Edwin L. Ferguson

decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo

Zygotic expression of the Drosophila TGF beta family member decapentaplegic (dpp) is required for the development of the dorsal embryonic structures. By injecting dpp transcripts into young embryos, we find that 2- to 4-fold increases in the concentration of injected RNA elicit progressively more dorsal cell fates: only low levels of dpp permit development of ventral ectoderm, intermediate dpp levels drive dorsal epidermal development, and high dpp levels drive cells to differentiate as the most dorsal pattern element, the amnioserosa. Localized dpp RNA injections into embryos that lack all known maternal and zygotic dorsal-ventral polarity indicate that dpp can both define embryonic polarity and organize detailed patterning within the ectoderm. We infer that dpp acts as an extracellular morphogen and that the graded activity of dpp specifies the pattern of ectodermal cell fates in the Drosophila embryo.

The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1

Mutations in the Drosophila tolloid (tld) gene lead to a partial transformation of dorsal ectoderm into ventral ectoderm. The null phenotype of tld is similar to, but less severe than decapentaplegic (dpp), a TGF-beta family member required for the formation of all dorsal structures. We have cloned the tld locus by P element tagging. At the blastoderm stage, tld RNA is expressed dorsally, similar to that described for dpp. Analysis of a tld cDNA reveals three sequence motifs: an N terminal region of similarity to a metalloprotease, two EGF-like repeats, and five copies of a repeat found in human complement proteins C1r and C1s. tld sequence is 41% identical to human bone morphogenetic protein 1 (BMP-1); the closest members to dpp within the TGF-beta superfamily are BMP-2 and BMP-4, two other bone morphogenetic proteins. These findings suggest that these genes are members of a signal generating pathway that has been conserved between insects and mammals.

The Xenopus Dorsalizing Factor noggin Ventralizes Drosophila Embryos by Preventing DPP from Activating Its Receptor

noggin is expressed in the Spemann organizer region of the Xenopus embryo and can promote dorsal cell fates within the mesoderm and neural development within the overlying ectoderm. Here, we show that noggin promotes ventral development in Drosophila, specifying ventral ectoderm and CNS in the absence of all endogenous ventral-specific zygotic gene expression. We utilize constitutively active forms of the dpp receptors to demonstrate that noggin blocks dpp signaling upstream of dpp receptor activation. These results suggest a mechanistic basis for the action of Spemanns organizer during Xenopus development and provide further support for the conservation of dorsal-ventral patterning mechanisms between arthropods and chordates.

Spatial bistability of Dpp-receptor interactions during Drosophila dorsal-ventral patterning.

In many developmental contexts, a locally produced morphogen specifies positional information by forming a concentration gradient over a field of cells. However, during embryonic dorsal–ventral patterning in Drosophila, two members of the bone morphogenetic protein (BMP) family, Decapentaplegic (Dpp) and Screw (Scw), are broadly transcribed but promote receptor-mediated signalling in a restricted subset of expressing cells. Here we use a novel immunostaining protocol to visualize receptor-bound BMPs and show that both proteins become localized to a sharp stripe of dorsal cells. We demonstrate that proper BMP localization involves two distinct processes. First, Dpp undergoes directed, long-range extracellular transport. Scw also undergoes long-range movement, but can do so independently of Dpp transport. Second, an intracellular positive feedback circuit promotes future ligand binding as a function of previous signalling strength. These data elicit a model in which extracellular Dpp transport initially creates a shallow gradient of BMP binding that is acted on by positive intracellular feedback to produce two stable states of BMP–receptor interactions, a spatial bistability in which BMP binding and signalling capabilities are high in dorsal-most cells and low in lateral cells.

Morphogen gradients new insights from DPP

The Drosophila TGFbeta family member Decapentaplegic (DPP) has been proposed to function as a morphogen to pattern cell fields in a number of developmental contexts. A series of recent reports add significantly to our knowledge of the mechanisms of DPP-gradient formation and interpretation. These reports identity additional genes and genetic circuitry necessary for this patterning system, and they highlight variations that might reflect developmental constraints within individual target cell fields.

The DSmurf Ubiquitin-Protein Ligase Restricts BMP Signaling Spatially and Temporally during Drosophila Embryogenesis

We identified Drosophila Smurf (DSmurf) as a negative regulator of signaling by the BMP2/4 ortholog DPP during embryonic dorsal-ventral patterning. DSmurf encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog MAD and likely promotes its proteolysis. The essential function of DSmurf is restricted to its action on the DPP pathway. DSmurf has two distinct, possibly mechanistically separate, functions in controlling DPP signaling. Prior to gastrulation, DSmurf mutations cause a spatial increase in the DPP gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, DSmurf mutations cause a temporal delay in downregulation of earlier DPP signals, resulting in a lethal defect in hindgut organogenesis.

Germline stem cell number in the Drosophila ovary is regulated by redundant mechanisms that control Dpp signaling

The available experimental data support the hypothesis that the cap cells (CpCs) at the anterior tip of the germarium form an environmental niche for germline stem cells (GSCs) of the Drosophila ovary. Each GSC undergoes an asymmetric self-renewal division that gives rise to both a GSC, which remains associated with the CpCs, and a more posterior located cystoblast (CB). The CB upregulates expression of the novel gene, bag of marbles (bam), which is necessary for germline differentiation. Decapentaplegic (Dpp), a BMP2/4 homologue, has been postulated to act as a highly localized niche signal that maintains a GSC fate solely by repressing bam transcription. Here, we further examine the role of Dpp in GSC maintenance. In contrast to the above model, we find that an enhancer trap inserted near the Dpp target gene, Daughters against Dpp (Dad), is expressed in additional somatic cells within the germarium, suggesting that Dpp protein may be distributed throughout the anterior germarium. However, Dad-lacZ expression within the germline is present only in GSCs and to a lower level in CBs, suggesting there are mechanisms that actively restrict Dpp signaling in germ cells. We demonstrate that one function of Bam is to block Dpp signaling downstream of Dpp receptor activation, thus establishing the existence of a negative feedback loop between the action of the two genes. Moreover, in females doubly mutant for bam and the ubiquitin protein ligase Smurf, the number of germ cells responsive to Dpp is greatly increased relative to the number observed in either single mutant. These data indicate that there are multiple, genetically redundant mechanisms that act within the germline to downregulate Dpp signaling in the Cb and its descendants, and raise the possibility that a Cb and its descendants must become refractory to Dpp signaling in order for germline differentiation to occur.

Conservation of dorsal-ventral patterning in arthropods and chordates

Dorsal-ventral patterning within the ectodermal and mesodermal germ layers of Drosophila and Xenopus embryos is specified by a system of genes that has been conserved over 500 million years of evolution. In both organisms, the activity of the TGF-beta family member DPP/BMP4 is antagonized by SOG/CHORDIN. A second Xenopus gene, noggin, has a similar biological activity to chordin. Analysis of the action of these genes indicate that Spemanns organizer promotes dorsal cell fates in Xenopus by antagonizing a ventralizing signal encoded by the Bmp4 gene.

Spatially Restricted Activation of the SAX Receptor by SCW Modulates DPP/TKV Signaling in Drosophila Dorsal–Ventral Patterning

Dorsal-ventral patterning within the embryonic ectoderm of Drosophila requires two TGFbeta ligands, DPP and SCW, and two type I TGFbeta receptors, TKV and SAX. In embryos lacking dpp signaling, increasing the level of TKV activity promotes progressively more dorsal cell types, while activation of SAX alone has no phenotypic consequences. However, SAX activity synergizes with TKV activity to promote dorsal development. Functional experiments suggest the two receptors have different ligands: DPP acts through TKV, and SCW acts through SAX. Furthermore, SOG, a negative regulator of this patterning process, preferentially blocks SCW activity. We propose that spatial regulation of the SAX pathway modulates TKV signaling to create positional information over the embryonic ectoderm.

Dorsal-ventral pattern formation in the Drosophila embryo: the role of zygotically active genes.

Publisher Summary This chapter describes the zygotically active genes that specify the dorsal, the ventral pattern of the Drosophila embryo. These genes respond to a gradient of maternal positional information that defines their realms of activity, and they, in turn, fix these domains and may direct subsequent patterning within these regions. Since these genes respond to and interpret maternal positional information, they are, in a sense, the dorsal–ventral counterparts of the anterior–posterior segmentation genes. Most of the zygotically expressed genes, known to be required for the dorsal–ventral patterning in the embryo, are defined in the saturation mutagenesis experiments. From the cuticular phenotypes of the differentiated mutant embryos, genes that are required to allow the normal development of specific regions of the dorsal–ventral pattern are identified. The analysis of the mutant phenotypes and the molecular characterization of the regulation and activity of the zygotically required dorsal–ventral patterning genes have provided important insights into the relationships between the cell position and differentiated fate. Three primary dorsal–ventral embryonic fields are defined in response to the maternal morphogen gradient. To establish these three fields, zygotically active dorsal–ventral patterning genes are transcribed locally in response to define the thresholds of maternal morphogen, and, in addition, the activities of their gene products are also essential in defining the extents of the three fields.