13 The Genetic Control of Development WORKING WITH THE FIGURES 1. In Figure 13-2, the transplantation of certain regions of embryonic tissue induces the development of structures in new places. What are these special regions called, and what are the substances they are proposed to produce? Answer: Special regions that induce development of specific tissues are called organizers because they can direct the development of surrounding tissues. The cells in the organizers are proposed to produce morphogens, which induce specific concentration-dependent developmental affects in surrounding tissue. 2. In Figure 13-5, two different methods are illustrated for visualizing gene expression in developing animals. Which method would allow one to detect where within a cell a protein is localized? Answer: Immunolocalization of protein expression allows fluorescent visualization of the protein in a cell and visual detection of where the protein is localized. In situ hybridization allows visualization of mRNA, but it does not provide information about the location of the protein because mRNA and the protein will not necessarily be in the same location. 3. Figure 13-7 illustrates the expression of the Ultrabithorax (Ubx) Hox protein in developing flight appendages. What is the relationship between where the protein is expressed and the phenotype resulting from the loss of its expression (shown in Figure 13-1)? Answer: Wild-type Ubx expresses a Hox protein in the developing hind wings that represses normal wing formation and leads to the development of halteres. A Ubx mutant lacks expression of the Hox protein in the hind wings, leading to their subsequent development as wings. 4. In Figure 13-11, what is the evidence that vertebrate Hox genes govern the identity of serially repeated structures? Chapter Thirteen 339 Answer: The normal number of lumbar vertebrae in mice is six. When the function of one Hox gene is lost, one lumbar vertabra is added, to make seven, and one sacral vertebra is lost. When the function of two Hox genes is lost, the number of lumbar vertebrae is increased to eight and two sacral vertebrae are lost. This indicates that Hox proteins govern the identity of the vertebral structures such that lack of Hox leads to formation of a lumbar vertebra in that segment. 5. As shown in Figure 13-14, what is the fundamental distinction between a pair- rule gene and a segment-polarity gene? Answer: Developing segments exist as pairs, and pair-rule mutants affect only one region of each segment pair, for example only the anterior or only the posterior member of the pair. In that way, pair-rule genes control double- segment periodicity. Segment-polarity genes have effects on all segments, in both members of the pair. 6. In Table 13-1, what is the most common function of proteins that contribute to pattern formation? Why is this the case? Answer: Most pattern formation proteins act as transcription factors. This allows them to coordinately regulate expression of many genes by binding specific sequences common to the enhancers of the different genes. 7. In Figure 13-20, which gap protein regulates the posterior boundary of eve stripe 2? Describe how it does so in molecular terms. Answer: The Giant protein, with a concentration curve shown in orange, regulates the posterior boundary of eve stripe 2. As the concentration of Giant increases, the expression of eve decreases. This produces the posterior boundary of stripe 2. From a molecular perspective, Giant is a transcription factor that binds with the stripe 2 enhancer of eve to repress transcription of eve posterior to stripe 2. 8. As shown in Figure 13-22, how many different transcription factors govern where the Distal-less (Dll) gene will be expressed? Answer: Six transcription factors, two Hox and four others, govern where Dll will be expressed. Mutations in both Hox binding sites cause full derepression of the Dll in A1-A7. Mutations in binding sites for other transcription factors, or combinations of transcription factors, cause different patterns of Dll derepression. 340 Chapter Thirteen 9. As shown in Figure 13-26, the Sonic hedgehog gene is expressed in many places in a developing chicken. Is the identical Sonic hedgehog protein expressed in each tissue? If so, how do the tissues develop into different structures? If not, how are different Sonic hedgehog proteins produced? Answer: Sonic hedgehog encodes a signaling protein and acts to induce other genes by activating transcription. Its developmental effect depends on the genes it induces, which differ from tissue to tissue. This leads to Shh activity promoting the development of feathers from a feather bud, or limbs from a limb bud. It would be possible for a gene involved in developmental regulation, such as Shh, to act differently in different tissues by producing different proteins. Alternate splicing of development gene mRNA could produce different isoforms in different tissues. The different isoforms could have tissue-specific effects. Sex determination in Drosophila is regulated through alternate splicing of genes involved in developmental pathways. BASIC PROBLEMS 10. Gooseberry, runt, knirps, and Antennapedia. To a Drosophila geneticist, what are they? How do they differ? Answer: These are names of genes that are required for normal Drosophila development. The mutant phenotypes associated with these genes provide clues to their different roles: knirps is a gap gene; runt is a pair-rule gene; gooseberry is a segment polarity gene; and antennapedia is a segment identity (homeotic) gene. 11. Describe the expression pattern of the Drosophila gene eve in the early embryo. Answer: The primary pair-rule gene eve (even-skipped) would be expressed in seven stripes along the A-P axis of the late blastoderm. 12. Contrast the function of homeotic genes with that of pair-rule genes. Answer: Pair-rule genes encode transcription factors expressed in repeating patterns of seven stripes. They are necessary to divide the embryo into the correct number of segments. Homeotic genes are also transcription factors, but they are essential for segment identity and do not affect segment number. 13. When an embryo is a homozygous mutant for the gap gene Kr, the fourth and fifth stripes of the pair-rule gene ftz (counting from the anterior end) do not form normally. When the gap gene kni is mutant, the fifth and sixth ftz stripes Chapter Thirteen 341 do not form normally. Explain these results in regard to how segment number is established in the embryo. Answer: Proper ftz expression requires Kr in the fourth and fifth segments, and kni in the fifth and sixth segments. Each of the seven stripes of pair-rule gene expression is controlled independently through distinct cis-acting regulatory elements and unique combinations of trans-acting transcription factors. 14. Some of the mammalian Hox genes have been shown to be more similar to one of the insect Hox genes than to the others. Describe an experimental approach that would enable you to demonstrate this finding in a functional context. Answer: A number of experiments could be devised. A comparison of amino acid sequence between mammalian gene products and insect gene products would indicate which genes are most similar to each other. Using cloned cDNA sequences from mammalian genes for hybridization to insect DNA would also indicate which genes are most similar to each other. The comparison of the gene and protein expression along the anterior-posterior axis in the two species may provide information relative to their deeply conserved functions. Finally, transgenic experiments could be performed to test functional equivalency. For example, when the mouse HoxB6 gene is inserted in Drosophila, it can substitute for Antennapedia. 15. The three homeodomain proteins ABD-B, ABD-A, and UBX are encoded by genes within the Bithorax complex of Drosophila. In wild-type embryos, the Abd-B gene is expressed in the posterior abdominal segments, Abd-A in the middle abdominal segments, and Ubx in the anterior abdominal and posterior thoracic segments. When the Abd-B gene is deleted, Abd-A is expressed in both the middle and the posterior abdominal segments. When Abd-A is deleted, Ubx is expressed in the posterior thorax and in the anterior and middle abdominal segments. When Ubx is deleted, the patterns of Abd-A and Abd-B expression are unchanged from wild type. When both Abd-A and Abd-B are deleted, Ubx is expressed in all segments from the posterior thorax to the posterior end of the embryo. Explain these observations, taking into consideration the fact that the gap genes control the initial expression patterns of the homeotic genes. Answer: If you diagram these results, you will see that deletion of a gene that functions posteriorly allows the next-most anterior segments to extend in a posterior direction. Deletion of an anterior gene does not allow extension of the next-most posterior segment in an anterior direction. The gap genes activate Ubx in both thoracic and abdominal segments, whereas the abd-A and Abd-B genes are activated only in the middle and posterior abdominal segments. The functioning of the abd-A and Abd-B genes in those segments somehow prevents Ubx expression. However, if the abd-A and Abd-B genes are deleted, Ubx can be expressed in these regions. 342 Chapter Thirteen 16. How can you tell if a gene is required zygotically and if it has a maternal effect? Answer: For zygotically required recessive mutations, crossing +/m +/m parents will produce offspring in the expected Mendelian ratio of 3 wild type : 1 mutant. Only homozygous mutant offspring will be mutant. For genes that exhibit a maternal effect, it is the genotype of the mother that determines the phenotype of the offspring. So, for the cross outlined above (+/m +/m), none of the offspring will be phenotypically mutant.