Dev Bio Exam 2

Maternal effect

Offspring's early phenotype depends on the mother's genotype because she deposits gene products into the egg

Syncytium

Many nuclei sharing one cytoplasm; the state of the early Drosophila embryo

How often do nuclei divide in the early syncytial fly embryo?

About every 8 minutes

When does the fly embryo become a cellular blastoderm?

After nuclear division cycle 13

Mid-blastula transition

When widespread embryonic transcription begins

Why does being a syncytium matter for patterning?

Proteins such as Bicoid and Nanos spread through the shared cytoplasm and reach different nuclei without crossing cell membranes

What changes at cellularization?

Plasma membranes form around the nuclei, so protein movement and signaling are limited by cell boundaries

Where Bicoid and Nanos act in the syncytium

Bicoid enters nuclei to regulate transcription; Nanos regulates translation in the cytoplasm

Receptor for Gurken in follicle cells

Torpedo

What does protein kinase A do in the oocyte?

Orients microtubules so their plus ends point posterior

Motor that carries bicoid mRNA to the anterior

Dynein (moves toward minus ends)

Motor that carries oskar mRNA to the posterior

Kinesin (moves toward plus ends)

Role of Oskar protein

Anchors nanos mRNA at the posterior pole

Posterior marker proteins in the oocyte

Par-1 and Staufen

Bicoid

Maternal protein that acts as both a transcription factor and a translational repressor; highest at the anterior

bicoid maternal mutant phenotype

No head or thorax; the anterior is patterned like the posterior

Is bicoid necessary and sufficient for the head?

Yes, bicoid mRNA is both

How to test requirement vs. sufficiency for Bicoid

Removing Bicoid tests requirement; ectopic expression tests sufficiency

Bicoid's two functions

Blocks caudal translation and turns on hunchback transcription

How does Bicoid block caudal translation?

Prevents translation initiation factors from binding (involves the 5′ cap and 3′ UTR)

Nanos

Maternal RNA-binding translational regulator at the posterior that blocks translation of maternal hunchback RNA

Caudal

DNA-binding transcription factor that promotes posterior patterning

Why is Caudal protein posterior when its RNA is everywhere?

Bicoid blocks caudal translation in the anterior

Hunchback

Transcription factor that helps establish anterior regions; has both maternal and zygotic contributions

Two ways Hunchback protein ends up anterior

Nanos blocks maternal hunchback translation posteriorly, and Bicoid activates zygotic hunchback transcription anteriorly

Proteins concentrated at the anterior

Bicoid and Hunchback

Proteins concentrated at the posterior

Nanos and Caudal

Do RNA and protein distributions always match?

No; caudal and hunchback RNAs are spread throughout, but translational repression restricts where their proteins are made

Blocking translation vs. blocking transcription of maternal RNAs

Blocking translation stops new protein from maternal RNAs; blocking transcription leaves the maternal RNAs available at first

C. elegans counterpart of Caudal

PAL-1

Meaning of "caudal"

Latin for "tail"

Maternal-to-zygotic transition

Shift from maternal RNAs and proteins to the embryo's own gene expression, along with clearance of many maternal RNAs

A-P patterning hierarchy in order

Maternal determinants, gap genes, pair-rule genes, segment polarity genes, Hox genes

One example from each of the first three hierarchy levels

Bicoid (maternal), Krüppel (gap), even-skipped (pair-rule)

Where in the hierarchy does zygotic transcription begin?

With the gap and pair-rule genes

Gap genes

Zygotic genes whose transcription factors define broad, overlapping regions along the A-P axis

Gap gene mutant phenotype

Loss of a block of neighboring segments

Examples of gap genes

hunchback, Krüppel, giant, knirps, tailless

Pair-rule genes

Zygotic genes expressed in seven alternating stripes that set up the repeated segment pattern

Pair-rule mutant phenotype

Parts of every other segment are missing

Examples of pair-rule genes

even-skipped, fushi tarazu, odd-skipped

Meaning of fushi tarazu

Japanese for "too few segments"

even-skipped mutant phenotype

Missing the even-numbered segments

Segment polarity genes

Organize the pattern within each repeated segmental unit

Segment polarity mutant phenotype

A-P patterning defects within every segment

What do homeotic (Hox) genes do?

Encode homeodomain transcription factors that assign regional identity along the A-P axis

Homeotic transformation

One body part develops as another (e.g., antenna becomes leg, haltere becomes wing)

Hox mutation vs. gap mutation

A Hox mutation changes a region's identity; a gap mutation deletes a region

Enhancer (cis-regulatory element)

DNA sequence where transcription factors bind to increase or restrict a target gene's transcription

Activators of the eve stripe 2 enhancer

Bicoid and Hunchback

Repressors that set the edges of eve stripe 2

Giant and Krüppel

How does an enhancer turn analog input into digital output?

Activators and repressors compete for overlapping sites, so each nucleus ends up on or off

Homeodomain (homeobox)

DNA-binding region shared by all Hox proteins

Collinearity

Hox gene order on the chromosome matches their order of expression along the A-P axis

Polycomb

Keeps Hox genes stably repressed through chromatin changes

Trithorax

Keeps Hox genes stably active through chromatin changes

Number of Hox clusters in mammals

Four (a–d)

Reciprocal cross for bicoid (b/b mother × +/+ father)

All embryos are +/b but lack normal anterior structures

Reciprocal cross for bicoid (+/+ mother × b/b father)

All embryos are +/b and develop normally

What the bicoid reciprocal crosses show

The same embryo genotype gives different phenotypes depending on the mother's genotype, so bicoid is a maternal-effect gene

Can a +/b mother supply bicoid to b/b embryos?

Yes, she loads functional product into the egg

Why can't the father's normal bicoid allele rescue the embryo?

The product has to be in the egg already, and the paternal copy can't supply it in time

Forward genetics

Phenotype first, then find the gene; discovery-based; no genome sequence needed

Reverse genetics

Gene first, then find the phenotype; hypothesis-based; needs some genome sequence

Is a large screen always forward genetics?

No; if each perturbation targets a known gene, it's reverse genetics

Mutagen used in Drosophila screens

EMS

F2 ratio in a recessive screen

1:2:1, with 25% showing the mutant phenotype

Zygotic-effect mutation

Phenotype depends on the embryo's own genotype

Maternal-effect mutation

Phenotype depends on the mother's genotype

Generation screened in a Drosophila maternal-effect screen

F4 (offspring of homozygous F3 mothers)

How do you find the gene in a forward screen?

Genetic mapping or sequencing, then confirming with rescue or an independent allele (e.g., pie-1)

Loss of function

Reduces or eliminates a gene's activity; tests whether the gene is necessary

Gain of function

Increases activity, creates new activity, or causes activity in a new place or time; an ectopic structure can show sufficiency

Rescue

Restoring normal activity in a mutant; recovery strengthens the link between the gene and the defect

What do loss of function and rescue together not show?

That the gene is sufficient, or that it directly specifies the fate

Independent allele

A second mutation in the same gene; giving the same phenotype supports causation

How are mouse knockouts made?

Homologous recombination in ES cells, then chimeric mice, then breeding through the germ line

Chimera

An organism made of cells with different genotypes

RNAi

Small RNAs guide sequence-specific silencing of a target RNA, reducing protein without deleting DNA

Why choose RNAi over a knockout?

Avoids a permanent deletion; useful when a knockout is lethal or when several related gene copies need targeting

Limitations of RNAi

Knockdown may be incomplete, vary between tissues, or hit unintended targets

CRISPR-Cas9

A guide RNA directs Cas9 to cut target DNA; repair can disrupt the gene or, with a donor, make a specific edit

CRISPR repair without a template

NHEJ, which makes random small indels

CRISPR repair with a template

Homologous recombination, which makes a precise edit

NHEJ

Non-homologous end joining

dCas9

Cas9 that can't cut; delivers attached proteins (e.g., histone modifiers) to specific loci

DNA repair needed for mouse knockouts and precise CRISPR edits

Homologous recombination

Cre-lox

Cre recombinase acts at loxP sites; if the sites face the same direction, the DNA between them is cut out

Floxed gene

A gene flanked by loxP sites

What happens to a floxed gene in cells without Cre?

It stays intact

How do you delete a gene in only one tissue?

Drive Cre with a tissue-specific promoter in an animal carrying a floxed gene

Is Cre excision permanent?

Yes, in that cell and its descendants, but existing RNA and protein can persist, so function isn't lost immediately

AID system

Auxin-inducible degron; targets a tagged protein for auxin-dependent destruction

Advantage of protein degradation over DNA deletion

Removes existing protein at a chosen time without waiting for old product to run out

TIR1

Plant ubiquitin ligase that only works when bound to auxin

What does ubiquitination do?

Marks proteins for destruction by the proteasome

GAL4-UAS

GAL4 is a transcriptional activator that binds UAS to turn on a linked gene; a tissue-specific GAL4 driver expresses that gene in one tissue

Molecule targeted by each method

CRISPR and Cre-lox act on DNA; RNAi acts on RNA; AID acts on protein

Cre-lox vs. AID

Both disrupt function in specific tissues; Cre-lox targets the gene, AID targets the protein

Which perturbations can be reversed?

Some RNAi and protein degradation effects; DNA deletion is generally permanent