DFF Lab Quiz 2

Deuterostomes after becoming fertilized:

-Cleavage -change in form often beings with gastrulation, involves cell movement -emergence of pattern -differentiation of cells -growth

Our experiment: Anitbody detecting ELISA

-Start with known antigen in all tests -is there an antibody in the sample that can bind to the antigen? -How do we determine if that binding occurred? -Enzyme-linked secondary antibody -addition of substrate detects presence of 2^o Ab+Enzyme

Features that vary in invertebrate phyla: Body systems:

-What body systems are present? -How does an animal survive without a given system (ex: without a circulatory system)?

All vertebrate animals dissected include the following features:

-anterior skull with a large brain -a rigid internal skeleton supported by the vertebral column -dorsal hollow nerve cord -well-developed circulatory system driven by contractions of a ventral heart.

Features that vary in invertebrate phyla: Tissue layers:

-are there distinct tissue layers? -how many are there in development? (not the same as how many tissue types!)

Invertebrate animal characteristics to examine

-body plan -food acquisition -locomotion -organ systems

ELISAs (Enzyme-Linked ImmunoSorbent Assay):

-can be set up to detect either an antigen or an antibody in a sample -both types of test rely on specific antigen-antibody binding -differ in order of reagents added

All vertebrates have:

-circulatory systems -digestive systems -gas exchange systems How do they differ/how have they evolved over time

Connective tissue

-consists of a sparse collection of cells scattered through an extracellular matrix (fibers embedded in fluid, gel, or solid foundation) -Function: connection of body tissues, structural framework for the body; protection of internal organs; energy storage Ex: cartilage, bone, adipose, blood 2 types: loose, fibrous

Fertilization What events occur after sperm and egg plasma membrane contact, but before first cleavage?

-fast block to polyspermy -calcium wave -Na-H exchange -slow block to polyspermy -pH changes -DNA protein synthesis activated -nuclear fusion -spindle formation

Epithelial tissue

-form sheets of densely packed and tightly connected cells that cover inner and outer body surfaces -functions: body protection, absorption, secretion, filtration, and transport; some are modified to detect chemicals

How do vertebrate systems differ/how have they evolved over time?

-jaws, teeth, paired fins evolved in sharks -bony skeleton, swim bladder/lung evolved in fish -lobe fins evolved for amphibians -terrestrial limbs and digits evolved for amphibians amniotic egg evolved for birds/reptiles

Antibodies

-produced by the immune system to inactive the foreign antigen

Muscle:

-specialized for contraction (shortening) -functions: rhythmic, involuntary contraction of hollow organs; voluntary movement of parts of the body such as long appendages and jaws Types: skeletal, smooth, cardiac

Nervous Tissue

-specialized to generate, send and receive electrochemical signals -functions: communication between different regions of the body; controlled, coordinated activity of the body; repeatable responses to external stimuli ex: neurons, neuroglia

Invertebrate animals to be dissected:

-sponge -comb jelly -hydra (live) -anemone -planaria (live) -earthworm -clam -roundworm (Ascaris) -crayfish -grasshopper -starfish -lancelet

Elisa uses: antibody detection

-virus testing by assaying for Ig molecules specific to viral antigens -Use known antigen to capture antibody, of present, in a sample

Sea Urchin Outcomes Regular seawater no sperm no chemical

1% fertilization envelope 1% first cleavage 167 observed

Sea Urchin Outcomes calcium free seawater no sperm no chemical

1% fertilization envelope <1% first cleavage 101 observed

Sea Urchin Outcomes low sodium seawater no sperm no chemical

1% fertilization envelope <1% first cleavage 81 observed

Features that vary in invertebrate phyla:

Tissue layers Symmetry body cavity body systems segmentation

Frog egg contains more yolk in the _____________ and affects __________.

Vegetal pole; cleavage

The evolution of segments: Segmentation:

Williston's Law (applied to phyla that have segmentation) - over time, segments tend to...... -reduce in total number -specialize function of remaining segments

Calcium wave triggers:

activation of sodium-proton pump exchange. protons are pumped out of the cell , pH starts to rise and then DNA/protein synthesis are activated.

Frog model Site of sperm entry

animal pole

Hormones:

are reseased from glands and travel through the blood stream, sometimes over long distances, to target organs.

NH3Cl is

base (to increase pH)

Removing free calcium by chelating agent (EGTA) _________ later steps.

blocks; (calcium rise is necessary)

EGTA is

calcium chelator (sequesters calcium out of solution)

A23187 is

calcium ionophore (which allows calcium to equilibrate over plasma membrane. calcium stores are in endoplasmic reticulum)

Features that vary in invertebrate phyla: Segmentation

can be obvious externally or internally

Vertebrates are a subgroup of ____________.

chordates

proximal

closer to the body or trunk

Holoblastic cleavage

complete ex: frog

ELISA this test is somewhat sensitive to the ______________ of the primary antibody.

concentration

Meroblastic cleavage

incomplete ex: fish

involution

individual cells moving inward (mesoderm, endoderm)

Calcium increase occurs even in calcium free water, so source of calcium must be ___________.

internal (from endoplasmic reticulum)

types of movements that occur during gastrulation:

invagination involution epiboly

Features that vary in invertebrate phyla: Body cavity:

is there a body cavity (lined with epithelium or not?) that contains digestive tract and other organs?

Fast block to polyspermy

membrane depolarization

Frog is cleavage always completed before the next cleavage starts?

no

In sodium free seawater, protons ________ pumped out and pH _________ rise. Protein synthesis _________ activated.

not; doesn't; isn't (pH rise is necessary)

A tube comprised of mesoderm, the ____________, is formed in the embryo.

notochord

ELISA Experimental Controls using positive and negative controls What reagent is purposely being included/left out of these samples:

primary antibody

Features that vary in invertebrate phyla: Symmetry:

radial vs bilateral

Adding NH3Cl with no sperm can ________ the egg pH and _________ protein synthesis

raise; activate (increase pH is sufficient)

slow block to polyspermy

release of cortical granules after Ca2+ "explosion"

pH rise and not __________ inflow is necessary and sufficient for protein synthesis activation.

sodium

Invagination

tissue of connected cells buckle inward due to changes in cell shape: endoderm

dorsal

toward the back (spine)

ventral

toward the belly

Anterior (cranial)

toward the head

medial (median)

toward the middle

posterior (caudal)

toward the rear

lateral

toward the side or away from midline

Frog The presence and distribution of what substance explains particular patters of cleavage in the frog?

yolk (controls how much cleavage because cleavage generally occurs in the animal pole where there is no yolk)

Multi-cellular heterotrophs

All animals, by definition are this. use internal processes to breakdown food from their environment.

ELISA no color change =

No enzyme present, no secondary antibody present, no primary antibody present in the suspect sample

what type of cleavage occurs in the frog?

holoblastic

A major regulator of the endocrine system in mammals is the ______________, a region of the brain.

hypothalamus

How our ELISA works:

1. add antigen to wells, wash 2. add sample which may contain antigen-specific primary antibody, wash 3. add secondary antibody, which is linked to an indicator enzyme, wash VERY WELL 4. add substrate and watch for color indicator appear

Sea Urchin Outcomes low sodium seawater no sperm chemical: A23187

17% fertilization 4% first cleavage 77 observed

Sea Urchin Outcomes calcium free seawater yes sperm Chemical: EGTA

19% fertilization envelope 6% first cleavage 87 observed

Sea Urchin Outcomes regular seawater yes sperm chemical: NH3Cl and EGTA

20% fertilization envelope 7% first cleavage 65 observed

Sea Urchin Outcomes calcium free seawater no sperm Chemical: A23187

23% fertilization envelope 4% first cleavage 101 observed

Sea Urchin Outcomes low sodium seawater yes sperm chemical: NH3Cl

24% fertilization envelope 11% first cleavage 79 observed

Sea Urchin Outcomes regular seawater yes sperm Chemical: EGTA

25% fertilization envelope 11% first cleavage 100 observed

Sea Urchin Outcomes Low sodium seawater yes sperm no chemical

26% fertilization envelope 8% first cleavage 165 observed

Sea Urchin Outcomes regular seawater no sperm chemical: A23187 and EGTA

31% fertilization envelope 4% first cleavage 87 observed

Sea Urchin Outcomes regular seawater no sperm chemical: A23187 and NH3Cl

35% fertilization envelope 5 % first cleavage 42 observed

Sea Urchin Outcomes regular seawater no sperm chemical: A23187

40% fertilization envelope 8% first cleavage 185 observed

Sea Urchin Outcomes calcium free seawater yes sperm no chemical

41% fertilization envelope 20% first cleavage 184 observed

Sea Urchin Outcomes regular seawater yes sperm chemical: NH3Cl

42% fertilization envelope 19% first cleavage 69 observed

Sea Urchin Outcomes regular seawater yes sperm no chemical

62% fertilization envelope 34% first cleavage 368 observed

Fertilization Effect of varying concentrations of chemicals in the seawater in the presence or absence of sperm:

Ca2+, Na+, A23187, EGTA, NH3Cl

Bastula undergoes gastrulation to form 3 germ layers:

ectoderm, endoderm, mesoderm

The ______________ system utilizes chemical messangers called _________

endocrine; hormones

ELISA color change to blue

enzyme was present secondary antibody was present primary antibody was present in the suspect sample

ELISA The secondary antibody has bound to it an ______________ (HRP, horseradish peroxidase) that will catalyze the reaction that turns substrate (TMB, tetremethylbenzidene) from __________ to ________.

enzyme; colorless; blue

distal

farther away from the body or trunk

Antigens

foreign particles (often proteins) that elicit an immune response

Artifical release of calcium by calcium transporter (A23187) can trigger similar downstream affects as __________.

sperm; (calcium rise is sufficient)

Epiboly

stretching and rearrangement of multiple layers of tissue into fewer layers over the surface of the embryo.

ELISA The secondary antibody will bind to

the primary antibody

The grey crescent is created by:

the rotation of the cortical cytoplasm relative to the inner cytoplasm.

Cleavage:

the special mitoses which begin immediately after fertilization