It is widely accepted that tissue differentiation and morphogenesis in multicellular organisms are regulated by tightly controlled concentration gradients of morphogens. How exactly these gradients are formed, however, remains unclear. Here we show that Fgf8 morphogen gradients in living zebrafish embryos are established and maintained by two essential factors: fast, free diffusion of single molecules away from the source through extracellular space, and a sink function of the receiving cells, regulated by receptor-mediated endocytosis. Evidence is provided by directly examining single molecules of Fgf8 in living tissue by fluorescence correlation spectroscopy, quantifying their local mobility and concentration with high precision. By changing the degree of uptake of Fgf8 into its target cells, we are able to alter the shape of the Fgf8 gradient. Our results demonstrate that a freely diffusing morphogen can set up concentration gradients in a complex multicellular tissue by a simple source-sink mechanism.

We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169(-/-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry.

Bone morphogenetic proteins (Bmps) and their roles during early dorsoventral patterning of the vertebrate embryo are well understood. The role and regulation of a more distant member of this family, the anti-dorsalizing morphogenetic protein (Admp), however, are less clear. Here, we report the isolation and characterization of zebrafish admp. Unlike other bmps, admp is exclusively expressed on the dorsal side. Expression starts at blastula stages in the region of the organizer, giving rise to anterior neuroectoderm and axial mesoderm. During the course of gastrulation, both the neuroectodermal and the mesodermal admp transcripts vanish in an anterior-posterior wave. The maintenance of admp expression is positively influenced by Nodal signaling and by Bozozok (Boz), an organizer-promoting homeodomain protein acting as a repressor of early bmp2b expression. Despite the positive effect of boz on admp expression, Boz and Admp have rather opposite effects on zebrafish patterning, as revealed in gain-and loss-of-function experiments. Upon overexpression, admp has Bmp-like activities causing a smaller organizer and enhanced ventral specification, very similar to the phenotype caused by the loss of boz function in mutant embryos. Antisense-based admp knockdown, on the other side, leads to an enlarged organizer and impaired ventral and posterior development, as observed in embryos after boz overexpression. This finding indicates that admp is required for the development of embryonic structures normally suppressed by organizer activities. The seeming discrepancy between the regulative and functional relationship of boz and admp is discussed, and models are proposed according to which Admp might be part of a negative feedback loop to pattern and confine the organizer region.

Many proteins are modified by conjugation with Sumo, a gene-encoded, ubiquitin-related peptide, which is transferred to its target proteins via an enzymatic cascade. A central component of this cascade is the E2-conjugating enzyme Ubc9, which is highly conserved across species. Loss-of-function studies in yeast, nematode, fruit fly, and mouse blastocystes point to multiple roles of Ubc9 during cell cycle regulation, maintenance of nuclear architecture, chromosome segregation, and viability. Here we show that in zebrafish embryos, reduction of Ubc9 activity by expression of a dominant negative version causes widespread apoptosis, similar to the effect described in Ubc9-deficient mice. However, antisense-based knock down of zygotic ubc9 leads to much more specific defects in late proliferating tissues, such as cranial cartilage and eyes. Affected cartilaginous elements are of relatively normal size and shape, but consist of fewer and larger cells.

Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.