The FGF-AKT pathway is necessary for cardiomyocyte survival for heart regeneration in zebrafish.

Zebrafish have a remarkable ability to regenerate the myocardium after injury by proliferation of pre-existing cardiomyocytes. Fibroblast growth factor (FGF) signaling is known to play a critical role in zebrafish heart regeneration through promotion of neovascularization of the regenerating myocardium. Here, we define an additional function of FGF signaling in the zebrafish myocardium after injury. We find that FGF signaling is active in a small fraction of cardiomyocytes before injury, and that the number of FGF signaling-positive cardiomyocytes increases after amputation-induced injury. We show that ERK phosphorylation is prominent in endothelial cells, but not in cardiomyocytes. In contrast, basal levels of phospho-AKT positive cardiomyocytes are detected before injury, and the ratio of phosphorylated AKT-positive cardiomyocytes increases after injury, indicating a role of AKT signaling in cardiomyocytes following injury. Inhibition of FGF signaling reduced the number of phosphorylated AKT-positive cardiomyocytes and increased cardiomyocyte death without injury. Heart injury did not induce cardiomyocyte death; however, heart injury in combination with inhibition of FGF signaling caused significant increase in cardiomyocyte death. Pharmacological inhibition of AKT signaling after heart injury also caused increased cardiomyocyte death. Our data support the idea that FGF-AKT signaling-dependent cardiomyocyte survival is necessary for subsequent heart regeneration.

A nontoxic fungal natural product modulates fin regeneration in zebrafish larvae upstream of FGF-WNT developmental signaling.

BACKGROUND: The regeneration of larvae zebrafish fin emerged as a new model of regeneration in the last decade. In contrast to genetic tools to study fin regeneration, chemical probes to modulate and interrogate regeneration processes are not well developed. RESULTS: We set up a zebrafish larvae fin regeneration assay system and tested activities of natural product compounds and extracts, prepared from various microbes. Colomitide C, a recently isolated product from a fungus obtained from Antarctica, inhibited larvae fin regeneration. Using fluorescent reporter transgenic lines, we show that colomitide C inhibited fibroblast growth factor (FGF) signaling and WNT/ß-catenin signaling, which were activated after larvae fin amputation. By using the endothelial cell reporter line and immunofluorescence, we showed that colomitide C did not affect migration of the blood vessel and nerve into the injured larvae fin. Colomitide C did not show any cytotoxic activities when tested against FGF receptor-amplified human cancer cell lines. CONCLUSION: Colomitide C, a natural product, modulated larvae fin regeneration likely acting upstream of FGF and WNT signaling. Colomitide C may serve as a template for developing new chemical probes to study regeneration and other biological processes.

A Rare Case of Recurrent Pituitary Collision Tumors.

Pituitary collision tumors are sporadically reported and rare. We present a case of pituitary collision tumors with nonfunctioning pituitary adenoma (NFPA) and craniopharyngioma. In order to look for any common activated pathway, we examined WNT/ß-CATENIN signaling activation, known to be involved in tumorigenesis in both craniopharyngioma and NFPA. We found nuclear accumulation of ß-CATENIN protein and expression of LEF1 protein, markers of active ß-CATENIN signaling in the craniopharyngioma but not in the pituitary adenomas. In our case, the NFPA is invasive macroadenoma, which is a frequently identified type of pituitary adenoma in collision tumor cases. Recurrence of this tumor was first observed after 8 years of follow-up. Based on this case, we suggest that pituitary collision tumors require long-term follow-up.

Development of the Proximal-Anterior Skeletal Elements in the Mouse Hindlimb Is Regulated by a Transcriptional and Signaling Network Controlled by Sall4.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.

Sall4 regulates neuromesodermal progenitors and their descendants during body elongation in mouse embryos.

Bi-potential neuromesodermal progenitors (NMPs) produce both neural and paraxial mesodermal progenitors in the trunk and tail during vertebrate body elongation. We show that Sall4, a pluripotency-related transcription factor gene, has multiple roles in regulating NMPs and their descendants in post-gastrulation mouse embryos. Sall4 deletion using TCre caused body/tail truncation, reminiscent of early depletion of NMPs, suggesting a role of Sall4 in NMP maintenance. This phenotype became significant at the time of the trunk-to-tail transition, suggesting that Sall4 maintenance of NMPs enables tail formation. Sall4 mutants exhibit expanded neural and reduced mesodermal tissues, indicating a role of Sall4 in NMP differentiation balance. Mechanistically, we show that Sall4 promotion of WNT/ß-catenin signaling contributes to NMP maintenance and differentiation balance. RNA-Seq and SALL4 ChIP-Seq analyses support the notion that Sall4 regulates both mesodermal and neural development. Furthermore, in the mesodermal compartment, genes regulating presomitic mesoderm differentiation are downregulated in Sall4 mutants. In the neural compartment, we show that differentiation of NMPs towards post-mitotic neuron is accelerated in Sall4 mutants. Our results collectively provide evidence supporting the role of Sall4 in regulating NMPs and their descendants.

Temporal changes of Sall4 lineage contribution in developing embryos and the contribution of Sall4-lineages to postnatal germ cells in mice.

Mutations in the SALL4 gene cause human syndromes with defects in multiple organs. Sall4 expression declines rapidly in post-gastrulation mouse embryos, and our understanding of the requirement of Sall4 in animal development is still limited. To assess the contributions of Sall4 expressing cells to developing mouse embryos, we monitored temporal changes of the contribution of Sall4 lineages using a Sall4 GFP-CreERT2 knock-in mouse line and recombination-dependent reporter lines. By administering tamoxifen at various time points we observed that the contributions of Sall4 lineages to the axial level were rapidly restricted from the entire body to the posterior part of the body. The contribution to forelimbs, hindlimbs, craniofacial structures and external genitalia also declined after gastrulation with different temporal dynamics. We also detected Sall4 lineage contributions to the extra-embryonic tissues, such as the yolk sac and umbilical cord, in a temporal manner. These Sall4 lineage contributions provide insights into potential roles of Sall4 during mammalian embryonic development. In postnatal males, long-term lineage tracing detected Sall4 lineage contributions to the spermatogonial stem cell pool during spermatogenesis. The Sall4 GFP-CreERT2 line can serve as a tool to monitor spatial-temporal contributions of Sall4 lineages as well as to perform gene manipulations in Sall4-expressing lineages.

Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules.

Isl1 is required for two processes during hindlimb development: initiation of the processes directing hindlimb development in the lateral plate mesoderm and configuring posterior hindlimb field in the nascent hindlimb buds. During these processes, Isl1 expression is restricted to the posterior mesenchyme of hindlimb buds. How this dynamic change in Isl1 expression is regulated remains unknown. We found that two evolutionarily conserved sequences, located 3' to the Isl1 gene, regulate LacZ transgene expression in the hindlimb-forming region in mouse embryos. Both sequences contain GATA binding motifs, and expression pattern analysis identified that Gata6 is expressed in the flank and the anterior portion of nascent hindlimb buds. Recent studies have shown that conditional inactivation of Gata6 in mice causes hindlimb-specific pre-axial polydactyly, indicating a role of Gata6 in anterior-posterior patterning of hindlimbs. We studied whether Gata6 restricts Isl1 in the nascent hindlimb bud through the cis-regulatory modules. In vitro experiments demonstrate that GATA6 binds to the conserved GATA motifs in the cis-regulatory modules. GATA6 repressed expression of a luciferase reporter that contains the cis-regulatory modules by synergizing with Zfpm2. Analyses of Gata6 mutant embryos showed that ISL1 levels are higher in the anterior of nascent hindlimb buds than in wild type. Moreover, we detected a greater number of Isl1-transcribing cells in the anterior of nascent hindlimb buds in Gata6 mutants. Our results support a model in which Gata6 contributes to repression of Isl1 expression in the anterior of nascent hindlimb buds.

Etv2-miR-130a-Jarid2 cascade regulates vascular patterning during embryogenesis.

Remodeling of the primitive vasculature is necessary for the formation of a complex branched vascular architecture. However, the factors that modulate these processes are incompletely defined. Previously, we defined the role of microRNAs (miRNAs) in endothelial specification. In the present study, we further examined the Etv2-Cre mediated ablation of DicerL/L and characterized the perturbed vascular patterning in the embryo proper and yolk-sac. We mechanistically defined an important role for miR-130a, an Etv2 downstream target, in the mediation of vascular patterning and angiogenesis in vitro and in vivo. Inducible overexpression of miR-130a resulted in robust induction of vascular sprouts and angiogenesis with increased uptake of acetylated-LDL. Mechanistically, miR-130a directly regulated Jarid2 expression by binding to its 3'-UTR region. Over-expression of Jarid2 in HUVEC cells led to defective tube formation indicating its inhibitory role in angiogenesis. The knockout of miR-130a showed increased levels of Jarid2 in the ES/EB system. In addition, the levels of Jarid2 transcripts were increased in the Etv2-null embryos at E8.5. In the in vivo settings, injection of miR-130a specific morpholinos in zebrafish embryos resulted in perturbed vascular patterning with reduced levels of endothelial transcripts in the miR-130a morphants. Further, co-injection of miR-130a mimics in the miR-130a morphants rescued the vascular defects during embryogenesis. qPCR and in situ hybridization techniques demonstrated increased expression of jarid2a in the miR-130a morphants in vivo. These findings demonstrate a critical role for Etv2-miR-130a-Jarid2 in vascular patterning both in vitro and in vivo.

Expression of Noggin and Gremlin1 and its implications in fine-tuning BMP activities in mouse cartilage tissues.

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1, BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin-LacZ and Gremlin1-LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin-LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1-LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671-1682, 2017.

Endoglin integrates BMP and Wnt signalling to induce haematopoiesis through JDP2.

Mechanisms of haematopoietic and cardiac patterning remain poorly understood. Here we show that the BMP and Wnt signalling pathways are integrated in an endoglin (Eng)-dependent manner in cardiac and haematopoietic lineage specification. Eng is expressed in early mesoderm and marks both haematopoietic and cardiac progenitors. In the absence of Eng, yolk sacs inappropriately express the cardiac marker, Nkx2.5. Conversely, high levels of Eng in vitro and in vivo increase haematopoiesis and inhibit cardiogenesis. Levels of Eng determine the activation of both BMP and Wnt pathways, which are integrated downstream of Eng by phosphorylation of Smad1 by Gsk3. By interrogating Eng-dependent Wnt-mediated transcriptional changes, we identify Jdp2 as a key Eng-dependent Wnt target, sufficient to establish haematopoietic fate in early mesoderm when BMP and Wnt crosstalk is disturbed. These studies provide mechanistic insight into the integration of BMP and Wnt signalling in the establishment of haematopoietic and cardiac progenitors during embryogenesis.

Gata6-Dependent GLI3 Repressor Function is Essential in Anterior Limb Progenitor Cells for Proper Limb Development.

Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod.

Cell migration during heart regeneration in zebrafish.

Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, pre-existing cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. Developmental Dynamics 245:774-787, 2016. © 2016 Wiley Periodicals, Inc.

Celf1 is required for formation of endoderm-derived organs in zebrafish.

We recently reported that an RNA binding protein called Cugbp Elav-like family member 1 (Celf1) regulates somite symmetry and left-right patterning in zebrafish. In this report, we show additional roles of Celf1 in zebrafish organogenesis. When celf1 is knocked down by using an antisense morpholino oligonucleotides (MO), liver buds fail to form, and pancreas buds do not form a cluster, suggesting earlier defects in endoderm organogenesis. As expected, we found failures in endoderm cell growth and migration during gastrulation in embryos injected with celf1-MOs. RNA immunoprecipitation revealed that Celf1 binds to gata5 and cdc42 mRNAs which are known to be involved in cell growth and migration, respectively. Our results therefore suggest that Celf1 regulates proper organogenesis of endoderm-derived tissues by regulating the expression of such targets.