retinal layering. Furthermore, two human polymorphisms that are in linkage with SERPINE3 are associated with eye-related traits. Together, these results suggest that SERPINE3 has a role in vertebrate eyes. More generally, by integrating comparative genomics with experiments in model organisms, we show that screens for specific phenotype-associated gene signatures can predict functions of uncharacterized genes.

CRISPR/Cas9-Based Split Fluorescent Protein Tagging.

Genetically encoded fluorescent tags such as green fluorescent protein fused to protein have revolutionized cell biology as they permit high-resolution protein imaging in live systems. Split fluorescent proteins, with a small fragment of 16 amino acids, can be inserted in the coding sequence to label proteins. We demonstrate successful integration of two bright and fast maturing split fluorescent proteins, mNeon green and sfCherry2, in zebrafish, and show that they are suitable for live imaging, including time-lapse series, and that they have a high signal-to-noise ratio. Furthermore, we show that CRISPR/Cas9 can be used to generate fluorescently tagged proteins in vivo.

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development.

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.

Subdivisions of the adult zebrafish pallium based on molecular marker analysis.

BACKGROUND: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species. RESULTS: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish ( Danio rerio) pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain. CONCLUSIONS: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish system as a model for neurobiological research and human neurodegenerative diseases.

Subdivisions of the adult zebrafish subpallium by molecular marker analysis.

The morphology of the telencephalon displays great diversity among different vertebrate lineages. Particularly the everted telencephalon of ray-finned fishes shows a noticeably different morphology from the evaginated telencephalon of nonray-finned fishes and other vertebrates. This makes the comparison between the different parts of the telencephalon of ray-finned fishes and other vertebrates difficult. Based on neuroanatomical, neurochemical, and connectional data no consensus on the subdivisions of the adult telencephalon of ray-finned fishes and their relation to nuclei in the telencephalon of other vertebrates has been reached yet. For tetrapods, comparative expression pattern analysis of homologous developmental genes has been a successful approach to clarify homologies between different parts of the telencephalon. In the larval zebrafish, subdivisions of the subpallium have been proposed using conserved developmental gene expression. In this study, we investigate the subdivisions of the adult zebrafish telencephalon by analyzing the expression pattern of conserved molecular marker genes. We identify the boundary between the pallium and subpallium based on the complementary expression of dlx2a, dlx5a in the subpallium and tbr1, neurod in the pallium. Furthermore, combinatorial expression of Isl, nkx2.1b, lhx1b, tbr1, eomesa, emx1, emx2, and emx3 identifies striatal-like, pallidal-like, and septal-like subdivisions within the subpallium. In contrast to previous models, we propose that the striatum and pallidum are stretched along the rostrocaudal axis of the telencephalon. Further, the septal nuclei derive from both the pallium and subpallium. On this basis, we present a new model for the subdivisions of the subpallium in teleost fish.

The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain.

BACKGROUND: Unlike mammals, zebrafish exhibits extensive neural regeneration after injury in adult stages of its lifetime due to the neurogenic activity of the radial glial cells. However, the genes involved in the regenerative neurogenesis response of the zebrafish brain are largely unknown. Thus, understanding the underlying principles of this regeneration capacity of the zebrafish brain is an interesting research realm that may offer vast clinical ramifications. RESULTS: In this paper, we characterized the expression pattern of cxcr5 and analyzed the function of this gene during adult neurogenesis and regeneration of the zebrafish telencephalon. We found that cxcr5 was upregulated transiently in the RGCs and neurons, and the expression in the immune cells such as leukocytes was negligible during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in altered proliferation and neurogenesis response of the RGCs. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection, we observed outcomes similar to the overexpression of the dominant negative cxcr5 variant. CONCLUSIONS: Thus, based on our results, we propose that cxcr5 imposes a proliferative permissiveness to the radial glial cells and is required for differentiation of the RGCs to neurons, highlighting novel roles of cxcr5 in the nervous system of vertebrates. We therefore suggest that cxcr5 is an important cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.

Fgf signaling is required for photoreceptor maintenance in the adult zebrafish retina.

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.

Interpretation of the FGF8 morphogen gradient is regulated by endocytic trafficking.

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.