Vertebrates Flashcards
what is developmental biology?
the study of the process by which animals and plants grow and develop, it involves the study of tissue homeostasis is adulthood
what are stem cells?
cells found in the embryo and adults that produce progeny (offspring), stem cells are studied in vitro to aid in regenerative medicine
what is regenerative biology?
the study of the innate ability of organisms to replace tissues or organs after they have been removed or damaged
what is meant by cell ‘fate’?
cells have a fate, this means the thing that it is destined to become after differentiation e.g. a cell can be fated to be a muscle cell
what determines a cell’s fate?
their location within a mass of cells after fertilisation and proliferation
what is the lineage tracing technique in cell fate maps?
cells are named according to their position and injected with coloured dyes, they are left for a few days and then analysed to identify which cells move where
what does it mean if a cell is committed to its fate?
it will give rise to the same cell type no matter where it is located
its fate is determined by intrinsic factors such as transcription factor
what does it mean if a cell’s fate is changeable?
it will give rise to a different cell type if it changes position
it can be reprogrammed and is controlled by signals that tell it to move locations
what is an ectopic signalling source?
a cell that induces the fate of other cells by releasing signals
what is experimental embryology?
experiments that involve the physical manipulation of the embryo
what are competent or receptive cells?
cells that express a specific receptor that receives a signal from another cell
what are permissive signals?
signals that allow or block cells from responding to other signals
what are chemo attractants?
signals that attract or repulse cells during migration
what are patterning cells?
identical cells that adopt different cell fates when stimulated by a signal
how is RNA in situ hybridisation used to analyse gene expression?
stains cells blue/purple to show if they express the RNA for a certain gene
how are antibodies used to analyse gene expression?
uses antibodies to detect where specific proteins are expressed in a cell and where they are located
what are the advantages and disadvantages of using zebra fish models?
advantages: vertebrate, large batches of embryos, relatively transparent, external fertilisation
disadvantages: complex genome with gene duplication, not inbred so high variation
what are the advantages and disadvantages of using mice models?
advantages: mammal, rapid generation time (8 weeks), inbred strains so low variability
disadvantages: internal embryos so poor access, small batches of embryos, expensive, ethical issues
what are the advantages and disadvantages of using frog models?
advantages: external fertilisation, large batches of embryos, large embryos and cells
disadvantages: long generation time (1+ year), yolky embryo so not transparent
what are the advantages and disadvantages of using chick models?
advantages: big embryo, tetrapod
disadvantages: not accessible early, limited genetics
what most an organism have to be classed as a vertebrate?
a vertebral column which protects the neural tube
a cranium (skull) that protects the brain
an endoskeleton
what is the conserved stage in early embryonic development?
the pharyngula stage
an embryo that has 5 pharyngeal pouches (which give rise to gills in fish) and somites (repeated structures called metameric structures down the trunk which form segments)
why is the pharyngula stage conserved in vertebrates?
due to a bottle-neck during development that is evolutionarily conserved because any mutations during early development of the embryo would cause it to be destroyed
what happens during early development in vertebrates?
development starts with fertilisation of the oocyte with sperm, the maternal and paternal nuclei fuse to form zygote and then there are rounds of cell division called cleavage
during cleavage the zygote divides to form blastomeres (early identical cells) and this goes on to form the blastula which is made up of blastomeres on outside forming blastoderm and an inside cavity called the blastocoel
what causes the start of cell division in the early embryo?
signalled by a wave of free calcium ions across the oocyte after the sperm enters, calcium ions are released from intracellular stores such as mitochondria
the calcium ions act on proteins that control the cell cycle to initiate cleavage and with each round of division there is a burst of calcium ions to synchronise divisions
what is unique about the cell cycle in early embryos?
there is only M phase, cytokinesis and S phase and no G1 or G2 phases
this means that cells RNA and proteins aren’t made because transcription happens in G1
how do embryos overcome the lack of G1 and G2 phase?
using maternal stores containing RNA and protein to provide the building blocks to make DNA
what happens when maternal stores in the early embryo run out?
the zygotic genome must be activated to produce its own RNA, when activated the cell cycle slows, loses its ability to divide rapidly and cells start to move around (gastrulation)
what is gastrulation?
the movement of cells to the inside of the embryo to form the endoderm and mesoderm layers, cells that remain on the surface form the ectoderm
the cells are specified into the layers and their cell fate becomes restricted
what cells are derived from each layer of the embryo?
neurons, glia, epidermis and pigment cells are derived from the ectoderm cells
muscle, cartilage and bone, dermis, kidney, heart and blood are derived from the mesoderm cells
lungs and the gut are derived from endoderm cells
what is the process of gastrulation?
a small pore called blastopore forms and cells change shape and move inside the pore and spread around to form a circular shape, the cells move up to form the anterior and posterior after gastrulation
the ectoderm moves around outside to encase the embryo
what are the features of the epithelial cells that arise from gastrulation?
highly structured
usually cuboidal
joined together by junctional complexes
what are the features of the mesenchymal cells that arise from gastrulation?
move around easily (because they are separated by extracellular matrix proteins) amorphous (no defined shape)
what is morphogenesis?
changing the shape of an embryo or part of an embryo
what can cause morphogenesis?
cytoskeletal rearrangements which change the shape of the cell
cell adhesion molecules that can hold cells together or repel them apart
cell migration that changes tissue shape localised cell proliferation which can cause elongation of limbs
cell death that causes tissue shape to change
what are somites?
temporary structures that form in the embryo and disintegrate
when they have formed they become specified to form different cell types
what is somitogenesis?
when somite cells form anterior to posterior after gastrulation
how does segmentation happen?
mesenchymal cells gather together dorsally and cells on the outside edge of the somite epithelialize to make it distinct from neighbouring tissue
the somites then disassemble and revert to mesenchymal cells
one somite gives rise to one vertebrae for segmentation but they will form the posterior of one vertebrae and the anterior of the adjacent vertebrae
what is dermomyotome?
a sheet of tissue that develops from the dorsal part of the somite and forms the dermis and skeletal muscle
what is the sclerotome?
a sheet of tissue that develops from the ventral part of the somite and forms vertebrae and ribs (bones)
what is neurulation?
formation of the neural tube (brain and spinal cord) from the ectoderm as it invaginates and folds up to join across the top down the posterior side
why are chicks the best model organism for limb development in vertebrates?
they share the same homologous skeletal features and have probably evolved from a common ancestor to us
they go through the same stages of development as us
what is the AER and how does it form?
the apical ectodermal ridge
located between dorsal and ventral running along the anterior-posterior axis
formed from epithelial cells bunching up
what is the progress zone and what happens when cells exit it?
an area containing proliferating mesenchymal cells that allows the limb to elongate
as cells exit the progress zone they differentiate into different tissues
how did scientists discover the role of the AER and progress zone?
they removed the apical ectodermal ridge (AER) and replaced it with epithelial cells
the chick embryos were incubated and their limbs were truncated (didn’t grow)
this showed that the AER ridge secreted something to stimulate the proliferating cells in the progress zone so the limbs can elongate
how did scientists discover the AER causes permissive growth?
they took an old AER and transplanted it onto a younger limb bud and observed that the limb developed normally
this showed that there is no instruction coming from the AER to elongate certain limbs it just tells cells to proliferate (permissive growth)
how did scientists discover that the genes Fgf8 and Fgf4 are redundant?
they knocked out the Fgf8 and Fgf4 genes in mice
in Fgf8 mutation the mice had a mild limb defect and in Fgf4 mutation the mice had no limb defects
this showed that Fgf8 and Fgf4 are redundant (they have overlapping functions so if one is removed the other takes over)
how did scientists discover the genes Fgf8 and Fgf4 are needed for limb development?
they grew embryos without both genes and observed the mice had no limbs proving that Fgf signalling is required for limb development
the mice without both Fgf genes had a similar phenotype to AER removal
how did scientists discover that the AER is a source of Fgf genes and Fgf are independently responsible for limb development?
they soaked beads in Fgf8 and substituted it with the AER in chick embryos, the limbs grew more as more beads were added suggesting that the AER is only needed as a source of Fgf
they implanted Fgf beads between the limb buds in the flank and embryos grew ectopic limbs, this showed that Fgf signalling alone is sufficient for limb development
what is the ZPA and what is its role in limb development?
zone of polarising activity
it polarises the tissue to form the anterior-posterior axis which is known as patterning identity
how did scientists discover the ZPA?
they took a region of tissue from a donor embryo and moved to a recipient to duplicate the region
the embryos were incubated and they observed the skeleton forming in a mirror image (the anterior and posterior were swapped)
how did scientists discover the ZPA was formed from mesenchymal cells?
they cut off the limb bud from an embryo to form the ectodermal jacket (skin) and they removed the mesodermal tissue
they mixed the cells up, centrifuged them and inserted them back into the limb and attached it to the embryo
the embryos had no ZPA and they observed toes forming distally without an anterior-posterior axis showing mesoderm are needed to polarise cells
how did scientists discover the ZPA secreted a signal to produce the anterior-posterior axis?
they separated mesoderm and packaged it back into the limb then they took the ZPA from a donor and inserted it into the anterior side
limbs grew but it had a reversed anterior-posterior polarity
this showed that a signal is secreted by the ZPA to give the anterior-posterior axis polarity
what controls digit formation?
there is a signal that diffuses out from the posterior end and establishes a concentration gradient to give different fates to cells at different concentrations
e.g. at a high concentration digit 4 is formed, at a medium concentration digit 3 is formed and at a low concentration digit 2 is formed
the concentration direct cells to the correct position
what is sonic the hedgehog?
SHH is a secreted ligand that is needed for the anterior-posterior axis formation
what was observed in mice with SHH mutations?
they had no limb defects but the limbs were mirrored like the ZPA mutants
what is the transcriptome?
all the genes that are actively transcribed into RNA
what does differential gene expression mean?
cells with different expression profiles
what are transcription factors?
proteins that interact with the major groove on the DNA backbone to form hydrogen bonds
they bind to the regulatory element but the bond formed is unstable so falls off easily
what are the 2 types of regulatory elements?
enhancers which are binding sites for transcriptional activators
silencers which are binding sites for transcriptional repressors
what is MyoD?
a transcription factor that is expressed only in muscle precursors and muscle cells
it controls the expression of genes for muscle differentiation and can maintain its own expression through a positive feedback loop
how did scientists discover MyoD is needed for differentiation?
they activated fibroblasts (that don’t usually express MyoD) to express it
they observed that the fibroblasts containing MyoD differentiated into muscle cells proving that MyoD is sufficient to direct muscle cell fate in fibroblasts
how did scientists discover MyoD is redundant?
scientists observed that mice that lacked the function of MyoD still developed with normal muscle showing it must be a redundant gene
Myf5 acts redundantly on MyoD
what is the process of muscle differentiation?
- mesodermal progenitors in somites receive signals such as the transcription factor mrf4 that start the differentiation pathway
- MyoD and myf5 are activated by mrf4 and cause myoblasts to form, growth factors can repress differentiation but if they aren’t present they start to express myogenin
- this causes multinucleate myotubes as muscle cells fuse together, myogenin directs transcription of muscle specific genes such as myosin II and the cells come together to form functional muscle fibres
what are satellite stem cells?
stem cells that line muscle fibres but aren’t differentiated into muscle cells yet
when stimulated by signals the they divide and differentiate into muscle cells
how are genes always written?
in all italics
what are germline and somatic mutations?
germline are inherited mutations carried in oocytes or sperm
somatic are random mutations that aren’t passed on to offspring
how are forward genetics used in labs?
creating high levels of mutations in model organisms (this process is called mutagenesis)
they raise mutagenic animals and cross them to look for interesting phenotypes
how are reverse genetics (gene knockout) used in a lab?
identifying a mutation in a gene of interest using gene knockout in mice, this is where the endogenous gene (gene inside the animal) is taken and genetically engineered to knock it out to remove it
how is reverse genetics (gene replacement) used in a lab?
gene replacement or knock in can also be used which is where the endogenous gene is changed slightly
how is reverse genetics (CRISPR) used in labs?
CRISPR is a different technology that can be used to knock in or out a gene and it can be used in any organism
how do mutations that change regulatory sequences affect genes?
changes binding site for transcription factors on DNA leading to reduced expression of a gene and production of a protein
how do mutations that change non-coding sequences affect genes?
changes to introns can affect RNA splicing, stability or translation so less protein is produced
how do mutations that change coding sequences affect genes?
changes in DNA that alters amino acid sequence of a protein affecting how it is folded and it could lose its function, could also create a premature stop codon leading to a truncated protein
what are the 3 functional groups of transcription factors and their roles?
a DNA-binding domain where DNA binds
a dimerization domain where dimers are formed from 2 identical proteins
a domain that regulates interactions
how are transcription factors activated?
the proteins dimerise and there is a conformational change causing it to be activated
what is a missense mutation?
a mutation where a single amino acid is substituted and nonsense mutations create premature stop codons
what is an amorphic mutation?
a missense mutation that inactivated the DNA binding domain
how do amorphic mutations affect heterozygous and homozygous animals?
in heterozygous animals there is haplosufficiency and redundancy of a gene so there is enough gene product from one wild-type allele
in homozygous animals (if two carriers of mutations are crossed) there will be a strong phenotype due to no transcriptional activation, this is recessive
what is a hypomorphic mutation?
a missense mutation in the DNA binding domain causing its activity to be weakened
how do hypomorphic mutations affect heterozygous and homozygous animals?
in heterozygous animals there is no phenotype as there is enough product from the wild-type allele
in homozygous animals there is a milder phenotype due to poor transcriptional activation, the dimer forms on DNA but often falls off, this is recessive
what is an antimorphic mutation?
a missense mutation that destroys the dimerization domain
how do antimorphic mutations affect heterozygous and homozygous animals?
in the heterozygote has a mutant form that binds to DNA but doesn’t dimerise to the wild-type so it doesn’t go through a conformational change and doesn’t become activated, however it still has some function but transcription is compromised and will only be activated if two wild-type proteins dimerise which is dominant
in the homozygote it is completely inactivated and there is no transcription
what is a hypermorphic mutation?
a missense mutation that results in activation that is independent of dimerization and the transcription factor is always in the active state
how do hypermorphic mutations affect heterozygous and homozygous animals?
in the heterozygote the mutant form binds to DNA and is active all the time (this is called constitutively active), this increases the overall activation of transcription and it is dominant
the homozygote is the same as the heterozygote
which mutations are loss-of-function and what are their phenotypes?
amorphic (recessive)
hypomorphic (recessive)
antimorphic (dominant)
which mutation if gain-of-function and what is its phenotype?
hypermorphic (dominant)
what are alleles?
different mutations of the same gene that result in the same phenotype
what are reporter constructs used for?
help us to study the activity of genes, cells or tissues in vivo
they use green fluorescent protein (GFP) which is found naturally occurring in jellyfish and can be cloned into other animals
fluorescence works as proteins can be excited using different wavelengths which are absorbed by the GFP to emit light at a different wavelength
how are GFP transgenic lines generated?
- fusion gene: dd the GFP sequence and add it to the last exon of the DNA by deleting the stop codon so GFP is fused to the end of the protein when it is translated
- reporter construct: remove exons and insert GFP in place of the protein, this won’t tell us where the protein is localised only which cells express the protein
- for both methods reintroduce the gene into the animal, usually the normal gene won’t be removed so there will be two copies of the gene
what are the 2 uses for GFP transgenic lines?
to follow the expression of a gene or to follow the behaviour of cells in vivo
to follow subcellular localisation of a protein
