BIOL 103 S21 Lectures 1-6

Gastrula

In triploblastic organisms, the gastrula is trilaminar ("three-layered"). These three germ layers are known as the ectoderm (outer layer), mesoderm (middle layer), and endoderm (inner layer)

Gastrulation in frog embryo

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.

4 steps to limb development

Limb bud initiation Outgrowth Patterning Chrondrogenesis and ossification

Bicoid, hunchback, nanos, caudal mRNAs

Maternally deposited. Bicoid is located at anterior portion, nanos located at posterior portion of fertilized egg. Hunchback and caudal mRNAs are uniformly distributed. Upon fertiliziation, all of these are translated into proteins. Bicoid inhibits translation of caudal mRNA and increases hunchback translation while Nanos inhibits translation of hunchback mRNA and increases Caudal translation.

Transcription initiation for Chromatin

Neg charged groups on DNA molecules interact with pos charged histone tails --> acetylation --> Interaction weakened when histone tails are acetylated, allowing access to transcription factors

Embryonic axis

Neural tube forms in association with notochord, laterally adjacent somites and mesodermal structures

Two types of transposable elements

Non-autonomous elements autonomous elements

Model organisms

Organisms with characteristics that make them useful for genetic analysis

Organogenesis

Organogenesis is the phase of embryonic development that starts at the end of gastrulation and continues until birth.

Cell-cell signaling

Permits cells to influence each other's fate and behavior via direct cell-cell contact via membrane-bound receptors (Notch) and ligands (Delta) or via secreted factors which can also be called inducers

Temporal regulation

Preferential segregation of cell fate determinants to one of two daughter cells during mitosis

Relaxed vs. Condensed Chromatin

Relaxed- DNA accessible Condensed- DNA inaccessible Only one of the two X chromosomes in somatic cells of mammalian females is transcriptionally active

Inducers/Secreted factor

Secretion of Shh from notochord and floor plate appears to generate a ventral-to-dorsal gradient of Shh activity that directs progenitor cell identity and neuronal fate in the ventral neural tube.

Histological staining

Staining sections of brain tissue to identify cell bodies in the brain viewed with a light microscope

Necessary vs. sufficient

Sufficient- of this molecule can be added to an entity that normally doesnt express or have this molecule and its presence is enough to trigger or induce a particular effect Necessary- if this sequence, when deleted or removed, disrupts the relevant function or effect

Homeodomain

The DNA-binding protein region of the Hox transcription factor is called the homeodomain, which is encoded by a segment of DNA called the homeobox.

Chinmo protein

The Drosophila Chinmo protein form a conc. gradient temportally which enables larval neuroblasts to generate different neural cells after each division

DNA Methylation

The addition of methyl groups to bases of DNA after DNA synthesis; may serve as a long-term control of gene expression.

Environmental factors affecting epigenetics

The chemical tags that turn genes on or off can be influenced by factors including diet, chemical exposure, and medication.

Cell fate

The fate of the cell describes what it will become in the course of normal development

Direct cell-cell contact

Example is Notch and Delta in neural precursor populations for competition of which one will become a NPC.

Which event marks the beginning of development?

Fertilization --> germ layers Zygote --> 8-cell stage --> blastula

Outgrowth

-AER is required, if removed replacement with FGF-expressing cells of FDF-coated beads can restore its function and lead to normal limb development -Produced FGF8 and seems to take over function from axial structures that initiate limb bud formation

Epigenetic regulation examples

-DNA Methylation -Euchromatin vs. Heterochromatin -Position-Effect Variegation (PEV) -Histone Modifications -RNA interference (RNAi) -Transposable Elements

Patterning

-ZPA (posterior mesoderm) responsible for establishing polarization along the a-p axis -Shh produced by ZPA sustained by FGF produced by AER -Grafting of additional ZPA led to mirror duplication of limbs

Basics of cell theory

-all life consists of cell -the basic unit of life -all cells come from preexisting cells

Limb bud initiation

-initially undifferentiated mesenchyme (mesoderm) w an epithelial (ectoderm) covering -paddle shaped -FGF10, when implanted ectopically, can induce the formation of accessory limbs

Gastrulation in mouse embryo

6.5 days after fertilization, primitive streak forms at the start of gastrulation at the future posterior end (inside cup is future dorsal side) Cells move through streak and spread forward/laterally between ectoderm and visceral endoderm to form 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).

Neural tube

A groove formed in the top layer of differentiated cells in the embryo that eventually becomes the brain and spinal cord. This is the site of neurogenesis in the spinal cord. The notochord (ventral, secretes Shh) and roof plate (dorsal), pattern the neural tube

Maternal effect gene

A maternal effect is a situation where the phenotype of an organism is determined not only by the environment it experiences and its genotype, but also by the environment and genotype of its mother.

Gastrulation

A phase early in the embryonic development of most animals, during which the blastula is reorganized into a multilayered structure know as the gastrula. Before gastrulation, the embryo is a continuous epithelial sheet of cells; by the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages, set up the basic axes of the body (e.g. dorsal-ventral, anterior-posterior), and internalized one or more cell types including the prospective gut.

Reporter gene function

A reporter refers to a molecule that scientists can use as a way to "report" this expression or activity so that we can actually detect it and measure it. GFP, thus, is an example of a fluorescent reporter which, as we learned in BIOL 101, can be fused to your protein-of-interest to track where it is expressed and when it is expressed. This is an example of a reporter that "reports" the expression of your protein-of-interest at the protein level. But, we can also design reporters such that they can "report" the expression of a gene-of-interest at the messenger RNA (mRNA) level and thus at the transcriptional level.

In situ hybridization

A technique using nucleic acid hybridization with a labeled probe to detect the location of a specific mRNA in an intact organism.

Signaling centers important for limb development

AER and ZPA etc.

Notch-Delta signaling

An example of lateral inhibition. Allows only one cells to become a neural precursor from a group of equivalent (equally competent) cells.

Phenotype

An organism's physical appearance, or visible traits.

Embryonic axes

Anterior: Animal pole Posterior: Vegetal pole Ventral: Sperm entry Dorsal: Future organizer

Numb protein

Asymmetric localization of the Numb protein in dividing precursor cells enables the two daughter cells to adopt different fates Numb loss and gain (symmetric distribution) cause the daughter cells to adopt the fate of their siblings Notch-mediated cell-cell communication is also required for the two daughter cells to adopt different fates after an asymmetric cell division

Dorsal lip secretes _______, which block epidermis formation. This allows the epidermal cells near the dorsal lip to adopt their default _____ state.

BMP antagonists, neural

_____ signal from the underling _______ induces the ectodermal cells to become ______ cells

BMP, mesoderm, epidermal

Catch it, break it, move it

Catch it: observe protein of interest --> where and when is it expressed? Break it: Distrupt your gene/protein of interest --> if you remove/reduce expression? if you mutate? LOF? Demonstrated necessity Move it: Moving gene/protein of interest to region/cell tissue that normally does not have it --> if you "add"? demonstrates sufficiency. GOF?

Asymmetric division

Cell divisions producing two daughter cells that adopt distinct fates are defined as asymmetric

What directly binds to BMP4

Chordin, Follistatin, Noggin

Early embryogenesis

Cut horizontally: one half is only animal pole, other half is entirely vegetal pole and neither develop normally Vertically: Each half contains some animal and some vegetal cells, develop normally but smaller (intrinsic)

CpG islands

DNA methylation occurs at CpG islands at promoters and silences transcription Heritable but also reversible, so pattern can be altered (in mammals, usually occurs in C residues that are adjacent to G residues or CpG islands)

Intrinsic property vs. extrinsic cue to induce cell fate

Determined due to gene expression profile of that cell would be intrinsic to that cell, whereas a secreted (diffusible) signal from outside of the cell that is able to induce that cell to become a particular cell type would be an extrinsic cue (animal cap dissociation assay demonstrated extrinsic cues play a role in cell fate decisions)

Parasegments

Developmental units, with each segment formed by the posterior half of one parasegment and the anterior half of the adjacent one

Forward genetics

Don't know your gene yet, but you have an interesting phenotype and now you want to determine the gene or genes responsible for that phenotype

What structure induces embryonic organization?

Dorsal lip of blastopore, contains "the Organizer" as Spemann and Mangold called it

"Gastrulation brings new neighbors"

During gastrulation, cells rearrange themselves and therefore will have new cells immediately next to them. Cell-cell signaling by secreted factors or direct contact is essential for certain cell fate decisions.

Pair-rule genes

The pair-rule genes are mostly transcription factors, expressed in regular stripes down the length of the embryo. These transcription factors then regulate the expression of segment polarity genes, which define the polarity of each segment. Boundaries and identities of each segment are later defined.

Position-effect variegation (PEV)

The position of the gene relative to heterochromatin influences its expression.So, if a gene is neighboring heterochromatin, it is more likely to be silenced, than a gene that is distant to heterochromatin.

Epigenome

The set of chemical tags that the genome is labelled with

ABC Model for Flower Development (MADS box genes)

These MADS-box genes of flowering plants have homeotic functions like the HOX genes of animals. The floral homeotic MADS-box genes participate in the determination of floral organ identity according to the ABC model of flower development.

Hox genes in mice versus MADS Box genes in plants

They are both homeotic genes, but MADS Box genes are not Hox genes, as they do not contain the homeobox sequence. So, even thought the MADS Box genes are homeotic transcription factors that work in the similar way to activate the expression of genes important for specifying the identity of a particular region (called a whorl in the flowering plant), they possess a different DNA-binding domain that is not a homeobox, called the MADS or MADS-box motif.

Fate determination via cell-cell signalling in C. elegans example

Three initial responding cells give rise to 22 cells that divide, move and fuse in a precise pattern to generate a structure made of 7 rings. Anchor cell produces a signal (LIN-3) that induces a primary fate and a secondary fate through interaction with the LET-23 receptor. The cell that adopts the primary fate, inhibits adjacent cells via lateral inhibition (via LIN-12)

Differentiation

To acquire specific cellular structures and functions (differential expression of genes)

Growth

To increase body and organ size (cell division and cell growth)

Morphogenesis

To organize and spatially distribute specialized cells (cell division, cell expansion, cell movement, and programmed cell death)

Determination

To set the fate of cells (i.e. what they ultimately will become) (differential expression of genes)

Spemann-Mangold Experiment

Took the dorsal lip of the blastopore from one gastrula and transplanted it onto another gastrula on the ventral side (opposite the endogenous dorsal lip). Both the endogenous lip and the transplanted lip became gastrulation sites, and each developed normally into a complete embryo. The result was two whole embryos joined belly-to-belly. Proved that the dorsal lip of the blastopore was capable of inducing the formation of an entire embryo --> primary embryonic organizer. The organizer has been studied extensively in recent years to determine the molecular mechanism of its unique capability.

Rhombomeres

Transiently divided segments

Autonomous elements

Transpose by themselves

Transposable elements (Transposons)

Transposons are mobile genetic elements. They are also referred to as jumping genes because seem to jump around randomly around the genome. So, this was discovered by McClintock in the 50s, where certain genes that are responsible for the variation in corn.

How can RNAi be used to study development or disease?

You could introduce specific RNA strands into the cell that target the mRNA sequence of your gene of interest to stop its production into protein.

Limb patterning by morphogens at the ZPA: The __________ produces the ___________ Shh, which makes a gradient on cells that will form limbs. The thumb forms when Shh is very __________ the little finger forms when there is __________ of Shh.

ZPA, morphogen, low, a lot

neomorphic mutation

a mutant expressing a new or novel function not seen in the wild type

Null mutation

a mutation that results in complete absence of function for the gene

Loss of function (LOF)

a mutation whose gene product adversely affects the normal, wild gene product within the same cell. Usually occurs if the product can still interact with the same elements as the wild-type product, but block some aspect of its function dominant negative gene mutation -random mutagenesis (chemical, transposable element) -targeted mutagenesis (knockout/editing, RNAi) when? random for forward genetics screen and targeted for reverse genetics screen

Theory of evolution

all living organisms are related to one another through decent, and evolution by natural selection is responsible for the diversity of adaptations found in living organisms

Germ layer

any of the 3 layers of cells differentiated in embryos following gastrulation

Unipotent

can give rise to only one type of cell

BMP4

causes cells to undergo apoptosis and die

Gap genes

define several broad ares and regulate pair rule genes

Hox genes

define the role of each segment

Morphogen concept (Lewis Wolpert)

described how a morphogen could subdivide a tissue into domains of different target gene expression. The morphogen provides spatial information by forming a concentration gradient that subdivides a field of cells by inducing or maintaining the expression of different target genes at distinct concentration thresholds.

Segment polarity genes

determine the boundaries and a-p orientation of each segment

Way to inhibit RNA from making final product

dsRNA can actually interfere with step to degrade mRNA that is produced from transcription

miRNA

endogenous, single stranded (i.e. made by cells themselves)

Positional cues

established along the embryonic body axes enable cells to choose different fates to become different parts of the central nervous system -activator instructs cells to choose neural fate -transformer specifies different regions in a concentration-gradient manner

siRNA

exogenous, double stranded (taken up by cells, e.g. viral infection)

Endogenous

from within the cell itself

Genotype

genetic makeup of an organism

Totipotent

have the potential to develop into a complete organism. extra embryonic and the embryo proper

Multipotent

have the potential to develop into all cell types of a particular lineage (e.g. blood lineage)

Pluripotent

have the potential to develop into all tissue of the embryo proper

Orthologs

homologous genes separated by a speciation event two genes in two different species that share a common ancestor

Paralogs

homologous genes within a single species two genes in the same genome that are a product of a gene duplication event of the original gene

Ways to determine expression pattern

in situ hybridization (RNA), immunostaining (protein), reporter (promoter fusion or biochemical assay)

RNA interference (RNAi)

introduction of double-stranded RNA into a cell to inhibit gene expression 2 pathways- exogenous and endogenous In cytoplasm, DICER cuts dsRNA into siRNAs antisense strand of dsRNA attaches to a protein complex RISC Antisense strand binds to the RNA, RISC will then cleave the mRNA strand Endogenous- starting material is miRNA, small hairpin and also ds. DICER recognizes it and chops into even smaller pieces.

Immunolocalization

labeled antibody binds proteins

Recessive mutation

loss of function

Mesoderm

middle germ layer; develops into muscles, and much of the circulatory, reproductive, and excretory systems

Chrondrogenesis and ossification

muscle, cartilage, and bone formation

Non-autonomous elements

need the presence of another transposable element encoding a transposase

Default state of the ectodermal cells

neural

Exogenous

originating from outside of the cell being acted upon

Ectoderm

outermost germ layer; produces sense organs, nerves, and outer layer of skin

Gain of function (GOF)

over-expression ectopic expression constitutively active

hypomorphic mutation

partial loss of function

Gene duplication

products of several types of errors in DNA replication and repair machinery

Apoptosis

programmed cell death

Histones

protein molecules around which DNA is tightly coiled in chromatin

Dominant mutation

results in mutant phenotype in diploid organism

Morphogen

substance whose non-uniform distribution governs the pattern of tissue development in the process of morphogenesis or pattern formation, one of the core processes of developmental biology, establishing positions of the various specialized cell types within a tissue. More specifically, a morphogen is a signaling molecule that acts directly on cells to produce specific cellular responses depending on its local concentration.

Segmentation

the division of some animal and plant body plans into a series of repetitive segments

Endoderm

the inner germ layer that develops into the lining of the digestive and respiratory systems

Development

the process by which a multicellular organism, beginning w a single cell, goes through a series of changes, taking on the successive forms that characterize its life cycle

Epigenetics

the study of how our DNA interacts with a multitude of smaller molecules found within cells which can activate or deactivate genes. So, at what step would epigenetic changes interfere with?

Homolog

the umbrella term for a genes that share origin.

Homeotic genes

transcription determines the developmental fate of each segment

Reverse genetics

you have a gene and you want to examine its role so you mutate it and observe the effect