Atsushi Kuroiwa

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 (PIP3), 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 PIP3-independent pathway.

Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development

The respiratory primordium is positioned and its territory is defined in the foregut. The visceral mesoderm of the respiratory primordium acquires the inducing potential that is necessary for endodermal budding morphogenesis and respiratory endoderm formation. Tbx4, a member of the T-box transcription factor gene family, was specifically expressed in the visceral mesoderm of the lung primordium. To analyze the function of Tbx4, we ectopically expressed Tbx4 in the visceral mesoderm of the foregut using in ovo electroporation. Ectopic Tbx4 induced ectopic bud formation in the esophagus by activating the expression of Fgf10. Conversely, interference of Tbx4 function resulted in repression of Fgf10 expression and in failure of lung bud formation. In addition, ectopic Tbx4 or Fgf10 also induced ectopic expression of Nkx2.1, a marker gene specific for the respiratory endoderm, in the underlying esophagus endoderm. When the border of the Tbx4 expression domain, which demarcates the respiratory tract and the esophagus, was disturbed by misexpression of Tbx4, formation of the tracheo-esophageal septum failed. These results suggested that Tbx4 governs multiple processes during respiratory tract development; i.e. the initial endodermal bud formation, respiratory endoderm formation, and septation of the respiratory tract and the esophagus.

Differential activities of Sonic hedgehog mediated by Gli transcription factors define distinct neuronal subtypes in the dorsal thalamus

The dorsal thalamus (DT) is a pivotal region in the vertebrate brain that relays inputs from the peripheral sensory organs to higher cognitive centers. It consists of clusters of neurons with relevant functions, called brain nuclei. However, the mechanisms underlying development of the DT, including specification of the neuronal subtypes and morphogenesis of the nuclear structures, remain largely unknown. As a first step to this end, we focused on two transcription factors Sox14 and Gbx2 that are expressed in the specific brain nuclei in the chick DT. The onset of their expression was found in distinct populations of the postmitotic cells in the prosomere 2, which was regulated by the differential activities of Sonic hedgehog (Shh) in a manner consistent with the action as a morphogen. Furthermore, both gain- and loss-of-function results strongly suggest that such distinct inductive activities are mediated selectively by different Gli factors. These results suggest that cooperation of the differential expression of Gli factors and the activity gradient of Shh signaling generates the distinct thalamic neurons at the specific locations.

Removal of vegetal yolk causes dorsal deficencies and impairs dorsal-inducing ability of the yolk cell in zebrafish

To examine the nature of cytoplasm determinants for dorsal specification in zebrafish, we have developed a method in which we remove the vegetal yolk hemisphere of early fertilized eggs (vegetal removed embryos). When the vegetal yolk mass was removed at the 1-cell stage, the embryos frequently exhibited typical ventralized phenotypes: no axial structures developed. The frequency of dorsal defects decreased when the operation was performed at later stages. Furthermore, the yolk cell obtained from the vegetal-removed embryos lost the ability to induce goosecoid in normal blastomeres while the normal yolk cell frequently did so in normal and vegetal-removed embryos. These results suggested that the vegetal yolk cell mass contains the dorsal determinants, and that the dorsal-inducing ability of the yolk cell is dependent on the determinants.

EXPRESSION OF MSX GENES IN REGENERATING AND DEVELOPING LIMBS OF AXOLOTL

Msx genes, homeobox-containing genes, have been isolated as homologues of the Drosophila msh gene and are thought to play important roles in the development of chick or mouse limb buds. We isolated two Msx genes, Msx1 and Msx2, from regenerating blastemas of axolotl limbs and examined their expression patterns using Northern blot and whole mount in situ hybridization during regeneration and development. Northern blot analysis revealed that the expression level of both Msx genes increased during limb regeneration. The Msx2 expression level increased in the blastema at the early bud stage, and Msx1 expression level increased at the late bud stage. Whole mount in situ hybridization revealed that Msx2 was expressed in the distal mesenchyme and Msx1 in the entire mesenchyme of the blastema at the late bud stage. In the developing limb bud, Msx1 was expressed in the entire mesenchyme, while Msx2 was expressed in the distal and peripheral mesenchyme. The expression patterns of Msx genes in the blastemas and limb buds of the axolotl were different from those reported for chick or mouse limb buds. These expression patterns of axolotl Msx genes are discussed in relation to the blastema or limb bud morphology and their possible roles in limb patterning.

FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9Eks) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.

The yolk syncytial layer regulates myocardial migration by influencing extracellular matrix assembly in zebrafish

The roles of extra-embryonic tissues in early vertebrate body patterning have been extensively studied, yet we know little about their function during later developmental events. Here, we analyze the function of the zebrafish extra-embryonic yolk syncytial layer (YSL) specific transcription factor, Mtx1, and find that it plays an essential role in myocardial migration. Downregulating the function of Mtx1 in the YSL leads to cardia bifida, a phenotype in which the myocardial cells fail to migrate to the midline. Mtx1 in the extra-embryonic YSL appears to regulate the embryonic expression of fibronectin, a gene previously implicated in myocardial migration. We further show dosage-sensitive genetic interactions between mtx1 and fibronectin. Based on these data, we propose that the extra-embryonic YSL regulates myocardial migration, at least in part by influencing fibronectin expression and subsequent assembly of the extracellular matrix in embryonic tissues.

A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish

Vertebrate endoderm development has recently become the focus of intense investigation. We have identified a novel sox gene, 226D7, which is important in zebrafish endoderm development. 226D7 was isolated by an in situ hybridization screening for genes expressed in the yolk syncytial layer (YSL) at the blastula stage. 226D7 is expressed mainly in the YSL at this stage and, during gastrulation, its expression is also detected in the forerunner cells and endodermal precursor cells. The expression of 226D7 is positively regulated by Nodal signaling. The knockdown of 226D7 using morpholino antisense oligonucleotides results in a lack of sox17-expressing endodermal precursor cells during gastrulation, and, consequently, lacks endodermal derivatives such as gut tissue. The effect is strictly restricted to the endodermal lineage, while the mesoderm is normally formed, a phenotype that is nearly identical to that of the casanova mutant (Dev. Biol. 215 (1999) 343). We further demonstrate that overexpression of 226D7 increases the number of sox17-expressing endodermal progenitor cells without upregulating the expression of the Nodal genes, cyclops and squint. Region-specific knockdown and overexpression of 226D7 by injection into the YSL suggest that 226D7 in the YSL is not involved in endoderm formation and 226D7 in the endoderm progenitor cells is important for endoderm development. Taken together, our data demonstrate that 226D7 is a downstream target of Nodal signal and a critical transcriptional regulator of early endoderm formation.

Zebrafish wnt11: pattern and regulation of the expression by the yolk cell and No tail activity

This study analyzed the spatial and temporal expression pattern of zebrafish wnt11 and the regulation of the expression during zebrafish early development, focusing on the interaction with the no tail (ntl) gene, a zebrafish orthologue of mouse Brachyury (T). Zygotic expression of wnt11 was first detected at the late blastula stage in the blastoderm margin, a presumptive mesoderm region. wnt11 expression coincided with mesoderm induction, and the expression was induced by mesoderm inducers such as the yolk cell (Mizuno, T., Yamaha, E., Wakahara, M., Kuroiwa, A., Takeda, H., 1996. Mesoderm induction in zebrafish. Nature 383, 131-132) or FGFs, indicating that, like ntl, wnt11 is one of the immediate-early genes in mesoderm induction. Initial expression domains of wnt11 and ntl overlapped, and these genes showed a similar response to mesoderm inducers. However, analysis of the ntl mutant embryos suggested that wnt11 and ntl are placed in distinct genetic pathways; the ntl mutation had no effect on wnt11 expression in the blastoderm margin. This was further supported by the result of RNA injection experiments showing that overexpression of Wnt11 did not affect ntl expression in the margin. Thus, wnt11 and ntl expression are induced and maintained independently in their initial phase of expression. In later stages, wnt11 was expressed in various organs, such as the somites, particularly in the developing notochord. Since no wnt gene has been reported to be expressed in the axial mesoderm, which is known to act as a signaling source that patterns the neural tube and somites, zebrafish wnt11 is the first wnt gene expressed in the notochord. Furthermore, in contrast to early expression, wnt11 expression in the notochord depended on Ntl activity. In the ntl mutant in which somite patterning is severely affected, wnt11 expression was completely lost, while another signaling molecule, sonic hedgehog is expressed in the mutant notochord precursor cells (Krauss, S., Concordet, J.-P., Ingham, P.W., 1993. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444). wnt11 expression in the somite also shows a characteristic pattern, correlated with the migration and differentiation of slow muscle precursors. These observations suggest a role for wnt11 in patterning the somites.

Fibroblast growth factor 10 gene regulation in the second heart field by Tbx1, Nkx2-5, and Islet1 reveals a genetic switch for down-regulation in the myocardium

During cardiogenesis, Fibroblast Growth Factor (Fgf10) is expressed in the anterior second heart field. Together with Fibroblast growth factor 8 (Fgf8), Fgf10 promotes the proliferation of these cardiac progenitor cells that form the arterial pole of the heart. We have identified a 1.7-kb region in the first intron of Fgf10 that is necessary and sufficient to direct transgene expression in this cardiac context. The 1.7-kb sequence is directly controlled by T-box transcription factor 1 (Tbx1) in anterior second heart field cells that contribute to the outflow tract. It also responds to both NK2 transcription factor related, locus 5 (Nkx2-5) and ISL1 transcription factor, LIM/homeodomain (Islet1), acting through overlapping sites. Mutation of these sites reduces transgene expression in the anterior second heart field where the Fgf10 regulatory element is activated by Islet1 via direct binding in vivo. Analysis of the response to Nkx2-5 loss- and Isl1 gain-of-function genetic backgrounds indicates that the observed up-regulation of its activity in Nkx2-5 mutant hearts, reflecting that of Fgf10, is due to the absence of Nkx2-5 repression and to up-regulation of Isl1, normally repressed in the myocardium by Nkx2-5. ChIP experiments show strong binding of Nkx2-5 in differentiated myocardium. Molecular and genetic analysis of the Fgf10 cardiac element therefore reveals how key cardiac transcription factors orchestrate gene expression in the anterior second heart field and how genes, such as Fgf10, normally expressed in the progenitor cell population, are repressed when these cells enter the heart and differentiate into myocardium. Our findings provide a paradigm for transcriptional mechanisms that underlie the changes in regulatory networks during the transition from progenitor state to that of the differentiated tissue.