ch 13 (lectures 26-28) Flashcards
What are the key questions asked about genes involved in development?
- Which genes are important for the development of an organism?
- Where in the developing animal (spatial expression) and when (temporal expression) are these genes active?
- How is the expression of developmental genes regulated?
- What molecular mechanisms do gene products use to affect development?
What are organizers and morphogens in animal embryos?
Organizers are specialized cells that guide the development of surrounding tissues.
* They exert their influence by releasing morphogens.
Morphogens are signaling molecules produced by organizers.
* They induce different developmental responses in nearby cells.
* The response depends on the morphogen concentration—different levels trigger different outcomes.
How are genes generally divided by function?
Housekeeping genes
* Encode proteins needed for essential cellular processes
* Active in all cells of the body
Developmental toolkit genes
* Encode proteins that guide development
* Involved in basic developmental decisions
* Highly conserved across species
What are maternal-effect and zygotic genes in developmental genetics?
Maternal-effect gene:
* Knockout causes a phenotype only when missing in the oocyte
* No effect if the mutation is inherited from the sperm
* Indicates the gene product is provided by the mother during early development
Zygotic gene:
* Knockout causes a phenotype regardless of parent of origin
* Required in the embryo’s own genome for proper development
How can we identify genes involved in development?
Use genetic tools (e.g., knockouts, reporters) to find genes that impact development
Determine the place of action for the gene:
* Where does the RNA localize?
* Where does the protein localize?
Expect RNA/protein localization to match the affected organ when the gene is perturbed
* Ask: Which organ fails to develop or develops abnormally when the gene or its expression is altered?
What methods are used to visualize gene expression during development?
Understanding where and when genes are turned on helps reveal their function in development
Visualization methods target either mRNA or protein expression
* Assumes mRNA and protein localize similarly
Assumption: mRNA and protein localize to the same region of the embryo or tissue
* This holds true for most genes, where mRNAs are translated and proteins remain in the same cell
Exceptions exist: e.g., translationally repressed mRNAs where mRNA is present but protein isn’t produced
Helps determine when and where genes are active in development
Useful for comparing wild-type and mutant gene expression
Two main approaches:
* mRNA detection (e.g., in situ hybridization)
* Protein detection (e.g., immunostaining)
These methods are often interchangeable
Most mRNAs are translated and their proteins stay in the same cell
What are the differences between in situ hybridization and immunolocalization?
In Situ Hybridization (for detecting mRNA)
* Express and isolate the mRNA of the gene of interest
* Create a complementary RNA or DNA probe
* Label the probe so that its presence produces a light signal (e.g., color or fluorescence)
* Apply the probe to tissue → it hybridizes to the specific mRNA if present
* Visualize gene expression by observing where the signal appears
Immunolocalization (for detecting protein)
* Express and isolate the protein of interest
* Inject the protein into an animal to generate a specific antibody
* Use the antibody, labeled to produce a light reaction, to bind the protein in tissues
* Visualize where the protein is localized based on where light signal appears
What phenotype is observed in bicoid mutants, and what does it tell us?
Bicoid is a maternal-effect gene
In bicoid mutant larvae, the anterior (head) region is missing
* This phenotype indicates that bicoid is required for anterior development
What are segmentation-gene mutants, and how do they affect embryo development?
Gap genes
* Affect the formation of contiguous blocks of segments
* Mutants show large missing regions of the body pattern
Pair-rule genes
* Act with a double-segment periodicity
* Each gene contributes to specific parts within every other segment
* Mutants show a loss of alternating segments
Segment-polarity genes
* Control patterning within individual segments
* Mutants disrupt anterior-posterior polarity within each segment
How is the anteroposterior (A-P) axis established during development?
The expression patterns of A-P patterning proteins correspond to the mutant phenotypes of their respective genes.
Gene expression becomes more refined over time:
* Gap proteins: expressed first in broad domains
* Pair-rule proteins: expressed in stripes about 3–4 cells wide
* Segment-polarity proteins: expressed in narrow stripes of 1–2 cells
The expression of these genes occurs in a developmental cascade:
* Bicoid (maternal)
* Gap proteins (zygotic)
* Pair-rule proteins (zygotic)
* Segment-polarity proteins (zygotic)
What types of genes contribute to A–P axis pattern formation in Drosophila?
Genes involved in anteroposterior (A–P) axis patterning are primarily:
* Transcription factors
* Ligand-mediated signal transduction pathway components
These genes help control spatial and temporal expression of other developmental genes.
What are homeotic genes and what is a homeotic transformation?
Homeotic transformation- A developmental phenomenon where one body part is replaced by another.
* Typically affects serially reiterated structures (e.g., digits, ribs, limbs, segments).
Causes of homeotic transformations:
* Loss of function mutation: gene is inactive where it normally acts.
* Gain of function mutation: gene is active where it should not be.
Homeotic genes:
* Regulate the development of anatomical structures.
* Highly conserved across species.
* Mutations often cause misplacement of body parts due to misexpression.
How were homeotic mutants of Drosophila melanogaster identified, and what are key examples?
Homeotic mutations were discovered through forward genetic screens.
Forward genetic screen:
* Mutagen is randomly applied to wild-type organisms.
* Researchers look for phenotypes of interest.
* The gene causing the phenotype is then identified (cloned).
Reverse genetic screen:
* Begin with a known gene sequence.
* Mutate it intentionally to observe the resulting phenotype.
Examples of homeotic mutants in Drosophila:
Ultrabithorax (Ubx):
* Acts in the developing hindwing.
* Loss-of-function: hindwing transforms into forewing.
* Gain-of-function: forewing transforms into hindwing.
Antennapedia (Antp):
* Dominant gain-of-function: causes leg structures to form in place of antennae.
What are Hox genes and how do they influence body part identity?
Hox genes- A group of eight loci that control the identity of segments and their associated appendages.
* Highly conserved across species.
Complete loss of any Hox gene:
* Results in early developmental lethality.
Homeotic transformations:
* Often caused by mutations in Hox genes.
* These mutations are usually dominant.
* Viable in heterozygotes because they still have one functional (wild-type) allele.
How do Hox genes in Drosophila compare to those in vertebrates?
Conserved Function & Sequence:
* Vertebrate Hox genes show striking similarity to Drosophila Hox genes.
* Their encoded Hox proteins share a conserved homeodomain (a DNA-binding region).
Colinearity:
* The order of Hox genes in the genome matches the order of body regions in which they’re expressed (anterior to posterior).
Evolution by Duplication:
* Mice (and other vertebrates) have four Hox loci (clusters).
What is the Homeodomain and why is it important in Hox proteins?
Homeodomain:
* A 60-amino acid DNA-binding domain.
* Found in many transcription factors, especially those involved in development.
Structure & Function:
* Forms a helix-turn-helix motif, a common DNA-binding structure.
* Binds DNA in a sequence-specific way, helping regulate target genes.
Hox Proteins:
* The homeodomain was first identified in Hox proteins, which control body patterning.
Related Examples:
* Other proteins with helix-turn-helix motifs include:
* Lac repressor (in bacteria)
* α2 and a1 transcription factors (yeast mating-type)
How does Dorsal protein contribute to dorsoventral (D-V) axis patterning?
Dorsal is a maternal-effect gene that encodes a transcription factor
* Dorsal is expressed in a gradient along the D-V axis, increasing toward the ventral side
Cells are exposed to different concentrations of Dorsal
* This leads to expression of different sets of zygotic genes that contribute to dorsoventral patterning
How are gap genes activated in the early Drosophila embryo, and what roles do Bicoid and the embryo’s syncytial structure play in this process?
Gap genes are activated by specific maternally provided proteins.
The early Drosophila embryo is a syncytium, meaning nuclei share a common cytoplasm.
* This syncytial structure allows proteins like Bicoid to diffuse and form a concentration gradient.
Hunchback, a gap gene, requires Bicoid binding to activate its transcription.
* The more Bicoid binding sites in a gene’s enhancer, the broader its expression pattern.
* More binding sites allow activation at lower Bicoid concentrations, enabling gene expression farther from the anterior (e.g., middle of the embryo).
How is individual pair-rule stripe formation regulated in the early Drosophila embryo?
Pair-rule stripe formation is controlled by combinations of maternal-effect and gap proteins.
* Each stripe is regulated independently by distinct cis-acting regulatory elements (enhancers).
A gene’s specific expression pattern results from different combinations of protein concentrations binding to these enhancers.
These enhancers integrate multiple inputs:
* Activators and repressors
* Maternal-effect proteins
* Gap proteins
Each enhancer binds several transcription factors that together determine when and where a stripe is formed.
How do Hox proteins regulate appendage formation in the Drosophila abdomen, and what roles do other genes play in this process?
Hox proteins repress appendage formation in the abdominal segments.
Hox gene expression is largely regulated by gap proteins.
Distal-less (Dll) is a homeodomain transcription factor that marks sites of future appendage development.
* Regulation of Dll involves multiple, independent, and modular cis-acting elements (enhancers).
Ultrabithorax (Ubx) is a Hox gene that represses Dll in the abdomen to prevent appendage formation.
Engrailed is a segment polarity gene involved in defining segment boundaries.
These modular enhancers allow rapid and independent evolution of regulatory control.
Why is Caenorhabditis elegans a useful model system for studying development?
Shares advantages with Drosophila:
* Rapid development
* Inexpensive to maintain
Has a highly invariant cell lineage (predictable and reproducible in every individual).
Contains fewer than 1000 somatic cells, making it ideal for studying cell fate and development.
Has a transparent body, allowing researchers to directly observe development in real time.
How can mRNA-binding proteins regulate translation to influence cell lineage decisions during development?
Development can be regulated by mRNA-binding proteins that repress translation.
These proteins often bind to specific sequences in the 3’ untranslated region (3’UTR) of target mRNAs.
GLD-1 is an mRNA-binding protein that binds to the 3’UTR of glp-1 at the spatial control region (SCR).
* This binding prevents translation of glp-1 in cells that express GLD-1 (specifically EMS and P2 cells).
This spatial control contributes to cell lineage specification.
glp-1 expression can be visualized using reporter genes like LacZ in experimental models.
What is an example of a microRNA that controls development, and how does it function?
MicroRNAs (miRNAs) can regulate development by binding to the 3’UTRs of mRNAs, repressing their translation.
let-7 is a well-known miRNA involved in developmental timing.
* let-7 is required to down-regulate cell division in hypodermal cells after the transition to the adult stage.
* It functions by binding to the 3’UTR of lin-41, repressing its expression.
* This regulation is important for timing developmental transitions.
let-7 and lin-41 are highly conserved across multicellular organisms, including humans.
What are the roles of the Sonic hedgehog (Shh) gene in development?
Toolkit genes like Shh can function at multiple stages of development.
Shh is expressed in different tissues at different times (e.g., posterior limb bud, developing feather buds).
* The Shh signaling pathway activates different target genes depending on the tissue context.
Shh is a vertebrate version of the segment polarity gene hedgehog.
It plays key roles in late developmental processes, including:
* Limb development
* Feather formation
* Brain development
* Eye development
