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Modern Genetic Analysis Chapter 16. The Genetic Basis of Development
Forming Complex Pattern: Establishing Positional Information

In this section and in subsequent sections about the early development of Drosophila, we summarize the results of a good deal of mutational analysis. See Genetics in Process 16-3 for a description of how these mutants were recovered and analyzed.

Cytoskeletal Asymmetries and the Drosophila Anterior Posterior Axis

As we will see here, not only is the Drosophila germ line established through a localized determinant anchored to microtubules, but the same is true for formation of the anteriorposterior (AP) axis of the soma. Positional information along the AP axis of the syncitial Drosophila embryo is initially established through the creation of concentration gradients of two transcription factors: the BCD and HB-M proteins. The BCD protein, encoded by the bicoid (bcd) gene, is distributed in a steeper gradient in the early embryo, whereas the HB-M protein, encoded by the hunchback (hb) gene, is distributed in a shallower but longer gradient (Figure 16-13). Both gradients have their high points at the anterior pole. In somewhat different ways, the gradients of both these proteins depend on the diffusion of protein from a localized origin: localized translation of two mRNA species, one tethered to microtubules at the anterior pole, and the other at the posterior pole of the syncitial embryo.

The origin of the BCD gradient is quite straightforward. The bcd mRNA, packaged during oogenesis into the developing oocyte, is tethered to the (minus) ends of microtubules, which are located at the anterior pole (Figure 16-14a on the next page). Translation of BCD protein begins midway through the early nuclear divisions of the syncitial embryo. The protein diffuses in the common cytoplasm of the syncitium. Because the protein is a transcription factor, it contains signals to become localized in nuclei. By diffusion, those nuclei nearer to the source of translation (the anterior pole) incorporate a higher concentration of BCD protein than do those farther away; this difference results in the steep BCD protein gradient (Figure 16-14b).

The origin of the HB-M protein gradient is more complex. The HB-M protein gradient is produced by means of post-transcriptional regulation. The hb-m mRNA is maternal in origin, being packaged during oogenesis into the developing oocyte, and is uniformly distributed throughout the oocyte and the syncitial embryo. However, translation of hb-m mRNA is blocked by a translational repressor proteinthe NOS protein product, encoded by the nanos (nos) gene. Like bcd mRNA, nos mRNA is maternal in origin. However, in contrast with bcd mRNA, nos mRNA is localized at the posterior pole, through its association with the + (plus) ends of microtubules (Figure 16-14c). When translation of nos mRNA begins midway through the syncitial stage of early embryogenesis, NOS protein becomes distributed by diffusion in a gradient opposite that of BCD. The NOS gradient has a high point at the posterior pole and drops down to background levels in the middle of the AP axis of the embryo (Figure 16-14d). NOS protein has the ability to specifically block translation of hb-m mRNA. Through this ability, the NOS translation repressor posterior-to-anterior gradient produces the shallow anterior-to-posterior gradient of HB-M protein.

MESSAGE
Localization of mRNAs within a cell is accomplished by anchoring the mRNAs to one end of polarized cytoskeleton chains.

How do the bcd and nos mRNAs get tethered to opposite ends of the polarized microtubules of the oocyte and early syncitial embryo? The answer is that there are specific microtubule-association sequences located within the 3 UTRsuntranslated regions of the mRNA 3 to the translation-termination codon. (Eukaryotic mRNAs always contain some sequence 5 to the translation-initiation codon, the 5 UTR, and some sequence 3 to the translation-termination codon, the 3 UTR. In some mRNAs, these regions are quite short, but, in others, they can be several kilobases long. We are learning that, in many cases, as here, specific regulatory functions are carried out by sequences within these 3 UTRs.)

The bcd mRNA 3 UTR localization sequences are bound by a protein that can also bind the ends of the microtubules. In contrast, the 3 UTR of nos mRNA has localization sequences that can bind other proteins, which also bind to the + ends of microtubules. (In actuality, there are more intermediary steps in anchoring nos mRNA at the posterior end, with the 3 UTR localization sequences of nos mRNA being anchored to the molecules in the polar granules, which are in turn anchored to the + end of the microtubules.)

How can we demonstrate that the 3 UTRs of the mRNAs are where the localization sequences reside? This has been determined in part by "swapping" experiments. For example, when a synthetic transgene that produces an mRNA with 5 UTR and proteincoding regions of the normal nos mRNA glued to the 3 UTR of the normal bcd mRNA is inserted into the fly genome, this fused nos-bcd mRNA will be localized at the anterior pole of the oocyte. This causes a double gradient of NOS: one from anterior to posterior (due to the transgene's mRNA) and one from posterior to anterior (due to the normal nos gene's mRNA). This procedure produces a very weird embryo, with two mirror-image posteriors and no anterior (Figure 16-15). This double-abdomen embryo arises because NOS protein is now present throughout the embryo and translationally represses hb-m mRNA (it also represses bcd mRNA, although it is not clear that this is its normal function in wild-type animals). For more detailed information about how AP positional information is generated, see Genetics in Process 16-4 on the next page.

MESSAGE
The positional information of the Drosophila AP axis is generated by protein gradients. The gradients ultimately depend on diffusion of newly translated protein from localized sources of specific mRNAs anchored through their 3 UTRs to ends of cytoskeletal filaments.

CellCell Signaling and the Drosophila DorsalVentral Axis

In the examples considered thus far, the determinants were intracellular products: mRNAs or larger macromolecular assemblies packaged into the oocyte. In many circumstances, positional information must depend on proteins secreted from a localized subset of cells within a developing field. These secreted proteins diffuse in the extracellular space to form a concentration gradient of ligand. The ligand then activates target cells though a receptorsignal transduction system in a concentration-dependent fashion.

An example of such a mechanism for position information is the establishment of the dorsalventral (DV) axis in the early Drosophila embryo. The proximate effect of the DV positional information will be to create a gradient of DL protein activity in cells along the DV axis. The DL protein is a transcription factor encoded by the dorsal (dl) gene. It exists in two forms: (1) active transcription factor located in the nucleus and (2) inactive protein located in the cytoplasm, where it is sequestered in a complex bound to the CACT protein encoded by the cactus (cact) gene. The concentration of active DL protein will determine cell fate along the DV axis. Both dl mRNA and DL protein are distributed uniformly in the oocyte and the very early embryo. However, late in the syncitial embryo stage, there develops a gradient of active DL protein, with its high point at the ventral midline of the embryo (Figure 16-16).

How does positional information generate the gradient of active DL protein? The key events take place in oogenesis, through an interaction between the oocyte itself and the layer of surrounding somatic cellsthe follicle cells (Figure 16-17). The follicle cells on the ventral side of the oocytefollicle cell complex synthesize and secrete some proteins that lead to a gradient of activation of a secreted precursor of the SPZ ligand, encoded by the spaetzle (spz) gene. The follicle cells also make the eggshell (shown in Figure 16-17), the inner boundary of which is the vitelline membrane (shown in Figure 16-18a). The SPZ ligand is temporarily bound to structures in the vitelline membrane, sequestering them until near the end of the syncitial stage of early embryogenesis, when they are released. Active SPZ ligand (with its highest concentration at the ventral midline) then binds to the TOLL transmembrane receptor, encoded by the Toll gene, present uniformly in the oocyte plasma membrane (Figure 16-18). In a concentration-dependent manner, the SPZTOLL complex triggers a signal transduction pathway that ends up phosphorylating the inactive DL and CACT cytoplasmic proteins of the DLCACT complex (Figure 16-18b). Phosphorylation of DL and CACT causes conformational changes that break apart the cytoplasmic complex. The free phosphorylated DL protein is then able to migrate into the nucleus, where it serves as a transcription factor activating genes necessary for establishing the ventral fates.

MESSAGE
Positional information can be established through cellcell signaling involving a concentration gradient of a secreted molecule.

The Two Classes of Positional Information

To summarize this section, the most important message is that the specific examples that we considered fall into two general categories of positional information (Figure 16-19).
Localization of mRNAs within a cell. This type of positional information can be utilized only in cases where the developmental field begins as a single cell. It is used to form gradients of positional information in unusual cases such as Drosophila early embryogenesis, because, at this time, the embryo is a unicellular syncitium. More generally, it is used as a way of asymmetrically distributing local determinants to progeny cells.
Formation of a concentration gradient of an extracellular diffusible molecule. This type of positional information can be employed in multicellular developmental fields, because the gradient is extracellular and therefore is not limited by the boundaries of the individual cells. Indeed, we now know of several cases where concentration gradients of secreted protein ligands that activate receptors and signal transduction systems fulfill the properties expected of classical developmental morphogensliterally, concentration-dependent determinants of form.

© 1999 by W. H. Freeman and Company. All rights reserved.