Developmental genetics tasks

What is actinomycin D? How does it act?

Actinomycin D inhibits RNA polymerase by binding to DNA, blocking transcription. It’s used to determine reliance of early development on maternal mRNA.

How would you determine types and quantities of mRNA in eggs of smoking vs. non-smoking women?

Extract total RNA and perform RNA sequencing (RNA-seq) to identify transcriptome. Use qRT-PCR for quantification.

Describe genetic crosses where reciprocal results differ.

Maternal-effect genes like bicoid cause differences where a homozygous bicoid mutant mother produces headless offspring regardless of zygotic genotype.

Analyze offspring geno- and phenotypes from crosses involving the mal gene of Drosophila.

Maternal-effect mutations like mal impair eye pigmentation due to defective maternal RNA or proteins, which zygotic genes cannot rescue.

Why do headless Drosophila embryos form?

Loss of bicoid, a maternal-effect gene, leads to absence of anterior structures, eliminating morphogen gradient and causing head/thorax loss.

How would you identify a human homolog of Drosophila hedgehog gene?

Use BLAST to compare hedgehog gene with human sequences, then clone and test function in a Drosophila mutant.

Identify observations that confirm bicoid as key gene for headless embryos.

Wild-type bicoid mRNA injection into mutant eggs restores head development, while exuperantia and swallow mutants impair bicoid localization.

Predict outcomes of reciprocal crosses involving gs mutant allele.

Crossing gs mutant female with wild-type male results in no pole cells; reciprocal ensures germ-line development.

How would you identify Drosophila genes for loading egg cytoplasm with yolk proteins?

Conduct mutagenesis screens and use immunostaining to examine yolk protein deposition.

Explain results of experiments on gurken and torpedo function.

Gurken signaling via torpedo receptor establishes dorsal fate; mutations result in ventralized eggs while overexpression leads to dorsalized eggs.

Describe technique for producing germ-line chimeras to study gurken gene requirements.

Transplant pole cells between mutant and wild-type embryos, analyze for phenotype corresponding to germ-line or somatic genotype.

Predict outcomes of complementation tests for mutations.

Cross heterozygous mutants; a wild-type phenotype indicates different genes, failure to complement suggests same gene.

How would you test if two mutations (m1 and m2) occur in the same or different genes?

Cross m1/+ with m2/+; if heterozygotes show wild-type phenotype, mutations affect different genes.

What is the fate of embryos from chimeric egg primordia with TollD mutations?

If mutation is in somatic cells, ventralization occurs; if in germ cells, dorsal-ventral polarity is unaffected.

What is the fate of an embryo from a mother homozygous for dorsal and an embryo homozygous for twist?

Dorsal mutants lack ventral and lateral structures; twist mutants fail to form mesoderm.

How would you determine the sequence of action for three pigmentation genes (a+, b+, c+)?

Create double mutants; epistatic interactions reveal functional order.

Why don’t atavistic traits result in more than one extra digit?

Developmental constraints and evolutionary selection limit atavistic traits due to functional redundancy or selective pressures.

How does absence of HOX-A4 gene lead to multiple atlases?

Loss of HOX-A4 causes homeotic transformation of vertebrae into cervical structures.

Why did no HOX-B8 knockout mice result from chimera experiments?

Knockout may cause embryonic lethality or nonviable chimeras affecting critical segmentation or neural development.

How would you determine whether ribs attached to cervical vertebra are inherited?

Conduct pedigree analysis and genetic mapping for inheritance patterns.

How does Sonic Hedgehog (Shh) signaling establish limb polarity?

Shh forms a concentration gradient, with high levels inducing posterior identities and low levels inducing anterior digits.

Describe the role of the Apical Ectodermal Ridge (AER) in limb development.

AER secretes FGF signals to maintain cell proliferation and differentiation in the distal limb.

Analyze effects of Lmx1b mutations on dorsal limb patterning.

Loss of Lmx1b transforms dorsal structures into ventral structures, as seen in Nail-Patella syndrome.

How do retinoic acid gradients interact with Shh signaling during limb development?

Retinoic acid induces Shh expression in posterior limb bud, integrating anterior-posterior and proximo-distal patterning.

What manipulations would you perform to masculinize a female rat?

Expose the rat to testosterone during critical developmental periods to masculinize the brain.

What fraction of progeny from a cross between XX; tra/+ females and XY; tra/+ males will be sterile males?

1/8 of progeny will be XX; tra/tra sterile males.

How can a fetus show a Barr body yet present as male externally?

Likely XXY (Klinefelter syndrome), where one X is inactivated, showing male genitalia with Barr body.

What might explain increased homosexuality rates in maternal relatives of gay men?

X-linked inheritance patterns or mitochondrial factors contribute to maternal-line inheritance of traits.