W2-L3+L4-Axis formation and segmentation in Drosophila melanogaster
when can you draw a fate map?
at the blastoderm stage -single cell embryo -amnioserosa:not part of embryo later
secondary pair rue genes
1-fushi tarazu 2-odd-paired 3-odd skipped 4-sloppy paired 5- paired
primary pair rule genes
1-hairy 2-even skipped 3- runt
Morphogens
=a protein that forms a gradient that has a source where concentration is highest -form a conc. gradient -present in a conc. gradient -source of morphogens is in the head (conc. highest) lowest toward posterior end
Segment polarity genes
=members of the wingless and hedgehog signal transduction pathway -engrailed -wingless -cubitus interruptus -hedgehog -fused -armadillo -patched -gooseberry -pangolin
Dorsal
=ventral morphogen =TF has to bind to mRNA in nucleus -responsible for the formation of the dorso-ventral axis -Amnioserosa: extra amniotic tissue that will not form an embryo later -Neurogenic ectoderm> trunk and CNS -Mesoderm=one of germ layers>muscle and connective tissue
wingless cuticle phenotype
wingless mutation: -very small larvae completely hairy>naked cuticle is missing -fusion of denticle belts -naked cuticle missing
5 morphogen gradients generated in the Drosophila egg
-5 morphogen gradients that act together to tell nuclei what cells to form -anterior to posterior gradients: 1-bicoid protein 2-hunchback protein -posterior to anterior gradients: 3-nanos protein 4-caudal protein -ventral to dorsal gradient: nuclear dorsal protein
Where is dorsal active?
-Dorsal is only active in the ventral part of the embryo -genes named after mutant phenotype -antibody stain for dorsal protein Dorsal protein Wild type: -protein is only present in ventral nuclei -only active and functional in ventral nuclei Dorsal mutant : if dorsal is forced on the dorsal side>become ventral -dorsal protein in all nuclei=ventralised embryo -dorsal cells are absent
what are gap gene expression patterns established and maintained by?
-Gap gene expression patterns are established by maternal morphogens and maintained by mutual repression -depending on conc. we will have activation of diff. TFs -naked DNA is easily accessible to TFs so they can bind easily>not possible in later cellular environment -need a system to maintain striped regions>genes inhibit each other=mutual inhibition -High levels of bicoid and hunchback(anterior morphogens) activate giant -low levels of hunchback(posterior region morphogen) activate kruppel -caudal activates knirps and giant>in the middle
Do morphologically visible segments coincide with pair rule expression pattern?
-No morphologically visible segments don't coincide with pair rule expression pattern -cant say that stripe 1=segment 1 -morphologically visible segment border forms exactly within each stripe -when pattern is projected onto later embryo we can see that pair rule expression is formed exactly where the segment border forms
What is pair rule gene expression regulated by?
-Pair rule expression is regulated by Gap proteins -gap genes either repress/activate the pair rule genes -need enhancers that are responsible for individual stripes and we need genes higher up cascade that activate or repress individual stripes
What is the other function of the wingless segment polarity genes?
-Wingless also determines the cell fate in epidermal cells anterior to engrailed expressing cells -genes can have many diff. functions in development -wingless and engrailed could have other functions during later development e.g. in Nervous system -wingless is responsible for formation of cells with naked cuticles
What results in the formation of segments in drosophila?
-a segmentation cascade results in the formation of segments 1-Maternal effect genes>morphogen gradients 2-Gap genes>subdivision of embryo into larger areas 3-Pair-rule genes>even further subdivision of embryo>1st stage when u can distinguish metameric units 4-segment polarity genes>pattern formation with individual segments and formation of pattern within segments
the pair rule gene expression patterns divide the embryo into precursors of the segmental units
-always show 7 stripes=14 segments -diff. kinds of pair rule genes 1-even skipped>form even number segments 2-fushi tarazu form odd number segments -cannot see structure of segment metameric gene expression> egg looks homogenous
signal transduction pathways
-at this stage of embryonic development we have a cellularised structure>cell membranes are separated individual nuclei -we have cellularised structure>DNA is not accessible>cant only work with TFs -cell communication occurs via signal transduction> one cell sends a signal and the other cell receives the signal> signal goes to nucleus> alters gene expression in nucleus -more complicated than previous stages -signal transduction pathways are impt. in many biological mechanisms and cancer
Anterior to posterior gradient: bicoid
-bicoid=head morphogens, 2 tailed -bicoid mutant egg: no thorax, no head and tails
Several morphogen gradients determine the cell fates along the anterior posterior axis
-by playing with gradient u can get diff. regions of embryo -in anterior end: high conc. of bicoid and low nanos>head structures formed -intermediate conc. of bicoid and nanos>more posterior -ratio of anterior posterior and posterior anterior morphogens determines which parts of embryo are formed
the early embryo of many insects consists of one large cell with many nuclei=Syncytium
-cytoplasm doesn't divide in most arthropods -syncytium=one large cell with many nuclei -stages have been subdivided depending on time and on temp differences -stages at 25C -17 stages-stage 17=preformed larvae -stages 1-5: 1-fertilization occurs 2-first mitotic division 3-4: further divisions-more nuclei-only nuclei divides 5-continous band=CM end of stage 5 cellularised embryo with single layer of cells 10-10th division nuclei migrate to periphery of egg CM appear between individual nuclei
odd skipped mutant phenotype
-deletion of denticle belts in odd-numbered abdominal segments -can have weaker phenotypes and stronger phenotype depending on where the mutation is -every other stripe is affected/missing -only half denticle belts are missing in weaker phenotypes
anerior-posterior and dorso-ventral morphogens subdivide the embryo into head, thorax, abdomen and dorsal/ventral side, respectively
-depends on conc. of morphogens in anterior posterior side and dorso ventral side
dorsal protein activates and represses different zygotic genes, depending on twist nuclear concentration
-dorsal inhibits/switches on other genes like twist
Dpp mutant embryos/larvae
-dorsal structures are missing -one of genes inhibited by dorsal -depending on severity of mutation>the phenotypic effect will be -where strong genes are affected>whole structure is missing -where weak genes are affected>slight part is missing -E87,hr56,hr4, hr93 and H46 are diff. mutant alleles(mild to severe). -All structures above the black lines are absent in the embryos/larvae of the Respective mutants.
formation of the anterior-posterior axis: the egg cytoplasm is not uniform
-embryo gets lots of info before fertilization - when egg cell is formed it gets info about dorsal and ventral -there are sections that are unevenly distributed in the cytoplasm of the cell
How do we define where the segment border forms?
-engrailed is activated in cells that show either high conc.s of Even skipped or Fushi tarazu -engrailed= a segment polarity gene that can be activated by ftz or even skippe gene>expressed in 14 stripes of embryo -wingless=a segment polarity gene that is only expressed in areas where ftz and even skipped are not expressed -wingless is expressed next to engrailed but in regions where Eve and Ftz are absent -ftz=odd skipped pair rule gene -eve=even skipped pair rule genes=6 expressed in alternating stripes -segmental border forms posterior to engrailed expressing cell
what happens in giant-deficient embryo?
-expression in stripe 5 expands -giant is required to repress even skipped expression outside small stripe 5 -when giant is absent>don't have small stripe
Nobel prize Physiology 1995- for the discovery of the genetic control of early embryonic development in Drosophila melanogaster
-found out which genes are present and how they form whole organism and embryo -how genes interact to form an embryo -Edward B. Lewis, Christiane Nusslein Volhard, Eric Wieschaus
Segments and parasegments
-gene expression subdivides into domains -refer to pair-rule stripes as parasegments -parasegments= not what we observe later in development of embryo -pair-rule segment is between posterior region of one section and anterior region of another segment -segment border forms in the middle of the pair rule stripe>pair rule genes are used to define where the segment border is
pattern formation: events that determine spatial organization in the developing organism
-going from a homogeneous structure to a more complex structure -diff. embryonic stages of drosophila -after 2 hours you can see trunk formation
Dpp mutant-weak phenotype
-if dorsal is mutated>embryo is dorsalized -can have diff. severity of phenotype -deletions of dorsal anterior structures> some missing parts not all
describe early embryonic development exhibited by many arthropods.E.g. Tribolium (beetle)
-in many arthropods(inc. humans) anterior-most segments form in a syncytium but remaining segments form sequentially>segments are formed one after another -AP and DV polarity is established in a cellular environment -growth zone is found at end of embryo -thoracic segments and everything else is formed much later sequentially -they use same genes as drosophila but have been altered in function and expression -use another system called Notch signalling which is also used by vertebrates -Notch signalling is involved in segment formation -Drosophila=very specific case> in drosophila all segments are formed at same stage>embryo is divided further and further at each level of development -different developmental mechanism exist between diff. species and phyla
what does the drosophila larvae show?
-it shows a complex pattern of hairs and denticles -they did a mutagenesis screen of mutagenized individual genes -could look at skin only -can clearly distinguish parts of larvae just by looking at cuticle of larvae you can figure out what genes are responsible for development of what part
life cycle of drosophila melanogaster
-larval form is generated in embryo form -larvae hatches from egg cells -3 larval stages> feeds and grows -prepupa=when larvae becomes mobilized and secretes a hard case that protects it -pupa: when metamorphosis occurs>worm like to more fly like
activation of the pathway depends on the concentration of Spatzle
-low concentration of Spatzle in the dorsal side -high concentration of Spatzle in the ventral side
What makes drosophila a good model system for developmental biology?
-mate within hours -we have next gen. within 2 weeks -1st developmental biology model organism -easy to work with and has easy genetics
Maternal effect genes
-messenger RNA is deposited into the unfertilized egg -mother deposits mRNA of morphogens in eggs -morphogen mRNA is anchored to the anterior side of the embryo -when egg is fertilized mRNA is translated>protein can then diffuse freely to the posterior end>forming a gradient
Dpp-strong phenotype
-more severe phenotypes -dorsal expansion of denticle belts up to denticle belts encircling the whole embryo -only patches of dorsal tissue present
where are morphogens deposited?
-morphogens are deposited into the egg in the ovary of the female fly -mother deposits morphogens before fertilization> they forms when eggs are formed in ovary
Genes downstream of Dorsal activate other genes that further subdivide ventral cell fate
-once dorsal is in the nucleus it activates other genes that are impt. in activation of other organ/tissue development -other structures are formed by trigger of dorsal being in nucleus -Dorsal inhibits proteins forming laterally/dorsal side -Dorsal=TF
Pair rule mutant:fushi tarazu
-only expressed in every other segment -first stripy patterns fushi tarazu mutation: -end up with small larvae -every other segment is missing
gap mutation:Kruppel P
-only segments in purple express gene -gene expressed in posterior embryo -area of gene action is absent in mutant>purple part will be missing>smaller larvae
4 enhancers drive expression of individual even skipped stripes
-only when TFs bind to all enhancer elements we can see all 7 stripes -TFs will bind to stripe 3 only=only stripe 3 is expressed -use enhancers that when activated only express genes in a specific stripe -coding region=ORF -fuse enhancer region with fuse gene (lacZ bacterial gene) -whole thing is injected into drosophila embryo -endogenous expression of even skipped stripes -use a staining method to detect bacterial gene
Segmentation cascade
-pair rule genes is when the first metameric patterns in the embryo
key events of embryogenesis occur in the syncytial blastoderm stage
-pole cell formation and migration (pole cells=germline cells; pass on the genetic info to the next generation)> germ line cells will move to gonads when developed -determination of the anterior-posterior and dorso-ventral axes -segmentation (subdivision of the embryo along the anterior-posterior axis)>genes expressed
repeated units
-present in some animals(inc. humans) -process of segmentation=further division of embryo into units
denticle belts
-protrusions you can see coming out of cells during early development
The DNA is injected into an embryo and the protein B-galactosidase is produced
-separate enhancers into individual stripes -inject into embryo -lacZ transcribed and then translated -gene product=B-Gal>can use antibody against B-Gal to see where bacterial gene is expressed in embryo -expressed only in first stripe
Hunchback cuticle phenotype
-still in blastoderm stage -hunchback mutant has 4 thoracic segments missing -3 thoracic segments absent (more anterior structures co-depend on bicoid) -dentical belts
what does the activation of gap genes depend on?
-the activation of gap genes depends on the conc. of morphogens -one set of genes higher up regulates lower genes in a hierarchal segmentation cascade -Bicoid and nanos genes are responsible for stripy second level genes
how is the expression of wingless and engrailed is maintained?
-the expression of wingless and engrailed is maintained by interactions between the products of these genes -set up expression of engrailed using pair-rule genes -set up expression of wingless and engrailed but we have to maintain this expression otherwise pair-rule genes will be switched off -wingless expressing cell and engrailed expressing cell communicate with each other
maintenance of wingless and engrailed expression
-wingless expressing cell sends signal(wingless protein) which binds to receptor on engrailed expressing cell> triggers a cascade of rxns which ends up in nucleus> leads to transcription of engrailed and hedgehog(secreted protein)> goes back to wingless expressing cell> signal transduction pathway back to nucleus> transcription of wingless protein again -expression is set up by pair rule genes>wingless and engrailed in anterior and posterior end -segmental border is formed posterior to engrailed expressing cell -wingless expressing cell tells engrailed expressing cell to keep on expressing engrailed -engrailed expressing cell tells wingless expressing cell to keep on expressing wingless
2 functions of segment polarity genes
1st function: -due to expression of wingless and engrailed we get morphologically visible segmental border=impt. function of segment polarity genes 2nd function: -important for morphology of epidermal cells -segment polarity gene define the pattern within the segment>responsible for anterior and posterior fates>expressed anteriorly -determine which structures are formed -denticle belts are found in anterior region>formed because specific segment polarity genes are expressed in the cells
Activation of the Toll receptors leads to degradation of cactus: Dorsal can enter the nucleus
Dorsal part of embryo: -low conc. of Spatzle protein -Cactus binds to dorsal>keeps dorsal in cytoplasm>cant enter protein Ventral part of embryo: -high conc. of Spatzle protein -Spatzle binds to Toll>signal transduction pathway>changes shape of Cactus>cant bind to dorsal>Dorsal can enter nucleus and change gene expression
evidence that bicoid is required for the formation of head and thorax
Exp 1: took sample of cytoplasm from head region in wild type egg and put it into head of bicoid mutant egg result= head structure formed in anterior end Exp2: took sample from head region of wild type egg and put it in middle of bicoid mutant egg results= head formed in the middle> source of morphogen is in the middle -experiments proves that bicoid morphogens is responsible for defect -same experiment for hunchback,nanos and caudal
diff. between primary pair rule genes and secondary pair rule genes
Primary pair rule genes are expressed first before secondary pair rule genes
What did the nobel prize winners discover?
The nobel prize winners discovered 15 genes of key importance in determining body plan and segmentation
What retains dorsal protein in the cytoplasm?
a complicated signal transduction pathway -results in dorsal entering the nucleus and altering gene expression
Gap phenotypes
mutations: Kruppel mutant embryo: -large area is missing Knirps mutant embryo: segments missing where gene is expressed
bicoid and nanos regulate translation of hunchback(head) and caudal (abdomen)
overlapping gradients inhibit each other and instruct nuclei to form different parts of embryo