Graded dorsal and differential gene regulation in the Drosophila embryo

Abstract

A gradient of Dorsal activity patterns the dorsoventral (DV) axis of the early Drosophila melanogaster embryo by controlling the expression of genes that delineate presumptive mesoderm, neuroectoderm, and dorsal ectoderm. The availability of the Drosophila melanogaster genome sequence has accelerated the study of embryonic DV patterning, enabling the use of systems-level approaches. As a result, our understanding of Dorsal-dependent gene regulation has expanded to encompass a collection of more than 50 genes and 30 cis-regulatory sequences. This information, which has been integrated into a spatiotemporal atlas of gene regulatory interactions, comprises one of the best-understood networks controlling any developmental process to date. In this article, we focus on how Dorsal controls differential gene expression and how recent studies have expanded our understanding of Drosophila embryonic development from the cis-regulatory level to that controlling morphogenesis of the embryo.

Figure 1.

Overview of the ventral signaling pathway. (A) Schematic of St. 10 egg chamber. The oocyte nucleus is located at the dorsoanterior cortex. Gurken, which is locally translated, is present in a protein gradient (green). (B) Cross section of schematic of St. 10 egg chamber. Gurken signaling (green) represses pipe expression (brown) in the follicle cells. (C) Schematic of syncytial blastoderm embryo. The ventral follicle cells, which had expressed pipe, deposit an unknown “chemical asymmetry” into the perivitelline space. (D) The chemical asymmetry results in a ventral-to-dorsal signaling gradient. (E) Cross section of schematic of syncytial blastoderm embryo. The signaling gradient is initially established within the perivitelline space, a small extracellular space between the embryo and an outer vitelline membrane. (F) Illustration of ventral signaling pathway in the early embryo. In the perivitelline space (PVS), a protease cascade (Ndl, Gd, Snk, Ea) eventually activates Spz, the ligand for the Toll receptor. The serine protease inhibitor, Spn27a, inhibits the activity of Ea. Activated Spz transduces the signal into the embryo through Toll, causing the degradation of Cactus and the nuclear translocation of the transcription factor Dorsal. The roles of Tube (T), Pelle (P), Weckle (W), and Myd88 (M) are relatively unknown, but participate in a signaling complex at the cytoplasmic tail of Toll.

Figure 2.

Illustration of early embryonic fate map. (A) Cross section of Stage 5 Drosophila embryo, fluorescently stained with an α-Dorsal antibody. (B) Cross section of Stage 5 Drosophila embryo, fluorescently stained by in situ hybridization to detect Dorsal target gene transcripts dpp, ind, vnd, sog, and sna. (C) Dorsal and Twist cooperate to specify both Type I and Type II Dorsal target genes. Dorsal functions together with Zelda to support expression of Type III (+ and −) target genes (See legend in part E). (D) Schematic of fate map. The Dorsal nuclear gradient divides the embryo into three main subtissues: mesoderm, neurogenic ectoderm, and dorsal ectoderm. The neurogenic ectoderm can be further divided into ventral and dorsal halves. (E) Groupings of Dorsal target genes. Type I genes are expressed in the ventral-most portion of the embryo, where Dorsal nuclear levels are the highest. Type II genes have dorsal borders in the middle of the neurogenic ectoderm. These genes are also repressed by Snail. Type III genes have their dorsal (+) or ventral (−) borders at roughly 50% DV axis, and contain sites for both Dorsal binding as well as a uniformly expressed activator, such as Zelda.

Figure 3.

Fruits of genomic approaches. (A) Dorsal gene regulatory network. The number of known Dorsal target genes increased roughly fivefold, from 10 to 50, through the use of genomic approaches. Careful study of the interactions among these genes allows for the construction of a network diagram. (Reprinted, with permission, from Levine and Davidson 2005, ©National Academy of Sciences.) (B) Network of repressors. Dorsal activity along the dorsoventral (DV) axis initiates a cascade of repressors (upper panel). Whole-mount embryo (lower panel) depicts the spatial organization of such genes by in situ hybridization using riboprobes to detect transcripts, with sna (red) on the ventral side, vnd (cyan) in the ventral neurogenic ectoderm (brk not shown), ind (dim, red) just dorsal of vnd, and schnurri (shn, green) in the dorsal ectoderm. msh (not shown) is expressed in a narrow stripe just dorsal to ind. Dashed connections are only hypothesized. It remains to be determined whether shn or another repressor functions in dorsal regions. (Image of embryo modified, with permission, from Stathopoulos and Levine 2005a, ©Elsevier.) (C) Cross talk between the Dorsal and AP patterning networks. ChIP-chip analyses with Dorsal, Snail, and Twist antibodies reveal strong binding peaks (left) for one or more of these proteins in several AP patterning genes. In situ hybridizations of reporter gene expression in whole-mount embryos (right) reveal DV asymmetries in these genes. (Reprinted, with permission, from Zeitlinger et al. 2007.)

Figure 4.

Dorsal target genes are integral components of signaling pathways that function to control gastrulation and differentiation. (A) Dorsal regulates the activation of the TGF-β, EGFR, and FGF signaling pathways by spatial regulation of pathway components. Dorsal target genes also include components of Notch, Insulin-like, TNF, and Wnt (not shown) pathways. Whether this results in regulated activation remains to be determined. (Modified, with permission, from Stathopoulos and Levine 2004, ©Elsevier.) (B) Shown are cross sections through an embryo with the indicated expression patterns of specific genes involved in the respective processes: invagination (the invaginated mesoderm has formed a tube [red] and ectodermal cells are at the surface [blue and yellow]), mesoderm migration (the ectoderm forms a surface on which the mesoderm migrates), and differentiation of the cardiac mesoderm (dorsal somatic lineages including heart precursors are induced when the mesoderm contacts Dpp-expressing cells). Expression of ths (Blue), htl and downstream of Fgf (dof) (Red), and dpp (Yellow). (Reprinted, with permission, from Stathopoulos and Levine 2004, ©Elsevier.)

Figure 5.

Revisiting the morphogen gradient model. (A) Depiction of classical morphogen gradient (purple). Horizontal dashed line denotes the concentration threshold that defines one cell fate boundary (shown below the graph). Gold curve denotes the same morphogen simulated with a 50% increase in morphogen production. The classical morphogen model predicts that the cell fate boundary, in response, will also shift by roughly 50% (arrow). This is an unacceptably sensitive system, and does not comport with experimental evidence. (B) Illustration of the affect of multiple inputs to cis-regulatory modules. In this hypothetical tissue, a primary morphogen (blue) initiates expression of several target genes (blue, orange, green, red) within the tissue. A secondary morphogen (red) is expressed in the red cells, but is repressed in the rest of the tissue by the primary morphogen, and acts as a repressor to other target genes (Inset). Consider a case in which the primary morphogen is present in a shallow gradient (dotted blue), at a concentration above green threshold, yet below the orange threshold, so that no secondary morphogen is present. The classical morphogen gradient model would predict all cells to turn green. However, because the secondary morphogen also serves as input to the target genes (and is not present in this case), it is possible that instead the orange gene is ubiquitously expressed.