Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells.

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.

Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/pinscher.

Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in 50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood. Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that dak(-/-) cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic(-/-) chondrocytes always act autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new model to explain the aetiology of HME.

The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell-cell adhesion.

BACKGROUND: Bone morphogenetic proteins (Bmps) are required for the specification of ventrolateral cell fates during embryonic dorsoventral patterning and for proper convergence and extension gastrulation movements, but the mechanisms underlying the latter role remained elusive. RESULTS: Via bead implantations, we show that the Bmp gradient determines the direction of lateral mesodermal cell migration during dorsal convergence in the zebrafish gastrula. This effect is independent of its role during dorsoventral patterning and of noncanonical Wnt signaling. However, it requires Bmp signal transduction through Alk8 and Smad5 to negatively regulate Ca(2+)/Cadherin-dependent cell-cell adhesiveness. In vivo, converging mesodermal cells form lamellipodia that attach to adjacent cells. Bmp signaling diminishes the Cadherin-dependent stability of such contact points, thereby abrogating subsequent cell displacement during lamellipodial retraction. CONCLUSIONS: We propose that the ventral-to-dorsal Bmp gradient has an instructive role to establish a reverse gradient of cell-cell adhesiveness, thereby defining different migratory zones and directing lamellipodia-driven cell migrations during dorsal convergence in lateral regions of the zebrafish gastrula.

Axon sorting in the optic tract requires HSPG synthesis by ext2 (dackel) and extl3 (boxer).

Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.

Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis.

Epithelial integrity requires the adhesion of cells to each other as well as to an underlying basement membrane. The modulation of adherence properties is crucial to morphogenesis and wound healing, and deregulated adhesion has been implicated in skin diseases and cancer metastasis. Here, we describe zebrafish that are mutant in the serine protease inhibitor Hai1a (Spint1la), which display disrupted epidermal integrity. These defects are further enhanced upon combined loss of hai1a and its paralog hai1b. By applying in vivo imaging, we demonstrate that Hai1-deficient keratinocytes acquire mesenchymal-like characteristics, lose contact with each other, and become mobile and more susceptible to apoptosis. In addition, inflammation of the mutant skin is evident, although not causative of the epidermal defects. Only later, the epidermis exhibits enhanced cell proliferation. The defects of hai1 mutants can be phenocopied by overexpression and can be fully rescued by simultaneous inactivation of the serine protease Matriptase1a (St14a), indicating that Hai1 promotes epithelial integrity by inhibiting Matriptase1a. By contrast, Hepatocyte growth factor (Hgf), a well-known promoter of epithelial-mesenchymal transitions and a prime target of Matriptase1 activity, plays no major role. Our work provides direct genetic evidence for antagonistic in vivo roles of Hai1 and Matriptase1a to regulate skin homeostasis and remodeling.

The proneural gene ascl1a is required for endocrine differentiation and cell survival in the zebrafish adenohypophysis.

Mammalian basic helix-loop-helix proteins of the achaete-scute family are proneural factors that, in addition to the central nervous system, are required for the differentiation of peripheral neurons and sensory cells, derivatives of the neural crest and placodal ectoderm. Here, in identifying the molecular nature of the pia mutation, we investigate the role of the zebrafish achaete-scute homologue ascl1a during development of the adenohypophysis, an endocrine derivative of the placodal ectoderm. Similar to mutants deficient in Fgf3 signaling from the adjacent ventral diencepahalon, pia mutants display failure of endocrine differentiation of all adenohypophyseal cell types. Shortly after the failed first phase of cell differentiation, the adenohypophysis of pia mutants displays a transient phase of cell death, which affects most, but not all adenohypophyseal cells. Surviving cells form a smaller pituitary rudiment, lack expression of specific adenohypophyseal marker genes (pit1, neurod), while expressing others (lim3, pitx3), and display an ultrastructure reminiscent of precursor cells. During normal development, ascl1a is expressed in the adenohypophysis and the adjacent diencephalon, the source of Fgf3 signals. However, chimera analyses show that ascl1a is required cell-autonomously in adenohypophyseal cells themselves. In fgf3 mutants, adenohypophyseal expression of ascl1a is absent, while implantation of Fgf3-soaked beads into pia mutants enhances ascl1a, but fails to rescue pit1 expression. Together, this suggests that Ascl1a might act downstream of diencephalic Fgf3 signaling to mediate some of the effects of Fgf3 on the developing adenohypophysis.

Fgf3 signaling from the ventral diencephalon is required for early specification and subsequent survival of the zebrafish adenohypophysis.

The pituitary gland consists of two major parts: the neurohypophysis, which is of neural origin; and the adenohypophysis, which is of non-neural ectodermal origin. Development of the adenohypophysis is governed by signaling proteins from the infundibulum, a ventral structure of the diencephalon that gives rise to the neurohypophysis. In mouse, the fibroblast growth factors Fgf8, Fgf10 and Fgf18 are thought to affect multiple processes of pituitary development: morphogenesis and patterning of the adenohypophyseal anlage; and survival, proliferation and differential specification of adenohypophyseal progenitor cells. Here, we investigate the role of Fgf3 during pituitary development in the zebrafish, analyzing lia/fgf3 null mutants. We show that Fgf3 signaling from the ventral diencephalon is required in a non-cell autonomous fashion to induce the expression of lim3, pit1 and other pituitary-specific genes in the underlying adenohypophyseal progenitor cells. Despite the absence of such early specification steps, fgf3 mutants continue to form a distinct pituitary anlage of normal size and shape, until adenohypophyseal cells die by apoptosis. We further show that Sonic Hedgehog (Shh) cannot rescue pituitary development, although it is able to induce adenohypophyseal cells in ectopic placodal regions of fgf3 mutants, indicating that Fgf3 does not act via Shh, and that Shh can act independently of Fgf3. In sum, our data suggest that Fgf3 signaling primarily promotes the transcriptional activation of genes regulating early specification steps of adenohypophyseal progenitor cells. This early specification seems to be essential for the subsequent survival of pituitary cells, but not for pituitary morphogenesis or pituitary cell proliferation.