Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.     

Role of zebrafish lbx2 in embryonic lateral line development

Background

The zebrafish ladybird homeobox homologous gene 2 (lbx2) has been suggested to play a key role in the regulation of hypaxial myogenic precursor cell migration. Unlike their lbx counterparts in mammals, the function of teleost lbx genes beyond myogenesis during embryonic development remains unexplored.

Principal Findings

Abrogation of lbx2 function using a specific independent morpholino oligonucleotide (MO) or truncated lbx2 mRNA with an engrailed domain deletion (lbx2^eh-) resulted in defective formation of the zebrafish posterior lateral line (PLL). Migration of the PLL primordium was altered and accompanied by increased cell death in the primordium of lbx2-MO-injected embryos. A decreased number of muscle pioneer cells and impaired expression pattern of sdf1a in the horizontal myoseptum was observed in lbx2 morphants.

Significance

Injection of lbx2 MO or lbx2^eh- mRNA resulted in defective PPL formation and altered sdf1a expression, confirming an important function for lbx2 in sdf1a-dependent migration. In addition, the disassociation of PPL nerve extension with PLL primordial migration in some lbx2 morphants suggests that pathfinding of the PLL primordium and the lateral line nerve may be regulated independently.     

Itch Is Required for Lateral Line Development in Zebrafish

The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous itch genes in zebrafish, itcha and itchb, with generalized expression patterns. By reducing itchb expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of cxcr4b and cxcr7b expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.     

Distinct contributions of CXCR4b and CXCR7/RDC1 receptor systems in regulation of PGC migration revealed by medaka mutants kazura and yanagi

Migratory pathways of PGCs to the gonad vary depending on the vertebrate species, yet the underlying regulatory mechanisms guiding PGCs are believed to be largely common. In teleost medaka embryo, PGC migration follows two major steps before colonizing in gonadal areas: (1) bilateral lineup in the trunk and (2) posterior drift of PGCs. kazura (kaz) and yanagi (yan) mutants of medaka isolated in mutagenesis screening were defective in the first and second steps, respectively. kaz^j2-15D was identified as a missense mutation in chemokine receptor gene cxcr4b expressed in PGCs. Embryonic injection of cxcr4b mRNA with vasa 3′ UTR rescued the PGC phenotype of kaz mutant, indicating a cell-autonomous function of cxcr4b in PGCs. yan^j6-29C was identified as a nonsense mutation in the cxcr7/rdc1 gene encoding another chemokine receptor. cxcr7 transgene with genomic flanking sequences rescued the yan mutant phenotype efficiently at the G0 generation. cxcr7 was expressed in somites rather than PGCs. cxcr7-expressing somitic domain expanded posteriorly with its margin immediately anterior of posteriorly drifting PGCs, as if PGCs were thrusted toward the gonadal area. kaz and yan mutants are also defective in lateral line positioning, suggesting combined employment of these receptor systems in various cell migratory processes.     

Sdf1/Cxcr4 signaling controls the dorsal migration of endodermal cells during zebrafish gastrulation

During vertebrate gastrulation, both mesodermal and endodermal cells internalize through the blastopore beneath the ectoderm. In zebrafish, the internalized mesodermal cells move towards the dorsal side of the gastrula and, at the same time, they extend anteriorly by convergence and extension (C&E) movements. Endodermal cells showing characteristic filopodia then migrate into the inner layer within the hypoblast next to the yolk syncytial layer (YSL). However, little is known about how the movement of endodermal cells is regulated during gastrulation. Here we show that sdf1a- and sdf1b-expressing mesodermal cells control the movements of the cxcr4a-expressing endodermal cells. The directional migration of endodermal cells during gastrulation is inhibited by knockdown of either cxcr4a or sdf1a/sdf1b (sdf1). We also show that misexpressed Sdf1 acts as a chemoattractant for cxcr4a-expressing endodermal cells. We further found, using the endoderm-specific transgenic line Tg(sox17:EGFP), that Sdf1/Cxcr4 signaling regulates both the formation and orientation of filopodial processes in endodermal cells. Moreover, the accumulation of phosphoinositide 3,4,5-trisphosphate (PIP(3)), which is known to occur at the leading edge of migrating cells, is not observed at the filopodia of endodermal cells. Based on our results, we propose that sdf1-expressing mesodermal cells, which overlie the endodermal layer, guide the cxcr4a-expressing endodermal cells to the dorsal side of the embryo during gastrulation, possibly through a PIP(3)-independent pathway.     

Xmrk‐induced melanoma progression is affected by Sdf1 signals through Cxcr7

Chemokine signals mediated by Sdf1/Cxcl12 through the chemokine receptor Cxcr4 are thought to play an instructive role in tumor migration and organ‐specific metastasis. We have used a small aquarium fish model to contribute to a better understanding of how the course of melanoma development is influenced by Sdf1 signals in vivo. We studied oncogene‐induced skin tumor appearance and progression in the transgenic medaka (Oryzias latipes) melanoma model. Similar to humans, invasive medaka melanomas show increased levels of sdf1, cxcr4, and cxcr7 gene expression. Stable transgenic fish lines overexpressing sdf1 exclusively in pigment cells showed a reduction in melanoma appearance and progression. Remarkably, diminished levels of functional Cxcr7, but not of Cxcr4b, resulted in strongly reduced melanoma invasiveness and a repression of melanoma. Our results thereby indicate that Sdf1 signals via Cxcr7 are able to constrain melanoma growth in vivo and that these signals influence tumor outcome.     

The CXCR4-CXCL12 axis promotes T cell reconstitution via efficient hematopoietic immigration

T cells play a critical role in immunity to protect against pathogens and malignant cells. T cell immunodeficiency is detrimental, especially when T cell perturbation occurs during severe infection, irradiation, chemotherapy, and age-related thymic atrophy. Therefore, strategies that enhance T cell reconstitution provide considerable benefit and warrant intensive investigation. Here, we report the construction of a T cell ablation model in Tg(coro1a:DenNTR) zebrafish via metronidazole administration. The nascent T cells are mainly derived from the hematopoietic cells migrated from the kidney, the functional homolog of bone marrow and the complete recovery time is 6.5 days post-treatment. The cxcr4b gene is upregulated in the responsive hematopoietic cells. Functional interference of CXCR4 via both genetic and chemical manipulations does not greatly affect T lymphopoiesis, but delays T cell regeneration by disrupting hematopoietic migration. In contrast, cxcr4b accelerates the replenishment of hematopoietic cells in the thymus. Consistently, Cxcl12b, a ligand of Cxcr4, is increased in the thymic epithelial cells of the injured animals. Decreased or increased expression of Cxcl12b results in compromised or accelerated T cell recovery, respectively, similar to those observed with Cxcr4b. Taken together, our study reveals a role of CXCR4-CXCL12 signaling in promoting T cell recovery and provides a promising target for the treatment of immunodeficiency due to T cell injury.     

Fgf and Sdf-1 Pathways Interact during Zebrafish Fin Regeneration

The chemokine stromal cell-derived factor-1 (SDF1) was originally identified as a pre-B cell stimulatory factor but has been recently implicated in several other key steps in differentiation and morphogenesis. In addition, SDF1 as well as FGF signalling pathways have recently been shown to be involved in the control of epimorphic regeneration. In this report, we address the question of a possible interaction between the two signalling pathways during adult fin regeneration in zebrafish. Using a combination of pharmaceutical and genetic tools, we show that during epimorphic regeneration, expression of sdf1, as well as of its cognate receptors, cxcr4a, cxcr4b and cxcr7 are controlled by FGF signalling. We further show that, Sdf1a negatively regulates the expression of fgf20a. Together, these results lead us to propose that: 1) the function of Fgf in blastema formation is, at least in part, relayed by the chemokine Sdf1a, and that 2) Sdf1 exerts negative feedback on the Fgf pathway, which contributes to a transient expression of Fgf20a downstream genes at the beginning of regeneration. However this feedback control can be bypassed since the Sdf1 null mutants regenerate their fin, though slower. Very few mutants for the regeneration process were isolated so far, illustrating the difficulty in identifying genes that are indispensable for regeneration. This observation supports the idea that the regeneration process involves a delicate balance between multiple pathways.     

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

Background

The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.

Results

By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis.

Conclusion

In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.     

Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.     

Medaka vasa is required for migration but not survival of primordial germ cells

vasa is essential for germline development. However, the precise processes in which vasa involves vary considerably in diverse animal phyla. Here we show that vasa is required for primordial germ cell (PGC) migration in the medakafish. vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of vasa RNA. Interestingly, vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. Importantly, vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcr4b in migratory tissues remained unchanged by vasa knockdown. By chimera formation we show that PGCs from vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in vasa-disrupted embryos. Furthermore, ectopic PGCs in vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival.     

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish

Background  

Development of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.     

Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line

The shape of most complex organ systems arises from the directed migration of cohesive groups of cells. Here, we dissect the role of the chemokine guidance receptor Cxcr4b in regulating the collective migration of one such cohesive tissue, the zebrafish lateral line primordium. Using in vivo imaging, we show that the shape and organization of the primordium is surprisingly labile, and that internal cell movements are uncoordinated in embryos with reduced Cxcr4b signaling. Genetic mosaic experiments reveal that single cxcr4b mutant cells can migrate in a directional manner when placed in wild-type primordia, but that they are specifically excluded from the leading edge. Moreover, a remarkably small number of SDF1a-responsive cells are able to organize an entire cxcr4b mutant primordium to restore migration and organogenesis in the lateral line. These results reveal a role for chemokine signaling in mediating the self-organizing migration of tissues during morphogenesis.     

A SNP associated with alternative splicing of RPT5b causes unequal redundancy between RPT5a and RPT5b among Arabidopsis thaliana natural variation

Background  

The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in Arabidopsis thaliana; RPT5a and RPT5b. We showed previously that RPT5a and RPT5b are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), RPT5b does not complement the effect of a strong rpt5a mutation in the male gametophyte, and only partially complements rpt5a mutation in the sporophyte. RPT5b ^Col-0 and RPT5b ^Ws-4 differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene.     

Fascin1-Dependent Filopodia are Required for Directional Migration of a Subset of Neural Crest Cells

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development.     

Coordinate regulation of DNA methyltransferase expression during oogenesis

Background  

Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of Dnmt3a and Dnmt3b, as well as a regulator of DNA methylation, Dnmt3L, in isolated female germ cells.