Lecture 26

Pattern formation along the dorsal-ventral axis of Drosophila

Recommended reading: 

1.  Martinez-Arias and Stewart. "Molecular principles of Animal Development".  Chapter 9, pages 278-281.

2.  Le Mosey et al. (1999).  Trends in Cell Biology 9, 102-107.

3.  Brickman JM, Adam M, Ptashne M.  Interactions between an HMG-1 protein and members of the Rel family.  Proc Natl Acad Sci U S A 1999 Sep 14;96(19):10679-83  

Introduction:

    The dorsoventral (D/V) axis is specified by a set of 18 genes that are separate from those that specify the anterior-posterior axis, and that work very differently.  All are maternal effect genes, function primarily or exclusively in establishment of the D/V axis, and they establish a gradient of a morphogen called Dorsal (Dl).  Like the A/P axis, the initial events occur in the ovary from an interplay between the oocyte and the follicle cells.  In contrast to the genes involved in anterior-posterior axis formation, loss-of-function mutations in different D/V genes have two opposite phenotypes.  Some mutants have dorsalizing effects, while others have ventralizing effects.   This led to the hypothesis that the genes involved in D/V specification all function in the same linear pathway.  Indeed, genetic analyses (epistasis) proved that this is so.

Table 1. Maternal effect genes affecting D/V axis formation in Drosophila

Class A (required for polarity of embryo and egg chamber)
Gene	Loss of function	Site of action	Product
fs(1)K10	dorsalizing	germline	nuclear protein (oocyte nucleus)
cappuccino	dorsalizing	germline
spire	dorsalizing	germline
gurken	ventralizing	germline	TGFa-like protein
cornichon	ventralizing	germline
torpedo	ventralizing	soma	Receptor Tyr kinase
Class B (required for polarity of embryo only)
Gene	Loss of function	Site of action	Product
pipe	dorsalizing	soma
nudel	dorsalizing	soma
windbeutel	dorsalizing	soma
gastrulation defective	dorsalizing	germline	serine protease
easter	dorsalizing	germline	serine protease
snake	dorsalizing	germline	serine protease
spatzle	dorsalizing	germline	Toll ligand
Toll	dorsalizing	germline	Receptor
pelle	dorsalizing	germline	ser/thr kinase
tube	dorsalizing	germline	novel (cortical cytoskeleton)
dorsal	dorsalizing	germline	bHLH Txn Factor
cactus	ventralizing	germline	HLH protein

    Localization of the Gurken (GRK) ligand, a member of the TGF-a family, requires the action of at least five genes.  The GRK ligand binds the torpedo (TOR) receptor in the dorsal, anterior follicle cells.  The TOR receptor, a receptor tyrosine kinase similar to the EGF receptor, most likely activates the ras-gap-raf-mek pathway.  The intervening events are not clear.  The end result of this Dorsal signal is to restrict expression of pipe to the ventral follicle cells.  The protein products of windbeutel, nudel, grastrulation defective, snake, easter and spatzle comprise a proteolytic cascade.  Briefly, each of the genes encodes a protease, or a gene required for the correct localization of a protease, that acts on the next protein in the pathway, activating it, so that it activates the next protein in the pathway.  The end result is to cleave a protein called spatzle (SPZ) which is the ligand for the Toll receptor.  SPZ is only activated in the ventral vintelline membrane and its cleavage allows it to diffuse across the space between the vitelline membrane and the egg membrane, where it is bound by Toll.

    Toll is uniformly distributed in the egg, however, its ligand is localized to the ventral surface, thus Toll is activated most strongly ventrally.  Toll activation falls off rapidly, probably because of the limited amount of ligand.  The result of Toll activation is to cause Dorsal, which is the morphogen, to leave the cytoplasm, and enter the ventral nuclei, to create a nuclear gradient of Dl that is ventral high and dorsal low.  Dl is inhibited from entering the nucleus by Cactus and two other proteins called Pelle and Tube.  Dorsal and cactus are homologues of the vertebrate NF-kB and I-kB.

From Gilbert's "Developmental Biology"

    The Dl gradient results in several outputs:  Dl activates twist (twi) and snail (sna) in the ventral mesoderm from low-affinity binding sites.  Lower Dl concentrations in the neuroectoderm activate rhomboid (rho) from high-affinity binding sites.   Dl represses the expression of decapentaplegic (dpp) in the dorsal epidermis and zerknult (zen) in the amnioserosa.  Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zen contains Dorsal binding sites as well as an essential element termed AT2. An AT2 DNA binding activity has been identified (called ZREB) and purified in embryos.

http://www-bier.ucsd.edu/dvpat.html

        DSP1 has opposite effects on the activity of Dorsal assayed with regulatory sequences excised from the twist and zen promoters. These experiments were performed by transiently transfecting mammalian cells in culture. Thus, reporters containing either a 180-bp fragment from zen (a fragment sufficient to mediate repression in Drosophila) or the entire regulatory region of twist (from -1,438 to +38) were activated by cotransfection with DNA encoding Dorsal. Cotransfection with DNA encoding DSP1 has just the opposite effects on this Dorsal mediated activation of the two promoters: activation from the twist promoter is stimulated 4-fold, whereas that from the zen promoter is inhibited 3-fold. DSP1's stimulation of Dorsal-mediated activation from the twist promoter can be mapped to the defined enhancer elements or VARs. Thus, DSP1 also stimulates Dorsal-mediated activation if the template bears, instead of the intact twist promoter, a cassette that contains the two VARs that drive ventral-specific expression of the twist gene in the Drosophila embryo. The two VARs together constitute approximately 300 bp and contain multiple Rel-protein-binding sites (Brickman et al., 1999).