Morphogen Concentration Gradients Research Articles

Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules

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.

Precision of the Dpp gradient

Morphogen concentration gradients provide positional information by activating target genes in a concentration-dependent manner. Recent reports show that the gradient of the syncytial morphogen Bicoid seems to provide precise positional information to determine target gene domains. For secreted morphogenetic ligands, the precision of the gradients, the signal transduction and the reliability of target gene expression domains have not been studied. Here we investigate these issues for the TGF-beta-type morphogen Dpp. We first studied theoretically how cell-to-cell variability in the source, the target tissue, or both, contribute to the variations of the gradient. Fluctuations in the source and target generate a local maximum of precision at a finite distance to the source. We then determined experimentally in the wing epithelium: (1) the precision of the Dpp concentration gradient; (2) the precision of the Dpp signaling activity profile; and (3) the precision of activation of the Dpp target gene spalt. As captured by our theoretical description, the Dpp gradient provides positional information with a maximal precision a few cells away from the source. This maximal precision corresponds to a positional uncertainly of about a single cell diameter. The precision of the Dpp gradient accounts for the precision of the spalt expression range, implying that Dpp can act as a morphogen to coarsely determine the expression pattern of target genes.

EFFECTS OF SOG ON DPP-RECEPTOR BINDING.

Concentration gradients of morphogens are known to be instrumental in cell signaling and tissue patterning. Of interest here is how the presence of a competitor of BMP ligands affects cell signaling. The effects of Sog on the binding of Dpp with cell receptors are analyzed for dorsal-ventral morphogen gradient formation in vertebrate and Drosophila embryos. This prototype system includes diffusing ligands, degradation of morphogens, and cleavage of Dpp-Sog complexes by Tolloid to free up Dpp. Simple and biologically meaningful necessary and sufficient conditions for the existence of a steady state gradient configuration are established, and existence theorems are proved. For high Sog production rates (relative to the Dpp production rate), it is found that the steady state configuration exhibits a more intense Dpp-receptor concentration near the dorsal midline. Numerical simulations of the evolution of the system show that, beyond some threshold Sog production rate, the transient Dpp-receptor concentration at the dorsal midline would become more intense than that of the steady state, before subsiding and approaching a nonuniform steady state of lower magnitude. The magnitude of the transient concentration has been found to increase by several fold with increasing Sog production rate. The highly intense Dpp activity at and around the dorsal midline is consistent with available experimental observations and other analytical studies.

Morphogens, their identification and regulation.

During the course of development, cells of many tissues differentiate according to the positional information that is set by the concentration gradients of morphogens. Morphogens are signaling molecules that emanate from a restricted region of a tissue and spread away from their source to form a concentration gradient. As the fate of each cell in the field depends on the concentration of the morphogen signal, the gradient prefigures the pattern of development. In this article, we describe how morphogens and their functions have been identified and analyzed, focusing on model systems that have been extensively studied.

Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans.

The signaling molecules Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) function as morphogens and organize wing patterning in Drosophila. In the screen for mutations that alter the morphogen activity, we identified novel mutants of two Drosophila genes, sister of tout-velu (sotv) and brother of tout-velu (botv), and new alleles of tout-velu (ttv). The encoded proteins of these genes belong to an EXT family of proteins that have or are closely related to glycosyltransferase activities required for biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation in any of these genes impaired biosynthesis of HSPGs in vivo, indicating that, despite their structural similarity, they are not redundant in the HSPG biosynthesis. Protein levels and signaling activities of Hh, Dpp and Wg were reduced in the cells mutant for any of these EXT genes to a various degree, Wg signaling being the least sensitive. Moreover, all three morphogens were accumulated in the front of EXT mutant cells, suggesting that these morphogens require HSPGs to move efficiently. In contrast to previous reports that ttv is involved exclusively in Hh signaling, we found that ttv mutations also affected Dpp and Wg. These data led us to conclude that each of three EXT genes studied contribute to Hh, Dpp and Wg morphogen signaling. We propose that HSPGs facilitate the spreading of morphogens and therefore, function to generate morphogen concentration gradients.

Gene transfer into chicken embryos by retrovirus vectors

While chickens have many properties that are advantageous for embryological studies, their genetic analysis has been restricted. However, by using retrovirus vector systems in combination with classical techniques of experimental developmental biology, it has recently become possible to analyze the function of genes involved in the development of this organism. Avian retrovirus vectors are unique in that they can be divided into two categories: replication-competent and replication-defective (replication-incompetent). By choosing the vectors correctly, there are many experimental applications of these vectors such as induction of constitutive (or regulated) gene expression in a restricted region of tissues, organs and embryos; cell lineage analysis; and formation of concentration gradients of morphogens in micromass cultures. In this paper, several retrovirus vectors available for the chicken will be introduced and their applications in developmental biology will be reviewed.

Mesoderm induction and morphogen gradients

Historical interest in the way cells respond to a concentration gradient of morphogen is outlined, with special reference to the work of Saxen and Toivonen. In vertebrate embryos, morphogen gradients have been must fully investigated in chick limb buds and in amphibian mesoderm formation. We summarize recent work on morphogen gradient analysis, using directional conjugates in which activin signalling spreads from a localized source across a field of competent cells. Dorsalization and community effects are inductive processes that promote the differentiation of mesoderm into notochord and muscle. We describe new results in which directional cell aggregates are used to analyse muscle and notochord differentiation.

Molecular approaches to vertebrate limb morphogenesis.

It has long been proposed that concentration gradients of morphogens provide cues to specify cell fate in embryonic fields. Recent work in a variety of vertebrate systems give bona fide evidence that retinoic acid, the biologically active form of vitamin A, is a candidate for such a morphogen. In the developing chick wing, for example, locally applied retinoic acid triggers striking changes in the pattern along the anteroposterior axis. Instead of giving rise to a wing with the normal 234 digit pattern, wing buds treated with retinoic acid develop a 432234 mirror-image symmetrical digit pattern. For this review, we focus on three aspects of limb morphogenesis. (1) We summarize the experimental evidence supporting the notion that retinoic acid is a candidate morphogen. (2) Limb buds contain high levels of cellular retinoic-acid-binding protein (CRABP). Using order of magnitude calculations, we evaluate how the concentration of CRABP might affect the occupancy state of the retinoic acid receptor. (3) We discuss the spatio-temporal expression pattern of homeobox-containing genes in the developing limb and speculate about the possibility that retinoic acid influences the pattern of expression of homeobox genes.