1.) Recently in C elegans, investigators have developed a technique for reverse genetics. Into the gonad of a hermaphrodite they inject ds RNA to the gene of interest and the progeny of this injected hermaphrodite do not express the gene of interest. The technique is called RNAi. Also the complete sequence of the C. elegans genome is known. Dr. N. Patel is interested in the evolutionary conservation of segmentation in animals. Using the sequence database of the C. elegans genome, he found that C. elegans has a gene that shares a high degree of amino acid similarity with Drosophila Hunchback. He injected RNAi to this hunchback homologue into the gonad.
Drosophila hunchback phenotype
a) What exciting phenotype do you think he saw in the progeny of the injected hermaphrodite? They were definitely not wild-type. (10)
b) What two additional results would you like to see demonstrated before this work can claim evolutionary conservation? Ask yourself what he has shown so far before answering the question. (10)
2.) The nematode worm C. elegans is a hermaphrodite. Hermaphrodism in C. elegans is due to the germ-line of the females worm undergoing spermatogenesis for a short time during gonadogenesis. The gonad first produces sperm and then oocytes. Therefore, hermaphrodism in C. elegans is the switch of the germ-line of an XX female to producing sperm for a short time. In C. elegans fem-3, fbf-1 and fog-2 are important factors in the sperm oocyte switch in XX hermaphrodites.
Genotype Phenotype of the hermaphrodite germ-line
wild-type sperm and oocytes
fem-3lf/fem-3lf oocytes only
fem-3gf/+ sperm only
fog-2lf/fog-2lf oocytes only
fbf-1lf/fbf-1lf sperm only
fem-3gf/+; fog-2lf/fog-2lf sperm only
fog-2lf/fog-2lf; fbf-1lf/fbf-1lf sperm only
fem-3lf/fem-3lf; fbf-1lf/fbf-1lf oocytes only
a) What do you conclude from the above? Indicate the activity states of the gene products during sperm and oocyte production and the interaction between the activities. (10)
b) fbf-1 encodes a component that binds to an RNA sequence element, PME, in the 3' untranslated region of fem-3 mRNA. fem-3 gain-of-function alleles are DNA sequence changes in the PME of fem-3. fbf-1 encodes a protein that is very similar to the protein Pumillo of Drosophila. Pumillo binds to the Nanos regulatory element (NRE) of the maternal hunchback mRNA. pumlf/pumlf mothers lay eggs where the embryo develops lacking the abdomen segments. Propose a model for what is occurring in the sperm oocyte switch. (10)
3.) In mammals, the major organ of excretion is the kidney, and in insects, the major organ of excretion is the malpighian tubules. These organs have not been thought of as homologous, because they have distinct developmental origins in mammals and insects. In mammals the kidneys are derived from the mesoderm, and in insects the malpighian tubules are derived from the ectoderm. However, the kidney and malpighian tubules are just specialized epithelia that contain a specialized array of ion pumps and pores that control excretion. Presently these organs are thought of as analogous and not homologous. You are going to do a series of experiments that is going to completely upset this archaic and boring dogma. In the first year of your PhD. you discover a gene that is specifically expressed in the mammalian kidney precursor cells using in situ hybridization with dig labeled probes and antibodies raised to the mouse protein. You sequence the mouse cDNA for this gene and find that the protein it encodes has a high degree of amino acid similarity to the PAX family of transcription factors; however, there is no obvious similarity to any one specific class of PAX protein, say like PAX6; hence, you have discovered a new subfamily that you have named KAX for kidney specific Pax. Your next experiment is to isolate homologous genes from three organisms Drosophila, the nematode C. elegans, and a flatworm. The gene products encoded by these genes show an incrediblely high degree of amino acid similarity to one another. There is only one KAX gene in all organisms studied. When you knock out the gene in mice, Kax-/Kax- mice develop lacking kidneys.
In Drosophila you have the following reagents
-a lethal allele that maps to the Drosophila Kax gene.
-a fly line that carries the P-element that contains the UASGAL4 regulatory sequences fused to the open reading frame (cDNA) of Drosophila Kax.
-a GAL4 driver line that expresses GAL4 specifically in the foregut; the normal malpighian tubules develop on the hindgut. The foregut and hindgut are ectodermal derivatives and the midgut is an endodermal derivative.
-dig labeled probes for in situ hybridization and antibodies to Drosophila KAX
-a fly line that carries the P-element that contains the UASGAL4 regulatory sequences fused to the open reading frame (cDNA) of mouse Kax.
a) Using these reagents propose the four experiments and describe the expected results of these experiments that led you to suggest that Kax is a evolutionarily conserved gene required for excretory organ development. Remember the mantra here! (20)
4.) Draw the canonical cell lineage for the following mutant situations. Use the letters A, B, and C to denote the cell fates. And assuming that each of the alleles is a loss-of-function allele, briefly describe, in each case, what the wild-type gene product is involved in doing.
5.) The product of the lin-44 gene is involved in determination of the polarity of asymmetric cell divisions. In lin-44 loss-of-function mutants the polarity of many asymmetric divisions are reversed. For example, note the reversal of polarity in the T blast cell lineage shown below. The T blast cell is located in the tail of C. elegans. The curled arrows indicate cell divisions with reversed polarity.
The lin-44 gene has been isolated, and the following experiment is performed. DNA containing the active, wild-type lin-44+ gene and the active, wild-type ncl-1+ gene are coinjected into the syncytial gonad. A freely segregating array containing the lin-44+ and ncl-1+ genes was isolated (i.e.. lin-44 and ncl-1 have not integrated into one of the chromosomes); this array is mitotically unstable. The lin-44 genetic locus is on chromosome I, and the ncl-1 genetic locus is on chromosome III. Loss of ncl-1+ activity results in enlarged nucleoli. A worm was constructed that contained this unstable array and that was homozygous for both lin-44 and ncl-1 loss-of-function alleles.
a) What analysis can the worm of the final genotype be used for? And what are the important aspects of the unstable array that allow this analysis to be done? (10)
Cell Ncl phenotype T lineage phenotype
example 1
T Ncl wild-type
hyp8/9 wild-type
hyp11 wild-type
example 2
T wild-type wild-type
hyp8/9 Ncl
hyp11 wild-type
example 3
T wild-type wild-type
hyp8/9 wild-type
hyp11 Ncl
example 4
T wild-type Lin (reversed polarity of
hyp8/9 Ncl asymmetric divisions)
hyp11 Ncl
b) What does example 1 tell us about lin-44+ activity with respect to determination of the polarity of T blast cell divisions? Why. (10)
c) The lin-44 gene is expressed (transcribed) specifically in the hyp8/9 and hyp11 cells. Does this information help explain the results in examples 2, 3, 4 and why? (10)
7.) The properties that a molecule needs to satisfy to be considered a morphogen are
1.) concentration dependent effects
2.) affect polarity
3.) determine multiple fates at different concentrations
4.) activity expressed as a gradient
5.) long range and direct action
Wingless is an important gene required for segmentation of Drosophila. Wingless mRNA as you should know by now is expressed at the posterior end of every parasegment. Without wingless (wg) there is no segmentation just a lawn of denticles. Peter Lawrence thinks that Wingless is working as a morphogen during segmentation in each segment. His evidence is as follows.
A.) Remember that Wingless mRNA is expressed in a line of cells at the posterior of every parasegment. The mRNA and protein expression for two parasegments (PS) are shown below.
B.) The following is a description of the structures associated with the T1 segment relative to the cells that express Wingless.
Below is a set of experiments in wg- embryos. First is the picture of a wg- embryo (homozygous for a null wg allele); a lawn of thoracic and abdominal denticles. Note, you see thoracic and abdominal denticles. Second is a wg- embryo expressing Wg protein from a UASGAL4wg gene and expressed uniformly using an armadillo-GAL4 driver (wingless is expressed in all cells). Note that you see only naked cuticle. Third is a wg- embryo expressing a temperature-sensitive Wg protein from a UASGAL4wgts gene expressed uniformly using an armadillo-GAL4 driver. This embryo was raised at a temperature where the Wgts protein has partial activity. Note that you see T1 beard denticles.
C) The interesting point of the above experiments is that no expression of Wg protein results in a short embryo due to the lack of segmentation, but uniform expression of Wg protein from the UASGAL4wg gene results in only naked cuticle and is also short due to a lack of segmentation. If Wg protein is expressed from the UASGAL4wg gene in a wg- embryo using a prd-Gal4 driver, you see the following phenotype. The prd-gal4 driver results in 7 stripes of GAL4 protein expression, and the area of GAL4 expression for two stripes in the abdomen and the denticle phenotype is shown below. The resulting embryo is significantly longer than the wg- embryos. Paired (prd) is a pair-rule gene and is expressed in 7 stripes early in development.
What do these experiments tell us about the morphgen properties that Wg has met and what properties/property are/is not addressed? (20)