A possible function of Nik-related kinase in the labyrinth layer of delayed delivery mouse placentas.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

SOX17-positive rete testis epithelium is required for Sertoli valve formation and normal spermiogenesis in the male mouse.

Seminiferous tubules (STs) in the mammalian testes are connected to the rete testis (RT) via a Sertoli valve (SV). Spermatozoa produced in the STs are released into the tubular luminal fluid and passively transported through the SV into the RT. However, the physiological functions of the RT and SV remain unclear. Here, we identified the expression of Sox17 in RT epithelia. The SV valve was disrupted before puberty in RT-specific Sox17 conditional knockout (Sox17-cKO) male mice. This induced a backflow of RT fluid into the STs, which caused aberrant detachment of immature spermatids. RT of Sox17-cKO mice had reduced expression levels of various growth factor genes, which presumably support SV formation. When transplanted next to the Sox17+ RT, Sertoli cells of Sox17-cKO mice reconstructed the SV and supported proper spermiogenesis in the STs. This study highlights the novel and unexpected modulatory roles of the RT in SV valve formation and spermatogenesis in mouse testes, as a downstream action of Sox17.

MAB21L1 modulates gene expression and DNA metabolic processes in the lens placode.

Mutations in human MAB21L1 cause aberrations in lens ectoderm morphogenesis and lead to congenital cerebellar, ocular, craniofacial and genital (COFG) syndrome. Murine Mab21l1-null mutations cause severe cell-autonomous defects in lens formation, leading to microphthalmia; therefore, Mab21l1-null mice are used as a mouse model for COFG syndrome. In this study, we investigated the early-onset single-cell-level phenotypes of murine Mab21l1-null lens ectoderms using electron microscopy and single-cell RNA sequencing (scRNA-seq). Electron microscopy and immunohistochemical analyses indicated endoplasmic reticulum stress at the 24- to 26-somite stage in Mab21l1-null lens placodes. scRNA-seq analysis revealed that 131 genes were downregulated and 148 were upregulated in Mab21l1-null lens ectoderms relative to the wild type. We successfully identified 21 lens-specific genes that were downregulated in Mab21l1-null cells, including three key genes involved in lens formation: Pitx3, Maf and Sfrp2. Moreover, gene ontology analysis of the 279 differentially expressed genes indicated enrichment in housekeeping genes associated with DNA/nucleotide metabolism prior to cell death. These findings suggest that MAB21L1 acts as a nuclear factor that modulates not only lens-specific gene expression but also DNA/nucleotide metabolic processes during lens placode formation.

Low retinoic acid levels mediate regionalization of the Sertoli valve in the terminal segment of mouse seminiferous tubules.

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.

Anatomical and histological characteristics of the hepatobiliary system in adult Sox17 heterozygote mice.

Biliary atresia (BA) is a rare neonatal disease characterized by inflammation and obstruction of the extrahepatic bile ducts (EHBDs). The Sox17-haploinsufficient (Sox17+/- ) mouse is an animal model of BA that encompasses bile duct injury and subsequent BA-like inflammation by the neonatal stage. Most Sox17+/- neonates die soon after birth, but some Sox17+/- pups reach adulthood and have a normal life span, unlike human BA. However, the phenotype and BA-derived scars in the hepatobiliary organs of surviving Sox17+/- mice are unknown. Here, we examined the phenotypes of the hepatobiliary organs in post-weaning and young adult Sox17+/- mice. The results confirmed the significant reduction in liver weight, together with peripheral calcinosis and aberrant vasculature in the hepatic lobule, in surviving Sox17+/- mice as compared with their wild-type (WT) littermates. Such hepatic phenotypes may be sequelae of hepatobiliary damage at the fetal and neonatal stages, a notion supported by the slight, but significant, increases in the levels of serum markers of liver damage in adult Sox17+/- mice. The surviving Sox17+/- mice had a shorter gallbladder in which ectopic hepatic ducts were more frequent compared to WT mice. Also, the surviving Sox17+/- mice showed neither obstruction of the EHBDs nor atrophy or inflammation of hepatocytes or the intrahepatic ducts. These data suggest that some Sox17+/- pups with BA naturally escape lethality and recover from fetal hepatobiliary damages during the perinatal period, highlighting the usefulness of the in vivo model in understanding the hepatobiliary healing processes after surgical restoration of bile flow in human BA.

Heterozygous mutation of the splicing factor Sf3b4 affects development of the axial skeleton and forebrain in mouse.

BACKGROUND: Splicing factor 3B subunit 4 (SF3B4) is a causative gene of an acrofacial dysostosis, Nager syndrome. Although in vitro analyses of SF3B4 have proposed multiple noncanonical functions unrelated to splicing, less information is available based on in vivo studies using model animals. RESULTS: We performed expression and functional analyses of Sf3b4 in mice. The mouse Sf3b4 transcripts were found from two-cell stage, and were ubiquitously present during embryogenesis with high expression levels in several tissues such as forming craniofacial bones and brain. In contrast, expression of a pseudogene-like sequence of mouse Sf3b4 (Sf3b4_ps) found by in silico survey was not detected up to embryonic day 10. We generated a Sf3b4 knockout mouse using CRISPR-Cas9 system. The homozygous mutant mouse of Sf3b4 was embryonic lethal. The heterozygous mutant of Sf3b4 mouse (Sf3b4+/- ) exhibited smaller body size compared to the wild-type from postnatal to adult period, as well as homeotic posteriorization of the vertebral morphology and flattened calvaria. The flattened calvaria appears to be attributable to mild microcephaly due to a lower cell proliferation rate in the forebrain. CONCLUSIONS: Our study suggests that Sf3b4 controls anterior-posterior patterning of the axial skeleton and guarantees cell proliferation for forebrain development in mice.

Early Crypt Formation Defects in the Uterine Epithelia of Sox17 Heterozygous Mice.

SOX17 activity in the uterine epithelium is essential for the implantation of mouse embryos. Previously, we demonstrated that female Sox17 heterozygous mutant mice are subfertile, and 2 active copies of Sox17 are required for the proper implantation of mouse embryos. To understand which implantation step is most sensitive to the Sox17 gene dosage, we comprehensively investigated the phenotypes and RNA transcriptomes of Sox17 heterozygous mutant mice. Uterine Sox17 expression drastically changed according to estrous cycle and during early pregnancy. The highest Sox17 expression was observed during the receptive period for blastocyst implantation. Sox17 heterozygous uterine epithelia showed ectopic high-level expression of SOX9, another SOX factor that is normally expressed in the uterine gland. Three-dimensional analysis of the uterus on day 5 of pregnancy revealed no crypt formation near the healthy blastocysts in the Sox17 heterozygous uterine epithelium, suggesting that early defects in embryo homing had occurred. Global transcriptional analysis revealed that the expression of Amphiregulin (Areg), a gene encoding a heparin-binding epidermal growth factor receptor ligand, was decreased drastically in Sox17+/- uterine epithelia. These data imply that full Sox17 activity is required to promote early crypt formation through proper regulation of SOX9 and AREG expression at the implantation site.

Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice.

In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7-10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.

Sox17 is essential for proper formation of the marginal zone of extraembryonic endoderm adjacent to a developing mouse placental disk.

In mouse conceptus, two yolk-sac membranes, the parietal endoderm (PE) and visceral endoderm (VE), are involved in protecting and nourishing early-somite-stage embryos prior to the establishment of placental circulation. Both PE and VE membranes are tightly anchored to the marginal edge of the developing placental disk, in which the extraembryonic endoderm (marginal zone endoderm: ME) shows the typical flat epithelial morphology intermediate between those of PE and VE in vivo. However, the molecular characteristics and functions of the ME in mouse placentation remain unclear. Here, we show that SOX17, not SOX7, is continuously expressed in the ME cells, whereas both SOX17 and SOX7 are coexpressed in PE cells, by at least 10.5 days postconception. The Sox17-null conceptus, but not the Sox7-null one, showed the ectopic appearance of squamous VE-like epithelial cells in the presumptive ME region, together with reduced cell density and aberrant morphology of PE cells. Such aberrant ME formation in the Sox17-null extraembryonic endoderm was not rescued by the chimeric embryo replaced with the wild-type gut endoderm by the injection of wild-type ES cells into the Sox17-null blastocyst, suggesting the cell autonomous defects in the extraembryonic endoderm of Sox17-null concepti. These findings provide direct evidence of the crucial roles of SOX17 in proper formation and maintenance of the ME region, highlighting a novel entry point to understand the in vivo VE-to-PE transition in the marginal edge of developing placenta.

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia.

The gallbladder excretes cytotoxic bile acids into the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos, but the molecular and cellular mechanisms underlying this remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in Sox17+/- embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severity of cholecystitis in individual Sox17+/- embryos. Embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia but not in fetal liver hepatoblasts. The Sox17+/- gallbladder also showed a drastic reduction in sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/- embryos, suggesting a potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia.

Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm.

The first binary cell fate decision occurs at the morula stage and gives rise to two distinct types of cells that constitute the trophectoderm (TE) and inner cell mass (ICM). The cell fate determinant, Cdx2, is induced in TE cells and plays an essential role in their differentiation and maintenance. Notch and Hippo signaling cascades are assumed to converge onto regulatory elements of Cdx2, however, the underlying molecular mechanisms are largely unknown. Here, we show involvement of Strawberry Notch1 (Sbno1), a novel chromatin factor of the helicase superfamily 2, during preimplantation development. Sbno1 knockout embryos die at the preimplantation stage without forming a blastocoel, and Cdx2 is not turned on even though both Yap and Tead4 reside normally in nuclei. Accordingly, Sbno1 acts on the trophectoderm-enhancer (TEE) of Cdx2, ensuring its robust and synergistic activation by the Yap/Tead4 and NICD/Rbpj complexes. Interestingly, this synergism is enhanced when cells are mechanically stretched, which might reflect that TE cells are continuously stretched by the expanding ICM and blastocoel cavity. In addition, the histone chaperone, FACT (FAcilitates Chromatin Transcription) physically interacts with Sbno1. Our data provide new evidence on TE specification, highlighting unexpected but essential functions of the highly conserved chromatin factor, Sbno1.

Mouse Sox17 haploinsufficiency leads to female subfertility due to impaired implantation.

Embryonic implantation comprises a dynamic and complicated series of events, which takes place only when the maternal uterine endometrium is in a receptive state. Blastocysts reaching the uterus communicate with the uterine endometrium to implant within a narrow time window. Interplay among various signalling molecules and transcription factors under the control of ovarian hormones is necessary for successful establishment of pregnancy. However, the molecular mechanisms that allow embryonic implantation in the receptive endometrium are still largely unknown. Here, we show that Sry-related HMG box gene-17 (Sox17) heterozygous mutant female mice exhibit subfertility due to implantation failure. Sox17 was expressed in the oviduct, uterine luminal epithelium, and blood vessels. Sox17 heterozygosity caused no appreciable defects in ovulation, fertilisation, blastocyst formation, and gross morphology of the oviduct and uterus. Another group F Sox transcription factor, Sox7, was also expressed in the uterine luminal and glandular epithelium relatively weakly. Despite uterine Sox7 expression, a significant reduction in the number of implantation sites was observed in Sox17 heterozygous mutant females due to haploinsufficiency. Our findings revealed a novel role of Sox17 in uterine receptivity to embryo implantation.

Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos.

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.

HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.

The role of angiomotin phosphorylation in the Hippo pathway during preimplantation mouse development.

The Hippo signaling pathway regulates a number of cellular events, including the control of cell fates in preimplantation mouse embryos. The inner and outer cells of the embryo show high and low levels of Hippo signaling, respectively. This position-dependent Hippo signaling promotes the specification of distinct cell fates. In a recent paper, we identified the molecular mechanism that controls Hippo signaling in preimplantation embryos. The junction-associated scaffold protein Angiomotin (Amot) plays a key role in this mechanism. At the adherens junctions of the inner cells, Amot activates the Hippo pathway by recruiting and activating the protein kinase large tumor suppressor (Lats). In contrast, Amot at the apical membrane of the outer cells suppresses Hippo signaling by interacting with F-actin. The phosphorylation of Amot inhibits its interaction with F-actin and activates Hippo signaling. We propose that Amot acts as a molecular switch for the Hippo pathway and links F-actin with Lats activity.

Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos.

BACKGROUND: In preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown. RESULTS: We show that a combination of cell polarity and cell-cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and angiomotin-like 2 (Amotl2) are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs), and cell-cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway. CONCLUSIONS: We propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell-cell adhesion by sequestering Amot from AJs. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates.

Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis.

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left-right asymmetric body plan in zebrafish, is generated from a cluster of ~20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left-right asymmetric body plan.

The Hsp90 capacitor, developmental remodeling, and evolution: the robustness of gene networks and the curious evolvability of metamorphosis.

Genetic capacitors moderate expression of heritable variation and provide a novel mechanism for rapid evolution. The prototypic genetic capacitor, Hsp90, interfaces stress responses, developmental networks, trait thresholds and expression of wide-ranging morphological changes in Drosophila and other organisms. The Hsp90 capacitor hypothesis, that stress-sensitive storage and release of genetic variation through Hsp90 facilitates adaptive evolution in unpredictable environments, has been challenged by the belief that Hsp90-buffered variation is unconditionally deleterious. Here we review recent results supporting the Hsp90 capacitor hypothesis, highlighting the heritability, selectability, and potential evolvability of Hsp90-buffered traits. Despite a surprising bias toward morphological novelty and typically invariable quantitative traits, Hsp90-buffered changes are remarkably modular, and can be selected to high frequency independent of the expected negative side-effects or obvious correlated changes in other, unselected traits. Recent dissection of cryptic signal transduction variation involved in one Hsp90-buffered trait reveals potentially dozens of normally silent polymorphisms embedded in cell cycle, differentiation and growth control networks. Reduced function of Hsp90 substrates during environmental stress would destabilize robust developmental processes, relieve developmental constraints and plausibly enables genetic network remodeling by abundant cryptic alleles. We speculate that morphological transitions controlled by Hsp90 may fuel the incredible evolutionary lability of metazoan life-cycles.

Canopy1, a novel regulator of FGF signaling around the midbrain-hindbrain boundary in zebrafish.

FGF signaling from the midbrain-hindbrain boundary (MHB, isthmus) plays a major role both in maintenance of the MHB and induction of the tectum and cerebellum. Since different levels of FGF signaling in the MHB result in a qualitative difference in inducing activity, FGF signaling in the MHB should be tightly regulated positively and negatively at multiple steps to ensure correct levels of FGF signaling. Factors that negatively regulate FGF signal around the MHB are reported. However, factors that ensure strong FGF signal in the MHB are largely unknown. Here we report the identification of Canopy1 (Cnpy1), a novel MHB-specific, Saposin-related protein that belongs to an evolutionarily conserved protein family. The cnpy1 gene was expressed specifically in the MHB of zebrafish embryos. Exogenous FGF8 induced expression of cnpy1 in the tectal primordial. Knockdown of cnpy1 resulted in MHB defects and impaired FGF signaling in a cell-autonomous manner. Cnpy1 is localized in the endoplasmic reticulum and interacts with FGFR1. This study highlights a positive-feedback loop between the FGFR pathway and Cnpy1 that may ensure the strength of FGF signaling in the MHB, leading to correct development of the tectum and cerebellum.