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Questions 1 (6 points) Predict both the molecular and morphological consequences of misexpressing the Abd-­‐B Hox gene throughout otherwise normal Drosophila embryos. Due to the phenomenon of posterior prevalence, Abd-­‐B protein will transcriptionally repress all the other Hox genes, so only Abd-­‐B will be expressed throughout the embryo. This can be observed in the morphological features of this fly: all segments will adopt a fate normally observed only in the A5-­‐8 segments. Notes for 2013: We didn’t talk about what particular A5-­‐8 segments are. Also, recall that this is the same phenotype as the PolyComb Repressor knockout fly. Question 2 (6 points) Why do Drosophila embryos that completely lack eve+ gene activity have no engrailed expression and no segment boundaries? Features of the boarder between eve-­‐expressing and -­‐nonexpressing cells generate coordinately regulated Hh and Wnt signaling that turns on engrailed in the eve+ cell. The activity of engrailed and other segment polarity gene products is required to regulate these signals and establish segment boundaries. Notes for 2013: We did not discuss segment polarity genes such as engrailed or the formation of segment boundaries. Don’t worry about this question. Question 3 (6 points) What is the consequence of expanding the Snail expression pattern on heart development in the Drosophila embryo? Be sure to provide both molecular and morphological details. Normally in these cells, the Dorsal target Twist would upregulate the FGF receptor gene heartless, activating FGF signaling to drive the heart fate. However, Snail transcriptionally represses FGF, the ligand required to activate this pathway. So, when Snail is ectopically expressed in presumptive heart cells, the FGF signaling pathway will remain inactivated, and heart cells will fail to differentiate. Notes for 2013: We did not discuss Drosophila heart development or Twist activity. We only know that Snail is activated by high nuclear concentrations of Dorsal (recall the Affinity Threshold Model here), and represses the other Dorsal targets rhomboid and Sog. Question 4 (6 points) In Drosophila, why does the misexpression of Oskar at the anterior pole cause the formation of ectopic pole cells AND the development of a second abdomen? oskar mRNA + Oskar protein forms RNPs (ribonuclear protein complexes) that define the polar plasm, which will be incorporated into pole cells to direct their fate. The Oskar RNP also recruits the maternal posterior determinant, nanos, to the Oskar RNP. Nanos regulates a gene, caudal, much the way Bicoid regulates Hunchback and Giant, starting a cascade that patterns the posterior portions of the embryo – for example, turning on discrete stripes of even striped. Notes for 2013: We discussed Oskar’s role in pole cell formation. However, we did not discuss recruitment or activity of nanos or caudal (I did mention them in section, but only briefly), so don’t worry about that. It would not be unreasonable, though, to expect you to propose a model along the lines of bicoid, hb + gt, Kr + Kn, etc. for establishment of posterior segments. Question 5 (6 points) Dorsal and Twist work synergistically to activate the transcription of a variety of target genes. Propose a general mechanism for this synergy. This is an example of a positive feedforward loop: Dorsal activates expression of Twist, and the two act together following this to upregulate other targets. Mechanisms of activator synergy that may be at work here include recruitment of the histone acetyltransferase CBP, the H3K4 methyltransferase Trx/SET, the histone-­‐displacing ATPase Swi/Snf (all of these make DNA more accessible to RNA Pol II), and/or Mediator (which directly recruits RNA Pol II). Notes for 2013: We did not discuss Twist, or this particular feedforward loop. You could be asked, though, about mechanisms of activator synergy (the four he presented in class, summarized above). For example, Bicoid and Hunchback can act in this fashion on certain even striped enhancers. Question 6 (6 points) Give examples of a maternally localized mRNA and its role in early pattern formation a) From Dorsal ventral patterning of Drosophila bicoid’s translation product regulates gap genes and subsequent pair-­‐rule gene expression to establish the anterior segments. oskar forms an RNP upon translation which recruits posteriorizing factors and defines the pole plasm inherited solely be the pole cells to maintain the germline. gurken localizes to the dorsally-­‐biased oocyte pronucleus, and (upon secretion and receptor binding) triggers the cascade that establishes the dorsoventral axis. b) From Xenopus vegT localizes loosely to the vegetal pole and 1) activates transcription of the endomesoderm inducer, Nodal (really, the Nodal genes Xnr1, 2, 4, 5, and 6, plus derriere) and 2) endoderm-­‐specific genes that prevent mesoderm induction in VegT+ cells (e.g., mix and bix genes) vg1 is transported to the dorsal side, and contributes to Nodal signaling in the Nieuwkoop Center by heterodimerizing with Nodal and increasing P-­‐Smad2/3 levels. This is important for inducing the organizer on the dorsal side. wnt11 is transported to the dorsal side, where it activates canonical Wnt/B-­‐catenin signaling. B-­‐catenin/TCF then act synergistically with P-­‐Smad2/3 to activate organizer genes, such as the BMP antagonists chordin, noggin, and follistatin, as well as Wnt antagonists in the head organizer, such as dkk and frzb. c) From Ciona, chicken, or mouse In Ciona, the macho-­‐1 RNA localizes (with the yellow crescent pigment) to the B4.1 blastomeres, whose progeny will adopt the tail muscle facte because Macho-­‐1 protein transcriptionally activates muscle-­‐specific genes, such as muscle myosin. Notes for 2013: This is a highly relevant question. We did not discuss examples of maternally localizes mRNA in chicken or mouse, though.