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Part B: Designing Experiments to Produce Predicted Outcomes…

Part B: Designing Experiments to Produce Predicted Outcomes (Adapted from Question 12) Now, you will design hypothetical experiments for each predicted outcome. For each predicted outcome below: Describe the treatment group(s) (e.g., the injected factor, genetic knockdown, or dominant-negative mutant). Specify any control group(s) needed to validate your conclusions (e.g., sham injections, wild-type embryos without manipulation, scrambled morpholino controls). Predict the expected morphological outcome (e.g., normal embryo, lack of dorsal structures, extra head) and a validation experiment to detect changes in expression. Provide a brief scientific rationale linking the molecular/genetic change to the phenotype. For each of these, answer: What steps would you take to carry out this experiment? Which controls are essential for a valid comparison? What phenotype (morphology, axis duplication, missing structures, etc.) do you expect to see? What changes in gene expression (increase in mRNA, etc) do you expect to see? Why does it happen at the molecular and developmental level? ——————————————————————————————————————————————— Experiment Design: Knockout of Chordin Gene Expression Using Morpholino Antisense Oligomers Below is a step-by-step outline describing how you might design and carry out an experiment to knock down chordin expression in amphibian embryos (e.g., Xenopus) and then analyze the resulting phenotype and gene-expression changes. 1. Treatment Groups Experimental (Chordin Morpholino Group): Inject chordin-specific morpholino antisense oligomers into one- or two-cell stage embryos. The morpholino is designed to bind the chordin mRNA near its start codon (or splice junction) to block translation (or correct splicing). 2. Control Groups Wild-Type (Uninjected) Embryos: Serve as a baseline for normal development and gene expression. Scrambled (or Non-Targeting) Morpholino Control: Inject embryos with a morpholino of the same nucleotide length and chemical composition but lacking a specific target in the Xenopus genome. Validates that any observed effect is not due to the injection procedure itself or non-specific morpholino effects. Expected Phenotype (Morphology) Ventralized Phenotype: The dorsal structures become smaller or disappear during development (e.g., the head becomes smaller, and the dorsal axis and notochord develop less). The anterior-posterior axis shows defects in formation when chordin expression reaches a certain critical level of reduction. The embryos show increased ventral characteristics because BMP signaling continues without Chordin’s inhibitory effect.   Changes in Gene Expression & Validation Experiment: Reduced or Absent chordin mRNA/Protein: In Situ Hybridization for chordin mRNA would show decreased or absent expression in dorsal organizer regions. qPCR (RT-PCR) for chordin transcripts would confirm reduced RNA levels compared to wild-type and scrambled morpholino controls. Western Blot (if antibodies are available) could confirm reduced Chordin protein. Altered Dorsal and Ventral Markers: Upregulation of BMP target genes (e.g., ventx, msx1) because BMP is no longer antagonized by Chordin. Downregulation of dorsal organizer markers (e.g., goosecoid, noggin, chordin itself) due to diminished dorsal-inducing activity. Validation: In situ hybridization or immunostaining for other known dorsal markers (e.g., goosecoid, sox2) can confirm a loss of dorsal identity. Western blot or immunohistochemistry for BMP target proteins can confirm enhanced BMP signaling. Scientific Rationale: Why Does It Happen? Molecular Mechanism: The secreted protein from the dorsal organizer binds and inhibits BMP, enabling dorsal fates to develop. The absence of Chordin allows BMP signaling to remain unchecked, leading to cell fate transformations toward ventral phenotypes. Developmental Outcome: The absence of Chordin leads to reduced dorsal structures because cells that should become notochord, somites, or other dorsal derivatives instead take on more ventral or lateral fates under high BMP signaling.