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Modern Genetic Analysis Chapter 16. The Genetic Basis of Development
Forming Complex Pattern: Utilizing Positional Information to Establish Cell Fates

When positional information has been provided, a system that can interpret the positional data must be in place. To use a geographical analogy, it is not sufficient to have a system of longitudes and latitudes; we also need equipment that can receive longitude and latitude information, whether it is through special instruments to read the positions of stars or through receivers that can triangulate signals transmitted from radio beacons. In the same way, the developmental positional information system requires that the signals transmitted be interpretable by elements within the cell.

The Initial Interpretation of Positional Information

As described earlier, two very different kinds of positional signal can be produced. However, both lead to the same outcome: a gradient in the amount of one or more specific transcription factors within the cells of the developmental field. In our examples of localized mRNAs in Drosophila AP axis development, this outcome is a direct consequence of the fact that the positional information gradients are of transcription factors themselves (BCD and HB-M). For diffusible extracellular sources of positional information, this outcome requires several intermediary steps. Such cases typically involve a gradient of a secreted protein ligand that binds to a transmembrane receptor in a concentration-dependent fashion. In turn, this binding activates a signal transduction pathway proportionately to the level of receptor activation. Eventually, this signal transduction pathway proportionately activates the key transcription factor(s) in the target cells. This is exactly what we saw in regard to the Drosophila DV axis, where the SPZ extracellular gradient leads to a graded activation of the DL transcription factor.

Given that positional information leads to a gradient of transcription-factor activities, we would naturally expect that the receivers are regulatory elements (enhancer and silencer elements) of genes whose protein products can begin the gradual process of specifying cell fate. This is exactly what we see. The genes targeted by the AP and DV transcription factors are zygotically expressed genes collectively known as the cardinal genes (Table 16-3) because they are the first genes to respond to the maternally supplied positional information. As an example, we will consider the AP cardinal genes. (The logic by which the DV axis is divided initially into three domains through the action of the DL transcription-factor activity gradient and then into numerous finer subdivisions is identical with that described for the AP axis.) Before considering cell fating of the AP axis, we need to review a bit of Drosophila embryology.

After the early embryonic syncitial stage, all somatic nuclei migrate to the surface of the egg and cellularize (Figures 16-20 and 16-21a). A few hours later, the first morphological manifestations of segmentation are apparent. At the end of 10 hours of development, the embryo is already externally divided into 14 segments from anterior to posterior3 head, 3 thoracic, and 8 abdominal segments (Figure 16-21b). By this time, each segment has developed a unique set of anatomical structures, reflective of its special identity and role in the biology of the animal. At the end of 12 hours, organogenesis occurs. At 15 hours, the exoskeleton of the larva begins to form, with its specialized hairs and other external structures. Only 24 hours after development began at fertilization, a fully formed larva hatches out of the eggshell (Figure 16-21c). Of special note in considering the AP pattern, the segmental arrangement of spikes, hairs, and other sensory structures on the larval exoskeleton makes each segment distinct and recognizable. Now let's return to a consideration of the AP cardinal genes.

The AP cardinal genes are also known as gap genes, because mutant flies lack a sequential series of larval segments, producing a gap in the normal segmentation pattern (look at the phenotypes of mutations in two gap genes, Krüppel and knirps in Figure 16-22). BCD or HB-M protein or both bind to enhancer elements of the promoters of the gap genes, thereby regulating their transcription. For example, transcription of one gene, Krüppel (Kr), is repressed by high levels of the BCD transcription factor but is activated by low levels of BCD and HB-M. In contrast, the knirps (kni) gene is repressed by the presence of any BCD protein but does require low levels of the HB-M transcription factor for its expression. These enhancer and promoter properties thereby ensure that the kni gene is expressed more posteriorly than is Kr (Figure 16-23a). By having promoters that are differentially sensitive to the concentrations of the transcription factors of the AP positional information system, the gap genes can be expressed in a series of distinct domains, and these domains then become different developmental fields. That is, the cells in the different domains become committed to different AP fates.

These commitments of the domains to different fates occur because each of the gap genes encodes a different transcription factor and thereby has the capability of regulating a different set of downstream target genes necessary to refine AP segmental fate.

Refining Fate Assignments through Transcription Factor Interactions

The gap gene expression pattern slices up the AP axis into several domains. However, gap genes are expressed too coarsely to allocate all the AP cell fates that are needed. Further, the end of the gap gene expression stage and the beginning of refinement closely coincide when the syncitial embryo becomes fully cellularized. The cytoplasm of each cell then contains a particular concentration of one or perhaps two adjacent gap geneencoded proteins. Essentially all further decisions are driven by the particular AP gap proteins present in the nucleus of a given cell.

The AP developmental pathway downstream of the gap genes bifurcates. Each of the two branches is instructive in regard to how pattern is refined. One branch establishes the correct number of segments. The other assigns the proper identity to each segment. (These different identities are manifest in the unique patterns of spikes and hairs on each segment of the larva, as described earlier.) The existence of two branches means that there are two different sets of target genes for regulation by the gap geneencoded transcription factors.

First, let's briefly consider the formation of segment number. (Refer to Figure 16-22 for a description of the mutant phenotypes that the different classes of segment-number genes produce.) The gap genes activate a set of secondary AP patterning genes called the pair-rule genes in a repeating pattern of seven stripes (Figure 16-23b). There are several different pair-rule genes, and each of them produces a slightly offset pattern of stripes. Additionally, there is a hierarchy within the pair-rule gene class. Some of these genes, called primarily pair-rule genes, are regulated directly by the gap genes, whereas others are activated by the primary pair-rule gene-encoded proteins, which are also transcription factors. The pair-rule genes then act combinatorially (several of their proteins are expressed within a given cell) to regulate the transcription of the segment-polarity genes, which are expressed in offset patterns of 14 stripes. Thus, the hierarchy of transcription-factor regulation extends all the way from the positional information system to the repeating pattern of segment-polarity gene expression. The products of the segment-polarity genes then permit the 14 segments to form and define the individual AP rows of cells within each segment.

MESSAGE
Through a hierarchy of transcription-factorregulation patterns, positional information leads to the formation of the correct number of segments. In the readout of positional information, transcription factors act combinatorially to create the proper segment-number fates.

How do the primary pair-rule genes become activated in a repeating pattern by the asymmetrically expressed gap proteins? The key is that the regulatory elements for the primary pair-rule genes are quite complex. For primary genes such as eve (even-skipped), separate enhancer elements regulate the activation of each eve stripe. The eve stripe 1 enhancer is activated by high levels of the HB-Z gap transcription factor, the eve stripe 2 enhancer by low levels of HB-Z but high levels of the KR gap transcription factor, and so forth.

MESSAGE
Regulatory element complexity of the primary pair-rule genes turns an asymmetric (gap gene) expression pattern into a repeating one.

Next, let's briefly consider the establishment of segmental identity. The gap genes target a clustered group of genes known as the homeotic gene complexes. They are called gene complexes because several of the genes are clustered together on the DNA. Drosophila has two homeotic gene clusters. The ANT-C (Antennapedia complex) is largely responsible for segmental identity in the head and anterior thorax, whereas the BX-C (Bithorax complex) is responsible for segmental identity in the posterior thorax and abdomen.

Homeosis is the conversion of one body part into another. Three examples of body-part-conversion phenotypes due to homeotic gene mutations are (1) the loss-of-function bithorax class of mutations that cause the entire third thoracic segment (T3) to be transformed into a second thoracic segment (T2), giving rise to flies with four wings instead of the normal two (Figure 16-24); (2) the gain-of-function dominant Tab mutation described in Chapter 14 (Figures 14-28 and 14-29) that transforms part of the adult T2 segment into the sixth abdominal segment (A6); and (3) the gain-of-function dominant Antennapedia (Antp) mutation that transforms antenna into leg (see the photograph on the first page of this chapter). Note that, in each of these cases, the number of segments in the animal remains the same; the only change is to the identity of the segments. By studying these homeotic mutations, we have learned much about how segment identity is established.

The domains of expression of the various gap proteins activate the target homeotic genes initially in a series of overlapping domains (Figure 16-25). These homeotic genes encode transcription factors of a class called homeodomain proteins. Homeodomain proteins interact with the regulatory elements of the homeotic genes in a specific pattern such that expression patterns of the homeotic genes become mutually exclusive. (We shall consider the structure and function relations within the homeotic gene complexes later in the context of evolution of developmental mechanisms.) These homeodomain proteins also regulate downstream target genes that are responsible for conferring the specific functions and identities to different regions of each segment.

MESSAGE
Segment identity is established through asymmetric gap gene expression patterns that deploy an asymmetric pattern of homeotic gene expression.

A Cascade of Regulatory Events

As we have seen, AP patterning of the Drosophila embryo occurs through a sequential triggering of regulatory events. Positional information establishes different concentrations of transcription factors along the AP axis, and target regulatory genes are then deployed accordingly to execute the increasingly finer subdivisions of the embryo, establishing both segment number and segment identity (Figure 16-26 on the following page).

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