Developmental Biology - Exam 1

Front 1

Autonomous Specification

Back 1

Specification by differential acquisition, independent and self differentiating.

- Differentiation of trochoblast cells of the Patella
- A trochoblast cell is isolated from the 16-cell stage and culture in vitro
- the isolated trochoblast cells divide and form their ciliated structures at the correct time in development
- The patella develops without ciliated trochoblasts

Front 2

Conditional Specification

Back 2

Specification by interactions between cells; relative positions are imporant.

- Normal back cells of a frog are removed from the blastula and transplanted into the belly region (and vice versa).
- The cell fate is determined by the interactions with neighboring cells.
- The transplanted cells will now form belly tissue instead of back tissue (and vice versa).

Front 3

Syncytial Specification

Back 3

Specification of body regions by interactions between cytoplasmic regions prior to cellularization of the blastomere; gradient of many nuclei.

- insect development; Bicoid (head), Nanos (rear end)
- Bicoid mRNA is placed into a Bicoid deficient egg.
- mRNA instructs competent cells to develop head structures at the position where the injection is made.
- The areas adjacent become thorax because the Bicoid protein that is produced diffuses in a gradient from the anterior structures to the posterior.

Front 4

Morphogenic Determinants

Back 4

Certain proteins or mRNA's that specify the cell type

- Early tunicate embryo
- Four blastomere pairs of an 8-cell embryo are dissociated.
- Each pair forms the structures it would have formed had it remained in the embryo due to morphogenic determinants that were apportioned as the embryo divided.
- A fate map shows that the left and right sides produce identical cell lineages.

Front 5

Morphogens

Back 5

Proteins that form concentration gradients and instruct cells what to do.

- Zebrafish Nodal protein is a morphagen.
- Nodal mRNA and a dye are injected into a single cell of a 256-cell zebrafish embry and is translated into Nodal protein.
- The dorsal cell fates are coordinately regulated by different amounts of Nodal protein.
- 4pg of Nodal mRNA injected, goosecoid gene was expressed in that cell and adjacent cells.
- Cells 5-6 cell diameters away the floating head gene was expressed.
- Cells 7-8 cell diameters away expressed no-tail gene.

**Flatworm Experiment**

Front 6

Cell and substrate adhesion during early development

Back 6

cell adhesion facilitates the aggregation of cells and the formation of tissues. Cadherins are the major cell adhesion molecule. The actin cytoskeleton helps to organize the cadherins so that they may form stable linkages between cells.

- Take an early developing sea urchin embryo and put it in artificial sea water lacking Ca2+. The cells will fall apart. Put back in seawater, the cells will clump back together.

Front 7

Evidence for Genomic Equivalence

Back 7

If each cell is identical to the zygote nucleus, then each cell should be totipotent (capable of directing all development).

Ex: A frog blastula cell is disrupted and its nucleus transferred via micropipette into an enucleated egg (the egg is activated by pricking it with a glass needle, then the nucleus is taken out when the 2nd polar body is being released). The blastula cell nuclei was able to direct development of complete tadpoles.

Note: most somatic cells lose the ability to direct development as they become differentiated, but some can.

Front 8

Cloning

Back 8

Ex: from the Mammary gland of a sheep

The cells were kept at the intact diploid stage of the cell cycle (G1). The nuclei were placed in enucleated oocytes from another strain of sheep. (The oocytes had to be in metaphase 2) The cells were fused using electric impulses then injected into the uteri of pregnant sheep. DNA analysis showed the lamb born was a clone of the donor nucleus (Dolly).

Front 9

In Situ Hybridization

Back 9

A labeled anti-sense mRNA probe is hybridized with mRNA in the organ. The anti-sense mRNA is labeled radioactively and then use autoradiography.

Ex: using rats, testing for Pax6 showed that it was found in the area of the developing retina, lens, and cornea.

Note: It is used to find where mRNA is located.

Front 10

Insert of new DNA or RNA into cell

Back 10

Techniques:
Microinjection - solution of cloned gene is injected into nucleus of cell of newly fertilized eggs.
Transfection - Incubating cells in solution with DNA fragments so that DNA is incorporated (usually DNA is spliced with antibiotic resistant gene so it can be separated using antibiotics)
Electroporation - high voltage pulse pushes DNA into cells
Retrovirus - viral packaging genes are removed and gene of interest is inserted.

Ex: An inner cell mass (of Embryonic Stem cells or ES cells) with the target gene is injected into another early stage embryo and will integrate into the host. A CHIMERIC MOUSE IS BORN. THE CHIMERAS MUST BE MATED TO A WILD TYPE, THEN THE HETEROZYGOUS OFFSPRING NEED TO MATE TO GIVE A HOMOZYGOUS TRANSGENIC (25%) WITH THE TARGET GENE.

Front 11

Knockout

Back 11

Replaces wild type gene with a mutant one to understand what is lost - breed with wild type, then homozygous transgenic.

Ex: BMP7 is isolated and cut using restriction enzymes and spliced into a bacterial gene for neomycin resistance. BMP7 loses its function this way. The genes were electroporated into stem cells where it sometimes replaced the normal gene with the mutant gene. The cells were placed in neomycin, the non-mutants died and the mutants ES cells were injected into mouse blastocysts. CHIMERAS WERE MATED TO WILD TYPE, AND THE HETEROZYGOUS OFFSPRING MATED TO GET HOMOZYGOUS (25%). The homozygous mice never developed eyes or kidneys, so BMP7 was responsible for eye and kidney development.

Front 12

Heterochromatin

Back 12

remains condensed throughout most of the cell cycle and replicates later than euchromatin.
The inactive X chromosome is heterochromatin.

Ex: Two activated X chromosomes resulted in ectodermal cell death and the absence of mesodermal formation.
Ex: Random X cell inactivation causes calico color in cats.

Front 13

DNA Methylation

Back 13

Methylated cytosine residues on inactive DNA prevents Transcription Factors (TF) from binding. Converts cytosine to 5-methylcytosine.

In VERTEBRATES the presence of methylated cytosines in promoter correlates with repression of transcription of the gene.

Ex: Transgenes with different methylation patterns were added to cells and showed that methylation of a promoter correlates with repression of gene transcription.
Ex: In developing RBC the DNA of the globin gene is almost completely unmethylated while in other cells the gene is highly methylated.

Front 14

Nucleosomes

Back 14

Basic unit of chromatin structure (beads on a string)

Ex: The H1 dependent conformation of nucleosome inhibits the transcription of genes in somatic cells by packing them so transcription factors and RNA polymerases can't gain access to the gene.

Ex: The default condition of chromatin is a repressed state and that tissue-specific genes become activated by local interruption of this repression.

Front 15

Histone Acetylation

Back 15

Destabilizes nuclesomes so that they come apart easily. Histone acetyl transferase opens the nucleosome while Histone deacetylases stabilize the nucleosomes and prevent transcription.

Ex: Acetylation of H3 and H4 plus methylation of lysine at H3K4 is associated with transcribing chromatin.

Front 16

Promoters

Back 16

Responsible for binding of RNA polymerase to initiate transcription and are located upstream from the site of RNA transcription. Most contain the TATA box. Basal Transcription Factors are needed to bind to promoters for them to work.

Ex: Protein encoding genes are transcribed by RNA Polymerase II and at least 6 TF's are needed for proper initiation of transcription. TFIID binds to the promoter, is stabilized by TFIIA, then TFIIB binds then RNA Polymerase II binds. Then TFIIE, F and H release RNA polymerase to unwind the DNA.

Front 17

Enhancers

Back 17

DNA sequence that controls the EFFICIENCY and RATE of transcription of a specific promoter: Tell where and when a promoter can be used, and how much of the gene product to make. Also can only activate cis-linked promoters. Need transcription.

Ex: A reporter gene (jellyfish green fluorescent protein gene) fused to a weak promoter will not initiate the transcription of a reporter gene without the help of an enhancer. This is integrated into an oocyte, where it integrates into the genome. If an enhancer is presented the reporter gene will fluoresce and the normal gene activated by the enhancer can be discovered.

Front 18

Transcription Factors

Back 18

Proteins that bind to enhancer and promoter regions and interact to repress or activate the transcription of a gene.

Ex: Pax6 is regulated by Pax6 TF. Mutations cause severe nervous system, pancreatic, and optic abnormalities.

Front 19

Differential Processing of mRNA

Back 19

Eukaryotic DNA has exons (regions of DNA that code for protein) and introns (useless junk)

To become an active protein an mRNA must be:
1. Processed into mRNA by removal of the introns.
2. Translocated from nucleus to cytoplasm.
3. Translated by the protein synthesizing apparatus.
4. Post-translationally modified to become activated.

Front 20

Differentiaton happens by...

Back 20

1. Different cells selecting different nuclear transcripts to be processed and sent to cytoplasm.
2. Splicing of mRNA precursors into messages for different proteins by using differing combinations of exons.

Ex: Sequences present in Sea Urchin blastula mRNA but absent in adult tissue mRNA were present in the nRNA. This showed that different cell types could be transcribing the same nRNA into different mRNA.

Front 21

Storage of mRNA

Back 21

Oocyte makes and stores mRNA for use after fertilization. Some encode cyclin proteins that regulate the timing of cell division.

Ex: In sea urchins early cell division rate and pattern do not require a nucleus, it requires protein synthesis from maternal store mRNA.
Ex: Bicoid and Nanos messages provide positional information in the Drosophila embryo.

Front 22

Competence

Back 22

Ability to respond to an inductive signal.

- competence to respond to the optic vesicle inducer can be conferred on ectodermal tissue by incubating it next to anterior or neural plate tissue.
- when the optic vesicle is placed beneath the ectoderm in another region of the same organism, the ectoderm will not respond, thus lens tissue will not be formed.

Front 23

Instructive Interaction

Back 23

Inducer cell instructs responding cell what to do.

- The optic vesicle inducer experimentally placed under the anterior or neural plate tissue sends a signal to the ectodermal tissue to form lens tissue.

Front 24

Permissive Induction (Genetic Specificity Induction)

Back 24

Transplantation of mesenchymal tissue from one species to another.

- Transplantation of flank ectoderm from early frog gastrula to early newt salamander gastrula destined to become the mouth.
- Similary flank ectoderm taken from the newt salamander gastrula was transplanted into mouth region of the frog.
- The frog larvae developed with salamander-like teeth and balancers.
- The salamander developed with frog-like mouths (adhesive glands)

Front 25

Specific Gene Action

Back 25

SRY gene: sex determination region of the Y chromosome. SRY is found in normal XY males as well as rare XX males. It is absent in XX females. It induces testis formation.

- Transgenic mice: DNA including the SRY gene microinjected into the pronuclei of fertilized mouse embryos.
- Several XX embryos developed testes, male accessory organs, and penises.

Front 26

Specific Endocrine Action

Back 26

Testosterone: hormone that masculinizes the fetus.

- causes differentiation of the Wolffian duct into the male internal genitalia.
- Testosterone is then converted into dihydrotestosterone (DHT) which cuases the morphogenesis of the penis and prostate gland.

Front 27

Specific Receptor Function

Back 27

- Individuals with mutations of receptors may have discordance between their primary and secondary sex characteristics.
- Androgen insensitivity syndrome individuals lack the receptor protein for testosterone and cannot respond to the testosterone produced by their testes.
- Respond to estrogen made by the adrenal gland and AMH from testes and develop the female phenotype.
- Individuals are sterile women with internal testes.

Front 28

Basic Component and Patterns of Development of the Human Reproductive System

Back 28

- Gonadal rudiment can develop into either overy or a testis via differentiation dictated by the genotype.
- First it has a bipotential or indifferent stage (4 weeks).
- Genital ridge shows expanded epithelium that gives rise to somatic cells of the gonad (6 weeks). Germ cells migrate into the gonad.

Front 29

Male Gonads

Back 29

- 8 weeks: somatic cells differentiate into Sertoli cells and they organize into testis chords. Testis chords in the medullary region of the testis form a network called the rete testis.
- Testis chords separate from epithelium and germ cells that enter the gonad will develop here.
- Sertoli cells secrete AMH that block female duct development.
- Interstitial mesenchyme cells differentiate into Leydig cells that make testosterone.
- At Puberty, the testis chord forms the seminiferous tubules where the germ cells migrate and differentiate into sperm.
- The sperm are transported form inside the testis via rete testsis and efferent ducts that link to the Wolffian duct.
-The Wolffian duct differentiates to become the epididymis and the vas deferesn.

Front 30

Female Gonads

Back 30

- Germ cells accumulate outside the gonad along with somatic cells.
- The germ cells will become ova and the cortical somatic cells will differentiate into granulosa cells.
- The mesenchyme cells of the overy become thecal cells.
- Granulosa and thecal cells form follicles that envelope a germ cell (oogonium) and secrete hormones.
- Mullerian duct remains intact and differentiates into the oviducts, uterus, cervix and upper vagina.

Front 31

Can environment or location determine sex?

Environment

Back 31

Yes, in some turtles and all crocodilians, temperature determines sex.

- Temperature induced change in sex ratios in red-eared slider turtles.
- Brood of eggs incubated below 28C produced male hatchlings.
- Brood of eggs incubated above 31C produced female hatchlings.
- At temperatures between, the brood gives rise to both sexes.

Front 32

Can environment or location determine sex?

Location

Back 32

Yes, the eichuroid worm, location of where larvae settle determines sex.

- If larva settles on the ocean floor, it develops into a 10cm long female.
- If larva is attracted to the female's proboscis, it enters the female and differentiates into a 1-3mm male symbiont that produces sperm.

Front 33

Primordial Germ Cells in Mammals (PGC)

Origin

Back 33

- Induced in embryo at the posterior proximal epiblast.
- Around day 6.5 of embryonic development, BMP4 and BMP8b give certain cluster of cells the ability to form germ cells. Within the cluster, about 20 cells express Blimp1. Blimp1 is a transcriptional inhibitor that may be critical for expressing PGCs. Cells that express Blimp1 are restricted to germ cell fate.

Front 34

PGC Migration

Back 34

- PGCs migrate directly into the endoderm from the posterior region of the primitive streak.
- These cells find themselves in the hindgut.
- At embryonic day 9, the PGCs exit the hindgut.
- The following day, PGCs migrate toward the genital ridges where paracrine factor Sdf1 is secreted.
- By day 11.5, the PGCs enter the developing gonads.

Front 35

PGC Development

Back 35

- Proliferation of the PGCs is promoted by the paracrine factors of Fgf7 and stem cell factor (SCF). SCF is produced by the cells lining the migration pathway and remains bound to their cell membraines - important for this factor's activity.
- PGCs proliferate from 10-100 cells to 2500-5000 present in gonads by day 12.

Front 36

Menstruation and Estrus

Back 36

1. FSH is released from anterior pituitary gland.
2. Stimulates Graafian follicle to release estrogen, which inhibits LH at low levels.
3. Secondary follicles start to develop and release inhibin, which makes FSH levels drop.
4. The estrogen levels rise sharply, which stimulates gonadotrophs in the brain and causes LH to be released.
5. LH triggers completion of Meiosis 1, the rupture of the follicular wall, and ovulation.
6. LH promotes progesterone secretion and the formation of the Corpus Luteum.
7. Progestrone levels rise and estrogen level falls.
8. Progesterone causes the endometrial lining to grow, enhances blood flow to the endometrium, and stimulates secretion of endometrial glands.
9. Corpus Luteum begins to degenerate. Progesterone levels drop, blood vessels in endometrium shrink and lining is sloughed off.
10. FSH is released as the progesterone and estrogen levels drop.

**Estrus is the same except is is controlled by light instead of being monthly. Light affects the pineal gland which sends GRH (gonadotrophin releasing hormone) to the pituitary.

Front 37

Significant Events in Gametogenesis

Back 37

- Sperm plasm formation and determination of PGCs
- Migration of PGCs into developing gonads.
- Process of meiosis.
- Spermatogenesis and oogenesis.

Front 38

Spermatogenesis

Back 38

- Meiosis initiated continuously in a mitotically dividing stem cell population.
- 4 gametes produced per meiosis.
- Meiosis completed in days or weeks.
- Meiosis and differentiation proceed continuously without cell cycle arrest.
- Differentiation occurs while haploid, after meiosis ends.
- Sex chromosomes excluded from recombination and transcription during first meiotic prophase.

Front 39

Oogenesis

Back 39

- Meiosis initiated once in a finite population of cells.
- 1 gamete, 3 polar bodies produced per meiosis.
- meiosis completion delayed for months or years.
- meiosis arrested at first meiotic prophase and re-initiated in a smaller population of cells.
- differentiation of gamete occurs while haploid, at first meiotic prophase.
- all chromosomes exhibit equal transcription and recombination during meiotic prophase.

Front 40

Yolk Formation

Back 40

- 3rd year of oogenesis formation in frog eggs.
- Vitellogenesis-accumulation of egg proteins.
- Occurs when oocyte reaches diplotene phase of meiotic prophase.
- Major component is 470-kDa protein - vitellogenin.
- Vitellogenin is synthesized in the liver and carried by the bloodstream to the ovary.

Front 41

Cell Cycle Controlled by Maturation

Back 41

- Oocyte maturation arrested at diplotene stage of first meiotic prophase by lack of mitosis promoting factor (MPF).
- Progesterone activates production of c-mos protein.
- C-mos protein initiates a cascade of phosphorylation that phosphorylates p34 subunit of MPF, allowing MPF to become active.
- MPF drives cell cycle through first cell division.
- Further division blocked by cytostatic factors (CTS).
- Upon fertilization, Ca ions released into cytoplasm are bound by calmodulin and are used to activate 2 enzymes; calmodulin-dependent protein kinase II and calpain II which inactivate and degrade CTS.
- Second meiosis is completed and the 2 haploid nuclei can fuse.
- Cyclin B is resynthesized allowing the first cell cycle of cleavage to begin.