BIOL 411 - 2023 Autumn - Exam 1 - Answer Key

BIOL 411 - 2023 Autumn Exam 1 3. As part of Unit 1, you had the opportunity to analyze the landmark paper “ macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis” by Hiroki Nishida and Kaichiro Sawada ( Nature 2001 409:724-729). 3a. This paper makes use of ascidian embryos ( Halocynthia roretzi ) as a model system. You are discussing the paper with a peer who has taken BIOL 355, but not BIOL 411, and is unfamiliar with H. roretzi as a model. You are asked to define three advantages and one disadvantage of H. roretzi as a model for analyzing muscle development compared to mammalian embryos. What do you say? (One complete sentence per bullet point) [2pts each/8pts total] Advantages [6pts] ● Multiple possible answers, e.g.: ○ Defined, invariant cell lineage ○ Externally developing embryos ○ Easy to inject mRNA/oligos ○ Simple body plan with well-described anatomy ○ Cheap, easy to culture in lab, etc. ○ It is a chordate making it closely related to vertebrates Disadvantage [2pts] ● Multiple possible answers, e.g.: ○ Genome may not contain factors relevant for mammalian muscle development ○ Mammalian embryos do not have yellow protoplasm, so mechanisms likely different ○ Great for studying autonomous development, but not the conditional development commonly used in mammalian systems Figure 3 legend: n–r, Embryos at the 110-cell stage fixed and in in situ hybridized with the muscle actin ( HrMA4 ) probe. n, Uninjected control. Actin expression is observed in ten precursor blastomeres of primary muscle cells. o, macho-1 -depleted embryos. p, q, Synthesized macho-1 mRNA was subsequently injected. Actin is ectopically expressed in endoderm ( p ) and epidermis ( q ) blastomeres. r, Subsequent injection of mutant mRNA has no effect. Figure 1 legend: b, Structure of macho-1 protein and constructs for in vitro mRNA synthesis. Macho-1 has five zinc fingers in the central part. The cDNA clone is 2,210 bp long and encodes a 556-amino-acid ORF. The wild-type construct encodes the full length, but the mutant construct lacks the zinc-finger domain. 3b. What do you conclude based on the results presented in Figure 3o ? (Limit: 1 sentence) [4pts] macho-1 is necessary for muscle actin ( HrMA4 ) expression at the 110-cell stage. Page 3

BIOL 411 - 2023 Autumn Exam 1 5. While studying at the health sciences library, you come across a back issue of the journal Development that contains an article analyzing the functions of the Drosophila gene staufen (stau) . Specifically, the authors perform a structure-function analysis to assess roles for the different domains of the Stau protein. Background: Full-length Stau (Stau full ) contains five (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. Methods: The authors assay rescue of a homozygous loss-of-function mutation in stau ( stau D3 /stau D3 ) with various transgenic constructs encoding the Stau protein forms shown below. You can assume that the Stau full transgene (T[Stau full ]) completely rescues the stau mutant phenotype and that the anti-Stau antibody used by the authors recognizes all of the Stau transgenes. Figure 1 legend: D, A diagram showing the structure of the Stau proteins encoded by the Stau full , Stau ΔN , Stau Δloop2 and Stau ΔdsRBD5 transgenes. The green boxes indicate the positions of the dsRBDs. The arrows and adjacent blue boxes indicate the N-termini of the amino acid sequences. Figure 2 legend: A–B, Localization of Stau protein and oskar ( osk ) mRNA as indicated by the dark staining in stage 9 (i and iii) and 10 (ii and iv) oocytes from stau D3 T[Stau full ]/ stau D3 females ( A ) or stau D3 T[Stau ΔN ]/ stau D3 females ( B ). 5a. Based on the data presented in Figure 2, what do you observe about the localization of the Stau ΔN protein within the oocyte? Note that you can ignore any differences in the intensity of the staining between panels A and B. (Limit: 1 sentence) [5pts] Similar to Stau full , Stau ΔN localizes to the posterior of the oocyte at stages 9 and 10. 5b. Based on the data presented in Figure 2, what do you conclude about the relationship between the structure of the Stau protein and osk localization within the oocyte? Note that you can ignore any differences in the intensity of the staining between panels A and B.(Limit: 1 sentence) The N-terminus of Stau is not necessary for localization of osk mRNA to the posterior of the oocyte at stages 9 and 10 [5pts] Page 6

BIOL 411 - 2023 Autumn Exam 1 A-P axis +2 for indicating HF position more posterior to WT +1 for having threshold Bcd concentrations at same level as WT 5e: +2 for drawing the Bcd concentrations for the T[Stau ΔdsRBD5 ]/ stau D3 embryos above WT across the A-P axis +1 for indicating HF position more posterior to WT +1 for having threshold Bcd concentrations at same level as WT +1 for having HF position more posterior compared to stau D3 T[Stau Δloop2 ]/ stau D3 embryos 6. Developmental biologists often order processes into step-wise pathways. A great example of this is the development of Drosophila oocyte polarity. Place the following items in order (1,2,3…) and put an “X” next to any false statement. Note that by convention, a name in lower-case italics refers to an mRNA (e.g., nanos ) and a non-italicized name with an upper-case first letter refers to a protein (e.g., Nanos). [10pts - partial credit possible] _ X _ Sperm entry cues initial asymmetry _ 4 _ Par-1 organizes microtubules with their plus and minus ends at the posterior and anterior ends of the oocyte, respectively _ 2 _ Gurken binds to Torpedo expressed by follicle cells _ X _ nanos is trafficked to the posterior pole along microtubules _ X _ Bicoid is trafficked to the anterior pole along microtubules _ 5 _ oskar is trafficked to the posterior pole _ 3 _ Follicle cells produce protein kinase A _ 6 _ Oskar binds to nanos at the posterior pole _ X _ Par-1 localizes to the anterior pole of the oocyte _ 1 _ gurken is synthesized by the nurse cells +4 for correctly identifying false statements (+1 for each correct identification) +6 for correctly identifying order (+1 is given for each correct step in the process, starting from step 1- “gurken is synthesized by the nurse cells”. So getting steps 1-2 right will fetch +2 points, getting steps 1-3 right will fetch +3 points, and so on). 7. Which of the following statement(s) are true about the transition from syncytial to cellular blastoderm in Drosophila ? [3pts] A. Nuclei migrate to the periphery of the blastoderm where they enter cell membrane pockets B. Transcription of zygotic genes occurs before cellularization, allowing diffusion of gene products within the blastoderm C. Nuclear divisions are synchronized and occur in waves across the blastoderm D. A and B E. A and C F. B and C G. All of the above H. None of the above Page 9