Molecular & Developmental Biology
(Complete course)
History and Basic Concepts
Developmental Biology is a fundamental aspect of biology.Development depends upon complex mechanisms and many layers of "biological information" that are superimposed one upon another.
As our techniques improve, developmental biology has the potential to be much better understood.
Once, developmental biology was mostly descriptive (ie. descriptive/comparative embryology).
Modern developmental biology is mostly experimental.
Recent advances in cell biology, genetics and molecular biology has
and will continue to further our understanding of development unlike any
time in the past.
Embryogenesis (embryo formation) determines the overall body plan.
Organogenesis (organ formation) determines subsections of the body (examples: vertebrate limb, Drosophila eye).
Often these processes share much more than is first obvious.
Many genes, proteins, signal transduction pathways and cell behaviours are common to both processes.
Major Developmental Biology Questions:
1) What processes happen during development?
2) What mechanisms control development?
3) How can we control development?
4) To what goals can we apply controlled developmental biology?
Xenopus Development (figure): A well characterized example of early development
Early Xenopus development: fertilizationThe unfertilized egg is a single large cell.
The animal pole, the upper part of the egg, has a pigmented surface.
The vegetal pole, lower region contains the yolk.
After fertilization, the male nucleus (from sperm) and female nucleus (from egg) fuse to form one nucleus.
After fertilization, cleavage begins without growth (mitotic division only).
Xenopus blastulation
After ~12 cycles of division make a layer of small cells surrounding a fluid-filled cavity (the blastocoel) that sits on top of the large yolk cells.
Three germ layers are mesoderm, endoderm and ectoderm
The mesoderm is located at the "equator" and becomes muscle, cartilage, bone, heart, blood, kidney
The endoderm is below the mesoderm and the ectoderm and becomes gut, lungs and liver
The ectoderm sits above the mesoderm and the endoderm and becomes the epidermis and nervous system
In the blastula, these layers are all on the surface and they interact!
Xenopus gastrulation & neuralation
Gastrulation is an extensive rearrangement of embryonic cells mesoderm and endoderm move to the inside of the embryo to give the basic body plan.
For the most part, the inside of the frog is now inside and the "outside" except for the skin is outside.
Notochord is a rod-like structure that runs from the head to the tail and lies beneath the nervous system.
Somites are segmented blocks of mesoderm form on either side of notochord which become muscles, spinal column and dermis (skin).
Neuralation occurs when ectoderm above the notochord folds to form neural tube (becomes spinal cord & brain).
The tailbud stage follows the completion of neuralation.
Epigenesis versus preformation
Epigenesis (~upon formation) is a theory of development that states that new structures arise by progressing through a number of different stages.Preformation theory suggests that all structures exist from the very beginning, they just get larger.
One preformation theory, the theory of the Homunculus suggested that a little human embryo was hidden in the head of every sperm.
[This theory has fallen out of favour.]
Error in Logic:
Since an embryo grows to be an adult and that adult produces another embryo and so on indefinitely,
according to the theory of preformation,
then the very first embryo must included within itself tiny copies of all the future embryos (the Russian Doll Conundrum).
Cell theory
Organisms are composed of cells, the basic unit of life.Both animals and plants are multicellular composites that arise from a single cell.
Therefore development must be epigenetic and not preformational since a single cell (the fertilized egg) results in many different types of cells.
Only the germ cells (egg and sperm) pass characteristics on to the offspring.
Somatic cells are not directly involved in passing on traits to the next generation and characteristics acquired during an animal's life are not passed onto the offspring.
To quote Samuel Butler: "A hen is only an egg's way of making another egg." Meiosis and fertilization
Meiosis is the reduction division that allows diploid precursor cells to generate haploid germ cells.
At fertilization, a diploid is reformed by joining two haploid germ cells.
The diploid zygote contains equal numbers of chromosomes from each of two parents.
Observations of sea urchin eggs revealed that after fertilization the egg contains two nuclei which fuse to form a single nucleus.
The nucleus must then contain the "physical basis of heredity."
Mosaic versus Regulative Modes of Development
Mosaic developmentdepends upon specific determinants in the one-celled zygote that are not divided equally between the daughter cells (asymmetric division).
Roux (1880's) destroyed one cell of a two-celled embryo (with a hot pin) to result in ~1/2 frog embryo.
Regulative development depends upon interactions between 'parts' of the developing embryo can result in causing different tissues to form (even if parts of the original embryo are removed).
Driesch destroyed one cell of a sea urchin embryo at the two cell stage and a normal appearing but smaller sea urchin larvae resulted. Regulatory development: induction
Induction is a type of regulatory development.
This is a process where one tissue directs the development of another tissue.
A classical experiment: Spemann & Mangold (1924) - graft of the blastopore lip of one newt onto another!
Note: The blastopore is the opening formed in early gastrulation through which cell migrate inside.
The Spemann Organizer can induce the formation of an ectopic axis (twinned embryo).
Model organisms in developmental biology
Although, we are mostly interested in human development (selfish reasons) many aspects of development are conserved in distantly related species.The major Model Organisms used to study the principles of development are...
nematode (Caenorhaditis elegans)
fruit fly (Drosophila melanogaster )
sea urchins
South African claw-toed FROG ( Xenopus laevis )
chick (Gallusgallus) (see Malpighi's drawings - circa 1673 - of the early and 2 day chicken embryos)
mouse (Mus musculis)
plant (Arabidosis thaliana)
Genetics has been key to Development
The developmental genetics of Drosophila melanogaster, Caenorhabditis elegans and mice are best known.Homologous genes identified in these organisms are found in other species.
Dominant (or semi-dominant) mutations: one copy of mutant gene produces mutant state.
Recessive mutations: two copies of a mutant gene gives the mutant state.
Mutants can arise spontaneously but induced mutation and screening has become the standard way to identify developmentally important genes. 5 Processes of Development
1. Cleavage Division: No increase in cell mass
2. Pattern Formation: A/P and D/V axes: Coordinate system
3. Morphogenesis: take 3D form, neural crest migrates far. 1 egg- 250 types
4. Cell Differentiation: cells become structurally and functionally different.
5. Growth: cell multiplication, increase in cell size, deposit extracellular material (bone, shell) growth can be morphogenetic.
5 Cell Behaviours
Cell Behaviour is the key link between Gene Expression and Development.
In brief, it is gene activity gives cell identity.
1. Cell-cell communiaction
2. Cell shape changes
3. Cell movement
4. Cell proliferation
5. Cell death (apoptosis)
Cell fate
Differential gene expression controls cell differentiation.
Positive and negative feedback mechanisms are important in establishing cell fates.
Common house-keeping genes do not cause cells to differ!
Developmentally specific transcription factors direct differential gene expression.
Technique: In situ hybridization
Purpose: To determine when and where a particular gene's mRNA is present.
Details: Complementary single-stranded nucleic acid strands (RNA or DNA), commonly known as "probes", will bind tightly (hybridize) to a specific mRNA.
This can be visualized if the probe is labeled (or tagged) with an isotope, a fluorescent dye or an enzyme that produces a coloured substance.
When embryos (or another tissue) are fixed and permeabilized then allowed to undergo such a hybridization reveal the pattern of gene expression.
Whole mount and tissue sections can be visualized.
Cell Fate is what cells should become (not differentiation).
Specification cells keep their fate even when isolated and is tested by transplantation (some cells change their fate).
Early embryonic cells are not narrowly determined, latter ones are!
transcription & processing nuclear export, translation & modification
Inductive interaction is the process by which one group of cells change the fate of another group of cells.
The information to cause induction passes from cell to cell in the form of ...
1. secreted diffusible molecule
2. surface molecule receptor
3. gap junction (channel)
Competence: the state of being able to respond to inductive signals due to the presence of receptor or transcription factors.
Positional information directs pattern formation
Positional information directs pattern formation by giving positional values to cells.
The French Flag model (blue, white and red stripes) refers to the assignment of positional values in response to a morphogenic gradient.
This biological information must first be specified and
Then the value must then be interpreted .
Morphogen varies in concentration and directs different fates at different concentrations.
[high] [low]
Generative program: Development depends upon a progressive series of instructions.
An embryo needs to have each action to be built upon the previous action and that on the one before.
Development instructions are not a "blue print" but is a structural list of actions.
Lateral Inhibition: Many structures are regularly spaced.
Cells that form a structure stop neighbouring cells from doing the same (feathers, compound eye faucets).
Lineage dependent fate: Cytoplasmic localization and asymmetric cell division control the fate of resulting cells.
Stem cells can produce daughter cells that are also stem cells.
However, some daughter cells become different & give rise to different lineages.
One last thing!
It is a really good idea to construct a glossary or mini-dictionary of Developmental words and phrases.
A/P axis: anterior ~ head; posterior ~ tail.
D/V axis: dorsal ~ upper or back; ventral ~ lower or front.
P/D axis: proximal ~ near; distal ~ far.
Lateral: to the side.
haploid ~ 1 set (of chromosomes) .
diploid ~ 2 sets (of chromosomes).
Model Organism: Drosophila melanogaster
Drosophila melanogaster: early embryogenesisThe Drosophila egg is the shape of a sausage.
It has a micropyle at the anterior end (site of sperm entry).
With fertilization, the fusion of nuclei is followed by rapid mitotic divisions (90 minutes) and no cleavage.
A syncytium is formed (many nuclei/common cytoplasm).
After nine divisions, nuclei move to the periphery to form the syncytial blastoderm (2 hours).
Drosophila melanogaster: embryogenesis
By 13 mitoses the membranes sprout to surround the nuclei to form cells (cellular blastoderm).
~15 cells at posterior (pole cells) are sequestered and become the germline.
During first ~3 hrs. large molecules such as proteins can move between nuclei until the cellularization occurs.
Single layer of cells give rise to all tissues.
Gastrulation starts at ~3 hrs.
1) Mesoderm forms from ventral tissue.
2) Midgut from endoderm at the anterior and posterior ends.
3) Ectoderm remains on outside.
Drosophila melanogaster: gastrulation
The mesodermal tube forms from ventral tissue then cells separate & move to internal locations under the ectoderm.
The mesoderm becomes muscle and connections tissues.
In insects, the nerve cord lies ventrally (vertebrates: dorsal).
Neuroblasts form a layer between mesoderm and outer ectoderm.
The midgut (anterior & posterior) grow from threads and fuse.
= anterior and posterior midgut
Ectoderm becomes epidermis.
No cell division occurs during gastrulation but division restarts afterward.
Drosophila melanogaster: segmentation
The germband (ventral blastoderm) is main trunk region.
The process of germ band extension pushes the posterior end over dorsal side.
The first signs of segmentation grooves appear to outline parasegments which give rise to segments.
Segments are formed from the posterior of one parasegment and the anterior of the next.
There are14 parasegments: 3 mouth, 3 thorax, 8 abdominal.
Drosophila melanogaster: larvae
The larvae hatch at 24 hrs post-fertilization.
The larval structures of note include ...
The anterior end is the acron.
The posterior end is the telson.
Along with the head, the larvae has 3 thoractic segments and 8 abdominal segments.
The ventral side of the larvae has denticle belts, alternating patches of denticle hairs and cuticle on each segment, used for locomotion.
Drosophila melanogaster: metamorphosis
Three instar stages of larval life are separated by molts.
1st instar -(Molt)-> 2nd instar -(Molt)-> 3rd instar
Third instar larvae forms pupae (pupation) to undergo metamorphosis.
The adult tissues arise from imaginal discs and histoblasts.
The imaginal discs are small sheets of epidermis (~40 cells each of cellular blastoderm) which grow throughout larval life.
6 leg, 2 wing, 2 haltere, 2 eye-antenna, plus genital, head discs and ~10 histoblasts (nest of cells in the abdomen which give rise to the abdominal segments).
Drosophila development: the body plan
Genes that control development in Drosophila are very similar to those that control development in vertebrates.
Drosophila is the best understood developmental system with great impact upon our knowledge of all development. (for example, Hox genes were first found in Drosophila.)
Bilateral symmetry is established by the A/P and D/V axes.
The larvae has an anterior acron, three thoracic and eight abdominal segments and a posterior telson.
Early patterning occurs in the syncytial blastoderm and it becomes multicellular at the beginning of segmentation.
Concentration gradients of proteins (transcription factors) can diffuse, enter nuclei & provide positional information.
Technique: Mutagenesis and genetic screening
Although mutants can arise spontaneously, induced mutation and screening has become the standard way to identify developmentally important genes.
To generate mutants in a specific gene, a chemical mutagen, such as ethyl methane sulfonate (EMS), is fed to a large number of male flies.
The sperm cells of these males are exposed to the mutagen.
The males are bred to females that carry a balancer chromosome of the gene of interest.
Individuals carrying a mutagenized chromosome and a balancer are isolated.
These are crossed to individuals carrying the balancer chromosome.
In the next generation, offspring that carry both the mutagenized chromosome and the the balancer chromosome (balanced heterozygotes) are crossed.
Homozygous progeny are examined and balanced heterozygous siblings are selected to maintain the line.
Drosophila development: maternal and zygotic genes
Maternal genes establish the body axes.
Maternal gene products, mRNAs and proteins are expressed in the ovary.
Zygotic genes are expressed by an embryo.
About fifty maternal genes set up the A/P and D/V axes: the framework of positional information (spatial distributions of RNA and proteins).
Zygotic genes respond to maternal gene expression.
First broad regions are established, then smaller domains (with a unique set of zygotic gene activities) in a hierarchy of gene activity.
Drosophila development: the A/P axis
Three classes of maternal genes set up the A/P axis
Maternally expressed genes distinguish the anterior from the posterior.
Maternal effect mutants result in females that can not produce normal progeny.
Three mutant classes are 1) anterior, 2) posterior and 3) terminal classes.
Anterior class: loss of head and thorax (sometimes replaced with posterior).
Posterior class: loss abdominal segments.
Terminal class: missing acron and telson.
bicoid, hunchback, nanos and caudal are key to A/P axis.
Drosophila development: maternal genes
bicoid is sequestered in the oocyte during oogensis.
bicoid sets a A/P morphogenic gradient and controls the first steps in embryo development and, thus, is essential to the developing organism.
bicoid mRNA is localized to the anterior end of the unfertilized egg.
After fertilization, the mRNA is translated and a concentration gradient is formed along the A/P axis.
bicoid was the first evidence of a morphogen gradient.
Drosophila development: clues to the role of bicoid
1) bicoid (bcd) females lay eggs that give rise to embryos missing the head and thorax (and have an anterior telson).
2) Embryos missing anterior cytoplasm resemble above.
3) bcd embryos rescued by anterior cytoplasm injections.
4) Anterior cytoplasm can induce ectopic head & thoracic segments by injection in the middle of a bicoid egg.
5) in situ hybridization shows bicoid RNA is at the anterior part of the unfertilized egg (attached to cytoskeleton).
6) Protein not in egg, forms A/P gradient after fertilization.
7) bicoid: transcription factor & morphogen.
8) other anterior-group (group 1) maternal genes are involved in bicoid localization and translational control.
The posterior pattern is controlled by nanos & caudal protein gradients (group 2)
nanos mRNA is localized to the posterior pole of the egg.
nanos is NOT a morphogen like bicoid but acts to suppress translation of another maternal gene, hunchback (hb).
hunchback is maternal (present at low levels in embryo) AND zygotic (the latter is activated by high bicoid levels).
nanos (and pumilio) bind hb mRNA to prevent translation.
caudal mRNA is distributed evenly.
The P-A gradient of caudal is established by inhibition of caudal protein synthesis by bicoid.
[bcd and hb run in A to P gradients & caudal runs P to A.]
Anterior and posterior extremes are specified by cell-surface receptor activation
Group 3 maternal genes specify the acron and telson regions.
torso mutants develop neither acron nor telson regions.
torso encodes a uniformly distributed receptor protein which is activated by ligand present only at the anterior and posterior parts of the vitelline membrane.
The ligand is released after fertilization.
torso (a receptor tyrosine kinase) signals to direct terminal zygotic gene expression.
D/V polarity is due to vitelline membrane proteins.
At fertilization, a protein deposited on the ventral vitelline membrane initiates a series of reactions which, in part, activates (cuts) spatzle: the ligand for the uniformly distributed receptor Toll.
dorsal provides positional information along the D/V axis
dorsal provides positional information along the D/V axis
In the syncytial blastoderm, dorsal (a transcription factor) is activated and enters nearby nuclei.
Dorsal is in highest concentration in ventral nuclei (little or none is present in the dorsal nuclei).
Toll signals the degradation of the maternal protein cactus.
Without Toll signal, cactus binds dorsal to keep it in the cytoplasm.
Dorsal and cactus are homologues of vertebrate NF-kappa-B and I-kappa-B.
Polarization of the body axes during oogenesis
Polarization of the body axes during oogenesis
In the germarium, a stem cell gives rise to 16 cells by four mitotic divisions which become the oocyte and 15 nurse cells, all which are connected by cytoplasmic bridges.
A sheath of somatic follicle cells surround the nurse cells and oocyte to form the egg chamber which secrete the vitelline membrane and egg shell.
Signals from older egg chambers act to polarize younger ones.
Some common signals are here.
A/P and D/V axes of the oocyte are specified by interactions with follicle cells
The oocyte induces follicle cells to adopt posterior fate & the anterior follicle cells are not in contact with the oocyte.
The signal from the oocyte to the follicle cells is the gurken protein, a member of the TGF-alpha family.
gurkin binds to torpedo, a receptor tyrosine kinase similar to the EGF receptor.
Follicle cells signal back to reorganize the oocytes cytoskeleton which direct bicoid mRNA to the anterior and oskar mRNA (which specifies germ plasm) and nanos mRNA to the posterior.
Later the D/V axis is set up by gurken (again) which signals to establish dorsal follicle cells (which do not produce the ventral follicle cell proteins needed for establishing ventral embryo fates).
A/P axis is divided into broad regions by gap genes
The gap genes, the first genes expressed along the A/P axis are transcription factors.
The gap genes are initiated by bicoid in the synctial blastoderm.
hunchback acts to help switch on the other gap genes (giant, Kruppel and knirps).
Mutants of gap genes have large sections of the body pattern missing.
Gap gene proteins are short lived (half-life of minutes) and extend only slightly outside of where the gene is expressed (bell-shaped concentration distribution.)
bicoid protein signals anterior hunchback expression
Zygotic hunchback expression is in the anterior half of the embryo.
Suppression in the posterior half, produces a gradient running A to P.
Anterior expression is switched on by high levels of bicoid.
Increased anterior bicoid expression will result in extending the hunchback gradient toward the posterior half of the embryo.
bicoid (homeodomain transcription factor) directly binds the hunchback promoter in several places.
hunchback activates and represses other gap genes
Kruppel is activated by a combination of bicoid and low levels of hunchback but is repressed by high levels of hunchback.
This locates Kruppel expression to the centre of the embryo.
knirps is repressed by high levels of hunchback .
In this way the initial gradients of morphogens can lead to the establishment of regions within the syncytial blastoderm which themselves lead to the beginning of segmentation.
Technique: Transgenic Drosophila
P element transformation is accomplished by cloning a sequence of interest (genomic region, cDNA or control region/reporter gene fusion) and a marker gene (often the white gene) into a cloned transposable P element.
The cloned DNA along with a source of transposase (helper plasmid) are injected into the pole plasm of an early embryo.
If incorporated into the germline, progeny of the injected individual that express the marker gene can be selected.
These will also carry the transgene.
Transgenesis allows manipulation of developmental processes segmentation: pair-rule gene activation
Technique: Targeted Gene Expression
One way to control gene expression is by fusing the heat-shock promoter to a given gene.
Another method involves the two-part Gal4/UAS system.
Gal4, a yeast transcription factor, is fused to Drosophila control sequences by
1) recombinant DNA cloning (and making transgenic Drosophila)
and 2) enhancer trap (random integration/selection)
to produce the transcription factor in a desired developmental pattern
and generate a driver line.
The responsive line is generated by cloning a coding region downstream of several copies of the UAS (up-stream activating sequence) and transgenesis.
By mating individuals of these lines, the targeted gene is expressed in the selected times and places.
Zygotic gene expression along D/V axis is controlled by dorsal protein
dorsal drives gene expression to activate and inactivate a number of genes by binding to the regulatory genes of many of genes it controls.
dorsal specifies the most ventral cells as prospective mesoderm.
High levels of dorsal activates twist and snail (required for mesoderm and gastrulation).
Low levels of dorsal activate rhomboid (which is suppressed by snail) to give rise to the neuroectoderm.
decapentaplegic (dpp), tolloid and zerknult are suppressed by dorsal and are restricted to the most dorsal regions.
zerknult specifies the amnioserosa.
Zygotic genes pattern the early embryo
The most ventral region become mesoderm (muscle and connective tissue).
Ventral ectoderm becomes neurectoderm (some epidermis and all nervous tissue).
Dorsal ectoderm becomes dorsal epidermis and the amnioserosa (an extra-embryonic membrane).
The endoderm from the terminal regions, give rise to the midgut.
dpp protein patterns the dorsal region
dpp is a member of the TGF-beta family of secreted growth factors.
After cellularization, dpp is expressed in cells that do not have dorsal in the nucleus.
It produces a gradient of activity by binding an inhibitory protein sog (short gastrulation).
sog is very similar to the vertebrate protein chordin.
Parasegments (PS) are the basic module of fly development
Parasegments arise first & each segment is made from the posterior part of one PS and the anterior of the next.
Parasegments are delimited by periodic expression pair-rule (PR) genes.
Transient grooves on embryo surface (after gastrulation) define the 14 PS.
Parasegments act as developmental units: "piece-meal fly".
Pair-rule genes delimit the parasegments
Pair-rule genes delimit the parasegments and are expressed in 7 transverse stripes (every 2nd parasegment).
Pair-rule expression determined by gap gene activity to interpret a series of broad expression patterns to make a repeated series of stripes.
Gap gene activity positions stripes of pair-rule expression
Pair-rule genes are expressed in alternate parasegments.
even-skipped defines odd parasegments.
fushi-tarazu define even parasegments.
Striped expression pattern of pair-rule genes begins just before cellularization.
After cellularization, each pair-rule gene is restricted to a few cells in seven stripes.
Some pair-rule genes define segment boundaries.
Stripes appear slowly, first fuzzy then later become sharply defined.
even-skipped is first expressed at low level in all nuclei but then redefines into stripes.
Each stripe is independently specified
The 2nd stripe of even-skipped (eve) requires bicoid & hunchback.
giant represses eve to form a sharp anterior border.
Kruppel represses eve to form a sharp posterior border.
Since each stripe is independently controlled by combinations of transcription factors (gap genes).
Each pair-rule gene has complex control regions with multiple binding sites for each of the different factors.
Some factors activate and other inactivate.
Some require the activity of the primary pair-rule genes (such as eve and hairy).
The 3rd and 4th stripes of eve are highly directed by the hunchback gradient.
Segment polarity (SP) (or "Segmentation"_ genes and compartments
Segment polarity/segementation genes are ...
1) a diverse group of genes (not just transcription factors),
2) are expressed in 14 stripes,
3) act after cellularization and
4) are activated by the pair-rule genes.
engrailed (a transcription factor) is expressed in the anterior of each parasegment to define a boundary of cell lineage restriction.
engrailed is a selector gene which confers identity by a duration of expression.
Technique: Genetic mosaics
A genetic mosaic is an individual that has some tissues that carry cells of different genetic constitutions.
Formally accomplished via X-rays.
Flies carrying a yeast recombinase (FLP) and target sequence (FRT), can be induced to form clones of mutant tissue in an otherwise normal individual.
Expression of engrailed delimits a cell lineage boundary and defines a compartment
engrailed is expressed throughout the life of the fly (not transient like gap and pair rule genes).
A parasegment is a compartment that cells do not move between (cell lineage restriction).
Compartments can be detected by marking cells and following the fates of the clones (cell's descendants).
engrailed defines anterior margin of parasegment and thus the posterior portion of the segment.
Compartment boundaries can be studied in the adult wing which is normally divided into anterior and posterior compartments.
In a mosaics, engrailed mutant cells do not respect the "A/P boundary" and lineages are not restricted .
Segment polarity genes pattern the segments and stabilize parasegment and segment boundaries
Each larval segment has an A/P pattern: the anterior part has denticles while the posterior part has naked cuticle.
In wingless & hedgehog mutants, the naked cuticle is converted to a mirror image duplication of the anterior part to give the "lawn of denticles" phenotype.
Segment polarity genes are expressed in a restricted subset of the cells of each parasegment.
wingless and hedgehog encode highly conserved proteins and are part of a number of signaling systems.
Parasegment boundary depends on the intercellular signaling between cells on either side of the compartment boundary involving segment polarity genes.
The patterning process is also apparent on the abdominal segments of adults.
Different mechanisms used by other insects for the body plan
Long germ band development develops all segments at once (Drosophila).
Short germ band development (Tribolium, the flour beetle), the anterior segments are formed in the blastoderm and the more posterior segments are added by growth of the posterior.
The mature germ bands appear to be similar (phylotypic stage, common to insects).
Although different growth processes are involved the same genes (i.e. Kruppel, wingless and engrailed) have conserved functions.
Segment Identity: selector and homeotic genes
Each segment has an unique identity.
Homeotic selector genes specify each segment to control other genes and maintain segment identity.
Two complexes [or a split complex] (Bithorax and Antennapedia: the HOM genes), together are homologous to the HOX gene complexes of vertebrates.
First identified by homeotic genes, mutations in which cause homeosis, the transformation of one structure into another structure.
Antenna to leg (Antennapedia) or haltere to wing (Bithorax)
Homeotic genes of the bithorax complex (BX-C) are responsible for the posterior segments
Bithorax complex (BX-C) consists of three homeobox genes (Ubx, abd-A & Abd-B).
Ubx is expressed from PS 5 and posterior.
abd-A is expressed in PS 7 and posterior.
Abd-B is expressed in PS 10 and posterior (and suppresses Ubx).
Expression is controlled by gap & pair-rule genes.
Larvae missing the complete bithorax complex, develop PS 5-13 as PS4, thus BX-C diversifies PS5-13 and PS4 is the default state modified by the BX-C proteins.
BX-C genes impose a new identity to the segments (selector genes).
Experiment: Replace BX-C components into embryos missing the complex.
BX-C absence: PS1-4, plus ten more PS4 like segments.
BX-C components were replaced by targeted gene expression.
Ubx only (missing abd-A and Abd-B): PS1-6 followed by seven more PS6-like segments.
Ubx plus abd-A (no Abd-B): PS1-9 plus 4 PS9 segments.
Parasegments must be acting in a combinatorial manner.
While gap and pair-rule genes control the original pattern of HOM gene expression, the polycomb and trithorax gene groups maintain the correct expression of these genes after first four hours.
The polycomb group maintain transcriptional repression of homeotic genes.
The trithorax group maintain expression of homeotic genes.
Antennapedia complex controls specification of anterior regions
Antennapedia complex (Antp-C) consists of 5 homeobox genes.
Antp-C control expression anterior parasegments in a manner similar to BX-C in the posterior segments (described above).
deformed mutants affect PS0&1.
Sex combs reduced mutants affect reduced PS2&3.
Antennapedia mutants affect PS4&5.
As with HOX genes in mammals, HOM gene expression order corresponds to the order of genes on the chromosome.
Vertebrate Development I: Life Cycles and Experimental Techniques
Model Organisms: VertebratesA few have been studied extensively, each has advantages and disadvantages.
Frog (Xenopus laevis): independent development but the known genetics is poor.
Chick (Gallus gallus): available, surgical manipulation and in vitro culture but poor genetics.
Mouse: good genetics (knockouts) but development is in utero. Please note the repeated pattern of segmentation. All vertebrate embryos undergo a similar pattern of development.
1) fertilization
2) cleavage
3) blastulation
4) gastrulation (where ectoderm covers embryo, endoderm and mesoderm are inside).
The Vertebrate Body Plan
The vertebrate body plan consists of the antero-posterior axis (segmented vertebral column and skull) and the dorso-ventral axis (including the ventrally located mouth).
All vertebrate embryos pass through the phylotypic stage when the embryos are all similar in appearance.
These embryos share 1) the head,
2) the neural tube which forms the brain and spinal cord (under which is the notochord, an early mesoderm structure along A/P) and
3) mesodernal somites (blocks of mesoderm flanking the notochord which form the muscles of the trunk & limbs).
The large eggs of fish, frogs and bird have large yolks that provide nutrients to the developing embryo.
Mammalian eggs are small and obtain nutrients from the ovoduct then the placenta.
Model Amphibian Embryo: Xenopus laevis
Xenopus laevis: eggThe egg is composed of an animal & a vegetal region, both covered by vitelline membrane.
Meiosis is stopped at 1st division with apparent 1 polar body (the 2nd polar body comes after fertilization).
After fertilization, the cortex (the layer below plasma membrane) rotates to determines future dorsal region at a position opposite to the site of sperm entry. Xenopus laevis : fertilization and early growth
1. one sperm enters animal region
2. completes meiosis
3. egg and sperm nuclei fuse
4. vitelline membrane lifts
5. yolk rotates to down (15 mins)
6. cortical rotation (60 mins). Cortex is layer below plasma membrane
-rotation determine future dorsal region (opposite sperm entry site)
7. 1st cleavage (90 mins) A/V
8. 2nd cleavage (110 mins) A/V 90 degrees to 1st
9. 3rd cleavage (130 mins) equatorial (4 small animal and 4 large vegetal= 8 blastomeres)
Xenopus laevis: blastulation & gastrulation
The blastula (after 12 divisions, thousand's cells) has radial symmetry.
The marginal zone will become the internalized mesoderm and endoderm.
Internalization of the mesoderm and endoderm starts at the blastopore.
1) mesoderm and endoderm converge and begin to move inwards at dorsal lip of the blastopore
2) this extends inwards along A/P axis
3) ectoderm spreads to cover embryo= EPIBOLY
4) dorsal endoderm separates mesoderm from the space between the yolk cells = ARCHENTERON (future gut cavity)
5) lateral mesoderm spread ventrally to cover inside of archenteron.
Xenopus laevis: late gastrulation
By the end of gastrulation...
1) dorsal mesoderm is beneath dorsal ectoderm
2) mesoderm spread to cover gut
3) epiboly- ectoderm covers embryo
4) yolk cells are internalized (food source)
5) dorsal mesoderm develops into a) notochord (rod along dorsal midline) and b) somites (segmented blocks of mesoderm along notochord).
Xenopus laevis: Neuralation
Neuralation or neural tube formation
1) The neural plate is the ectoderm located above notochord and somites.
2) The edge of the neural plate forms neural folds which rise towards midline.
3) The folds fuse to form neural tube.
4) The neural tube sinks below epidermis.
The anterior neural tube becomes brain. Mid and posterior neural tube becomes spinal cord.
The somites...
The dorsal part of somites become dermatome (future dermis).
The rest of each somite becomes vertebrae and trunk muscles (and limbs).
Lateral plate mesoderm becomes heart, kidney, gonads and gut muscles.
Ventral mesoderm blood-forming tissues.
Also at this stage, the endoderm gives rise to the lining of the gut, liver & lungs.
Xenopus laevis: early tail bud stage
After gastrulation, the early tail bud stage occurs.
In the anterior embryo,
a) the brain is divided,
b) eyes and ears form,
c) 3 branchial arches form (anterior arch later becomes the jaw).
In the posterior embryo, the tail formed last from dorsal lip of blastopore by extension of notochord, somites and neural tube.
Xenopus laevis : Neural Crest Cells
Neural crest cells come from the edges of the neural folds after neural tube fusion.
They detach and migrate as single cells between the mesodermal tissues.
to become:
1) sensory and autonomic nervous systems
2) skull
3) pigment cells
4) cartilage
Model Fish Embryo: Zebrafish
The zebrafish is quickly being established as the model for fish development.The short life cycle of ~12 weeks and the transparent 0.7 mm embryo are great advantages.
Similar to the chick, cleavage does not involve the yolk and results in a zygote forming on top of the yolk. Technique: Large-scale mutagenesis in zebrafish
Large transparent embryos can be examined for developmental abnormalities.
Male fish are treated with mutagen and mated to wild type females.
F1 males are backcrossed to wild type females.
F2 males and females are crossed and examined for homozygous mutant progeny (~25% or 3:1) .
Alternatively, F1 females mated with heavily irradiated sperm will develop haploid offspring.
Although, haploids do not survive for long, they do go through early development.
Mutants can be recovered by rebreeding selected F1 females to wild type males.
Model Avian Embryo: Chick (Gallus gallus)
Chick embryo: the blastodiscThe blastodisc arises through cleavage and is formed within 20 hours of fertilization.
The chick blastodisc can be divided into two areas:
1) the area pellucida (a light area) surrounded by
2) the area opaca (a dark ring).
The posterior marginal zone (PMZ) forms at the junction of the area pellucida and the area opaca and defines the dorsal side and posterior end of the embryo.
The hypoblast (the source of extra-embryonic tissues) develops as a layer on top of yolk and develops from cells from the posterior marginal layer and the overlying cells of the blastoderm. Chick embryo: the primitive streak
The primitive streak is a slit or line on the disk which lays down the A/P axis.
This structure begins to form from the posterior marginal zone and extend to a point in the central region of the disk.
Cells move towards the streak and mesoderm & endoderm internalize at this site.
When the primitive streak reaches its greatest length, the anterior end begins to regress back to the posterior end.
The anterior end of the regressing streak is known as Hensen's Node.
Chick embryo: gastrulation
As Hensen's Node moves toward the posterior, several structures form behind it.
1) The head fold (from ectoderm and endoderm)
2) The notochord and somites (from mesoderm)
3) The neural tube forms above the notochord (from ectoderm)
(The anterior structures are formed first while the posterior structures are completed last.)
4) Neural folds fuse at the dorsal midline and neural crest cells migrate away
5) Finally, the head fold separates, gut forms and heart pieces fuse to form heart.
Model Mammalian Embryo: Mouse
Mouse embryo: fertilizationFertilization of the mouse ovum occurs in the oviduct.
Cleavage also occurs in oviduct: 1st at 24 hours and every 12 hours after that to form the morula (ball of cells).
Blastomere compaction happens at 8 cell stage and results in smooth inner membranes and outer membranes covered with microvilli.
The trophectoderm becomes extra-embryonic tissues.
The inner cell mass (ICM) becomes the embryo plus some extra-embryonic tissues. Mouse embryo: blastocyst
The morula (~32 cell stage) has 2 cell fates: inner 8 cells (Inner Cell Mass) and outer ~20 cells (trophectoderm).
In the blastocyst (~3&1/2 days), the trophectoderm and ICM are established.
Fluid is pumped in to expand cavity and increase the size of the blastocyst.
The preimplantation blastocyst (3&1/2 - 4&1/2 days)
The surface of ICM will become the primitive endoderm while the remaining becomes primitive ectoderm (= epiblast).
Implantation occurs and the zona pellucida is discarded and blastocyst attaches to uterine wall.
Mouse embryo: post-implantation
In the first two days post-implantation, the mural trophectoderm (cells that are not in contact with the ICM) gives rise to polyploid trophoblast giant cells.
The rest of trophectoderm becomes the ectoplacental cone and the extra-embryonic ectoderm which give rise to the placenta.
Primitive endoderm migrates ...
1) to cover inner surface of mural trophectoderm to become the parietal endoderm and
2) to cover egg cylinder and epiblast to become the viseral endoderm
By six days after fertilization, the epiblast is cup-shaped (~1000 cells).
Mouse embryo: gastrulation
By 6&1/2 days after fertilization ...
The primitive streak forms at the start of gastrulation at the future posterior end! (Inside cup is future dorsal side)
Cells move through the streak and spread forward and laterally between the ectoderm and the visceral endoderm to form the mesoderm.
Later, the definitive endoderm (from epiblast) will replace the visceral endoderm.
The primitive steak first elongates, then at the anterior tip of the primitive streak, the node forms.
Then notochord and somites form anterior to the node.
Cells migrate through mesoderm to form endoderm (gut).
Mouse embryo: late embryogenesis
By 8 &1/2 days after fertilization,
1) the neural folds form at anterior and dorsal and
2) the embryonic endoderm internalizes to form the gut.
Between 8&1/2 and 9&1/2 days, the mouse embryo undergoes a complex conformational change and turns to be completely enclosed in the protective amnion and amniotic fluid.
Finally, by 9 days after fertilization is gastrulation is complete.
Technique: Generation of transgenic "knock-out" mice (insertional mutagenesis)
Injection of inner cell mass cells from one mouse blastocyst to another will contribute to many tissues to produce a chimera.
Injection of genetically modified embryonic stem cells (ES cells) into a mouse blastocyst allows formation of transgenic chimeras.
A targeting vector is constructed that has the central (functional) region of a gene replaced with a drug resistance gene.
This is transfected into ES cells and selected by drug exposure.
By homologous recombination, a fraction of the transformants will have one copy of the original gene replaced with the altered (non-functional) form.
These cells are injected into the inner cell mass of a blastocyst.
Resultant chimeric mice give rise to heterozygous mutants which can be bred to generate a "knock-out" mutant mouse strain.
prof premraj pushpakaran writes -- 2018 marks the 100th birth year of Edward Butts Lewis!!!
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