Sandra van de Water

Repression of smoothened by patched-dependent (Pro-)Vitamin D3 secretion

The developmentally important hedgehog (Hh) pathway is activated by binding of Hh to patched (Ptch1), releasing smoothened (Smo) and the downstream transcription factor glioma associated (Gli) from inhibition. The mechanism behind Ptch1-dependent Smo inhibition remains unresolved. We now show that by mixing Ptch1-transfected and Ptch1 small interfering RNA–transfected cells with Gli reporter cells, Ptch1 is capable of non–cell autonomous repression of Smo. The magnitude of this non–cell autonomous repression of Smo activity was comparable to the fusion of Ptch1-transfected cell lines and Gli reporter cell lines, suggesting that it is the predominant mode of action. CHOD-PAP analysis of medium conditioned by Ptch1-transfected cells showed an elevated 3β-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3β-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3β-hydroxysteroid (pro-)vitamin D3 to the medium effectively inhibited Gli activity. Vitamin D3 bound to Smo with high affinity in a cyclopamine-sensitive manner. Treating zebrafish embryos with vitamin D3 mimicked the smo –/– phenotype, confirming the inhibitory action in vivo. Hh activates its signalling cascade by inhibiting Ptch1-dependent secretion of the 3β-hydroxysteroid (pro-)vitamin D3. This action not only explains the seemingly contradictory cause of Smith-Lemli-Opitz syndrome (SLOS), but also establishes Hh as a unique morphogen, because binding of Hh on one cell is capable of activating Hh-dependent signalling cascades on other cells.

Characterization of zebrafish primordial germ cells: Morphology and early distribution of vasa RNA

Research into germ line development is of conceptual and biotechnologic importance. In this study, we used morphology at the level of light and electron microscope to characterize the primordial germ cells (PGCs) of the zebrafish throughout embryonic and larval development. The study was complemented by the detailed analysis of mRNA expression of a putative germ line marker vasa. By morphology alone PGCs were identified at the earliest at the 5‐somite stage in the peripheral endoderm in contact with the yolk syncytial layer. Subsequently, they move from lateral to medial positions into the median mesoderm and from there by means of the dorsal mesentery into the gonadal anlage at day 5 postfertilization (pf), to establish gonads with mesenchymal cells by day 9 pf. Ultrastructural analysis of the 4‐day‐old zebrafish larvae demonstrates the presence of the germ line‐specific structures, nuage, and annulate lamellae. vasa RNA‐positive cells can be followed during zebrafish embryogenesis from the 32‐cell stage onward (Yoon et al., 1997 ). Upon completion of gastrulation, the RNA is exclusively present in the cells of the hypoblast, which as a consequence of convergence and extension movements first arrange themselves in a V‐shaped string‐like conformation to end up, by late somitogenesis, as a string of cells on each side of the midline. We show that the localization of maternal vasa RNA in the ovary changes from cytoplasmic, in the previtellogenic oocytes, to cortical in the vitellogenic oocytes, to concentrate at the boundary of the yolk and cytoplasm in the one cell stage zygote. These results demonstrate that the cortical vasa RNA localization precedes its cleavage furrow‐associated localization in the embryos and is presumably cytoskeleton dependent. vasa RNA localization changes from asymmetric subcellular at the sphere stage, to become entirely cytoplasmic at the dome stage. These data suggest a close resemblance in modes of segregation of the germ plasm in the frog and vasa mRNA in the fish during cleavage stages. Based on the significantly larger size and the stereotype and similar position of morphologically distinct cells, presumed to be PGCs, and their vasa RNA‐positive counterparts, we conclude that vasa RNA‐positive cells are the PGCs and vasa RNA represents a definitive germ line marker in the fish. Dev Dyn 1999;216:153–167.

The novel gene asb11: a regulator of the size of the neural progenitor compartment

From a differential display designed to isolate genes that are down-regulated upon differentiation of the central nervous system in Danio rerio embryos, we isolated d-asb11 (ankyrin repeat and suppressor of cytokine signaling box–containing protein 11). Knockdown of the d-Asb11 protein altered the expression of neural precursor genes sox2 and sox3 and resulted in an initial relative increase in proneural cell numbers. This was reflected by neurogenin1 expansion followed by premature neuronal differentiation, as demonstrated by HuC labeling and resulting in reduced size of the definitive neuronal compartment. Forced misexpression of d-asb11 was capable of ectopically inducing sox2 while it diminished or entirely abolished neurogenesis. Overexpression of d-Asb11 in both a pluripotent and a neural-committed progenitor cell line resulted in the stimulus-induced inhibition of terminal neuronal differentiation and enhanced proliferation. We conclude that d-Asb11 is a novel regulator of the neuronal progenitor compartment size by maintaining the neural precursors in the proliferating undifferentiated state possibly through the control of SoxB1 transcription factors.

Apc1-mediated antagonism of Wnt/beta-catenin signaling is required for retino-tectal pathfinding in the zebrafish.

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/beta-catenin pathway. We examined the effects of an Apc1 loss-of-function mutation on retino-tectal axon pathfinding in zebrafish. In apc mutants, the retina is disorganized and optic nerves portray pathfinding defects at the optic chiasm and do not project properly to the tectum. Wild-type cells, transplanted into mutant retinae, acquire retinal ganglion cell fate and project axons that cross at the mispositioned optic chiasm and extend to the contralateral tectum, suggesting a function of apc1 in axon pathfinding. These defects are caused mainly by stabilization of beta-catenin. These data demonstrate that Apc1 function is required for correct patterning of the retina and proper retinal ganglion axon projections.

Apc1 is required for maintenance of local brain organizers and dorsal midbrain survival

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/beta-catenin pathway, which is vital for induction and patterning of the early vertebrate brain. However, its role in later brain development is less clear. Here, we examined the mechanisms underlying effects of an Apc1 zygotic-effect mutation on late brain development in zebrafish. Apc1 is required for maintenance of established brain subdivisions and control of local organizers such as the isthmic organizer (IsO). Caudal expansion of Fgf8 from IsO into the cerebellum is accompanied by hyperproliferation and abnormal cerebellar morphogenesis. Loss of apc1 results in reduced proliferation and apoptosis in the dorsal midbrain. Mosaic analysis shows that Apc is required cell-autonomously for maintenance of dorsal midbrain cell fate. The tectal phenotype occurs independently of Fgf8-mediated IsO function and is predominantly caused by stabilization of beta-catenin and subsequent hyperactivation of Wnt/beta-catenin signalling, which is mainly mediated through LEF1 activity. Chemical activation of the Wnt/beta-catenin in wild-type embryos during late brain maintenance stages phenocopies the IsO and tectal phenotypes of the apc mutants. These data demonstrate that Apc1-mediated restriction of Wnt/beta-catenin signalling is required for maintenance of local organizers and tectal integrity.

Protein kinase A is involved in the induction of early mesodermal marker genes by activin.

In this study we have investigated the role of cAMP-dependent protein kinase A (PKA) in the induction of the early mesodermal marker genes goosecoid and no tail by activin in zebrafish embryos. We show that upon treatment with activin, zebrafish blastula cells exhibit a rapid and transient increase in PKA activity. In these cells, activin rapidly induces the expression of the immediate early response genes goosecoid and no tail. Stimulation and inhibition of PKA by activin, respectively, enhances and reduces the induction of goosecoid and no tail mRNA expression. Similar effects of PKA stimulation and inhibition on the induction by activin of a 1.8 kb zebrafish goosecoid promoter construct were observed. The induction by activin of a fragment of the zebrafish goosecoid promoter that mediates an immediate early response to activin is blocked by inhibition of PKA. Activation of PKA alone has no effect in these experiments. Finally, inhibition of PKA in whole embryos by overexpression of a dominant negative regulatory subunit of PKA reduces the expression of no tail and goosecoid, whereas the expression of even-skippedl remains unaltered. Overexpression of the catalytic subunit of PKA in embryos does not affect expression of goosecoid, no tail or even-skippedl. These data show that in dissociated blastulae, PKA is required, but not sufficient for activin signalling towards induction of goosecoid and no tail. In intact zebrafish embryos, PKA contributes to induction of goosecoid and no tail, although it is not required or sufficient.