ZOO- 3050 (Midterm)

how can we identify actively dividing cells?

*marker varies throughout development, a lot of division in embryo but not in adults - Thymidine replaced by BrdU (bathe organism in BrdU) - We have an antibody that binds to BrdU - BrdU incorporated as DNA strand is produced - Add antibody into cell (ensure it goes into nucleus), then cells that have BrdU will be marked

how do we induce pluripotent stem cells (iPSC) in the lab

- isolate adult multipotent stem cells - add transduction/transcription factors to make them differentiate into three types of tissue control normal gene expression in the adult cell via methylase inhibitors, deacetylase inhibitors, cell dependent

2 factors controlling cleavage patterns

1) amount of yolk - yolk platelets inhibit division 2) orientation of spindles - centriole from each sperm divide before mitosis to form an aster at each pole of the mitotic spindle - these asters determine the plane of cleavage - spindle orientation can change during embryogenesis

germ cell determination in nematodes where do they come from?

1) chromosome diminution - set of chromosomes deleted in the animal hemisphere, leads to gene loss 2) suppression of part of genome, not physical removal

2 cytoskeletal mechanisms of mitosis

1) contractile ring - actin microfilaments in between future 2 cells 2) microtubules - pulling things to each cell

2 basic patterns of mammalian ovulation

1) copulation activated - results in the release of the second block - fertilization success (males + females acc meeting) - induced by gonadotropin release from pituitary 2) periodic pattern - seasonal patterns - photoperiod - estrus - signals being released by females to induce copulation (30 day pattern, not in humans)

determination vs differentiation

1) determination is a reduction in the potential 2) differentiation is when the cell gains its identity these two processes are temporally separated

oogenesis in amphibia - 2 differences how long does the process take?

1) eggs are produced continuously 2) oogenesis takes multiple years - massive mature oocyte due to vitellogenin which is a yolk protein - vitellogenic phase is a phase where yolk proteins are incorporated - results in cohorts of oocytes - 1st arrest - prophase I oocyte growth takes 3 months maturation @ 16 months

2 developmental processes of indirect life cycle

1) embryo 2) larva (which gives rise to juvenile)

differentiation is happening at what two levels and in what manner/state

1) embryogenesis (totipotent state) 2) adults (differentiated organism), some cells maintain puripotency (germ line), transcription factors + hormones involved

migration routes 1) fish and amphibians 2) birds + reptiles 3) mammals

1) fish and amphibians - PGC's arise in vegetal pole - Follow ECM to genital ridge 2) birds + reptiles - PCG's arise in the extraembryonic membranes - Move through blood circulation to genital ridge 3) mammals - PGC's arise in extraembryonic membranes - use filopodia to migrate

3 concepts of the central paradigm

1) genomic equivalence - all cells contain the same genes bc exact copies after division (not everything is expressed) 2) transcription - DNA to mRNA which gets transported to the cytoplasm 3) translation

three things that happen during human embryogenesis

1) polar bodies degenerate (17-24 hours) 2) compaction (8-16 cell stage) --> cells cluster together 3) cavitation --> essential for blastocyst formation (last stage before implantation

oocyte activation in amphibians

1) progesterone activates MPF (mitosis promoting factor) mitosis promoting factor is responsible for first block (diplotene block) release (meiosis I completion), leading to GVB, first polar body 2) ovulation leads to the start of meiosis II (secondary oocyte --> triggers 2nd block "metaphase block) - second block maintained by CSF (cytostatic factor) and is released by a complex cascade (ca2+ flux after fertilization) p34 and cyclin are the main proteins regulating that cycle

major differences between spermatogenesis and oogenesis (2)

1) unequal cell division - point to this is size + energy, optimize resources to one egg 2) pause in meiotic cycle - human males: pause when spermatogonia develop into mature sperm (at very end) -human females: arrest when oogonia develops into primary oocyte, meiosis I, prophase I, 12-15 years (first block) meiosis II is the second arrest

what does the egg contain

1) yolk - protein 2) cellular machinery - all the things that make growth + cleavage possible (ribosomes, RNA, mitochondria) 3) informational molecules - mRNA, protein

mammalian oogenesis (general time line)

3 to 7 months after embryonic development - (10s of thousands of oocytes) overproduction 7 months - # of oocytes starts dropping, apoptosis around birth - few hundred left then arrest just these oocytes to work with until menopause where oocyte reserves are depleted

holoblastic cleavage patterns

A) Isolecithal (sparse + evenly distributed yolk) 1) Radial - 2 meridonal divisions 2) Spiral - first set of blastomeres shifted/rotated 3) Bilateral - one side of embryo mirrors other side 4) Rotational - humans, axis changes after 1 division B) Mesolecithal (moderate vegetal yolk disposition) - displaced radial cleavage

meroblastic cleavage patterns

A. Telolecithal (dense yolk throughout most of cell) - top section is yolk free that gets divided 1) Bilateral 2) Discoidal B. Centrolecithal (yolk in center of egg) - birds and fruit flies - the embryo is separate from the yolk

factors that decide whether stem cells will divide or not

CHANE 1) hormonal signalling (juxtacrine vs autocrine) 2) asymmetric cell division (localization of factors in cytoplasm) 3) neurotransmitters 4) ECM - cell adhesion processes, integrans are receptors that can connect the cytoplasm to ECM 5) cell adhesion (communication)

meiosis I

cell division that reduces chromosome number by one half 4 phases: 1) prophase I - synapsis to create tetrad - crossing over - aster fibers - centrioles visible - tubulin fibers (across cell) - aster fibers (microtubules, at the end) 2) metaphase I - contractile ring formation 3) anaphase I - separation of homologous chromosomes 4) telophase I

cleavage

cleavage pattern - geometry of how the cell is divided, determines how adult is going to look like sets up cell lineages, multiple lineages work together on one tissue

spermatogenesis condensation how long does the process take

condensation - a lot of structures compacted, highly specialized - nucleus, mitochondria, specialized membranes 1 primary spermatocyte makes 4 sperm process takes 65 days (complete renewal of sperm production)

transition from spermatogonia to spermatid

condensation of nucleus where the entire cytoplasm is reduced/compacted process is not dependent on gene expression, mRNAs already present so simply translation is taking place acrosome - tip of spermatozoa allows for the fertilization to happen

stem cell research critical for what and depends on what

critical to disease and depends on differentiation as a factor

yolk distribution patterns (4)

isolecithal - sparse, evenly distributed yolk mesolecithal - moderately dense, uneven distribution, concentrated at one end telolecithal - very dense, concentrated at one end centrolecithal - very dense, concentrated at center of egg

lampbrush chromosomes

lampbrush chromosomes can be seen with a marker during xenopus oogenesis (during prophase I, when most RNA is produced)

differences in oogenesis in mammals vs sea urchins + frogs

mammals: - primary oocytes produced before birth - small number of oocytes are produced - prophase I arrest sea urchins, frogs: - continuous reproduction - cohorts of eggs produced from stem cell pop'ns throughout reproductive life span

two types of divisions

meridonal equatorial (divides animal and vegetal pole)

oogenesis general

more variable between species and within variation reflects developmental schedule determined by requirements of the egg - human eggs are small, chicken eggs are big - # of eggs, how many did you produce -- more eggs means less energy per egg

organogenesis in frogs

morphogenesis - cells from different layers interact to form tissues and organs neurla - notocord and neural tube form after gastrulation

polytene chromosomes

stage in which transcription occurs, chromosomes are unraveled + accessible allows for gene mapping have to look at these to observe transcription

germ cell determination in fruit fly

syncytial blastoderm (common cytoplasm) 9th division - nuclear migration occurs - 5 migrate to posterior which gives rise to pole cells - pole plasm exists around these pole cells - eventually migrate to gonads after arrest and metamorphosis

sperm morphology + labelling what allows to propel it forward

tail - cytoskeleton + tubulin (red) head - primarily DNA (blue) middle - mitocondria (yellow) axoneme structure - 9 + 2 filaments allows to propel it forward

what gives rise to sperm and eggs what decides? (difference between mammals and turtles + crocodiles)

the same PGCs, influenced by the environment as a determinant (genital ridge) - bipotential sex determination system decides!: mammals - sex of gonads (genotype) - y chromosome decides - by default we are all female, lack of signal means eggs will develop turtles, crocodiles - environmental sex determination - temperature -- linked to breeding season, global warming

how do we study differential gene expression in development

there is tissue specific transcription so we can use a GFP fusion to promoter

what happens to the germ line at the beginning of division

these cells are set aside usually and they stop dividing

what is different between the stem cell and committed cell

transcription factors, cytoplasmic components (get unequally distributed)

which animals go through a transdifferentiation step and which have an early separation of somatic and germ cells?

transdifferentiation: echinoderms (sea urchins), flatworms, tunicates, chidarians early separation: insects, roundworms, vertebrates

IPSC for curing human diseases

*Ie sickle cell anemia 1) Harvest fibroblast cells from mutant strain + clone them in a dish 2) Infect with transcription factors (Oct4, Sox2, Klf4, and c-Myc) 3) Correct sickle cell mutation in iPS by specific gene targeting - colony selection 4) Differentiate into embryoid bodies (don't have differentiation agents, never differentiate, 3D constructs of stem cells) 5) Transplant corrected hematopoetic progenitors back into irradiated mice

types of stem cells

*as we go down there is a reduction in potency *self renewal can occur at any point Potential - cell, source Totipotent - zygote, zygote Pluripotent - ESC, blastocyst (inner cell mass) Multipotent - multipotent stem cell, embryo + adult brain Limited differentiation potential - neuronal progenitor, brain or SC Limited division potential (unipotent) - differentiating neuronal precursors, regions of the brain Functional nonmitotic neuron - differentiated cells, specific areas of the brain

cloning requires understanding of what are the two things needed to be understood

internal and external regulation of development factors (egg with the right combination of factors needs to be picked) & cell division (early divisions are critical)

ISH purpose and method

ISH is to visualize gene expression - Spatial and temporal expression - Gene interaction can be looked at (coexpression) - Expression of specific alleles 1) Build a probe - RNA, antisense RNA allowing hybridization to target RNA - Attach a label to it to visualize (DIG usually bc not naturally occurring in animals) - Conjugate it to an alkaline phosphatase which transforms from yellow to blue

spermatogenesis where is gamete development happening what two cell types assist and what are their functions?

PGCs inhabit the testes gamete development is happening in the semineferous tubules sertoli cells provide factors (hormones) for maturation process leydig cells in connective tissue and provide testosterone

2 examples of unipotent stem cells

intestine (old cells shed on daily basis) basal layer (skin cells) - humans replace 1.5g of skin/day - stem cells are underneath epithelium

extraembryonic membranes

amnion, chorion, allantois, yolk sac

difference in yolk distribution in amphibians vs sea urchins

amphibians: - unequal yolk distribution - animal pole (smaller cells, complete division) - vegetal pole (larger cells, incomplete division) sea urchins: - little yolk but homogenous distribution

2 areas being differentiated

animal cells and vegetal cells

leapord frog gastrulation

archenteron: primitive gut, forms the mouth shrinking of the blastocoel by the archenteron which then forms the blastopore cells ingress through the blastopore into the blastocoel which is decreasing in size blastocoel differentiates into the germ layers: endoderm, mesodern, ecdoterm

animal examples for telolecithal cleavage types

bilateral - cephalopods discoidal - birds, fish

cytokenesis

blastomere formation, splitting of cytoplasm

neuronal stem cells (adults)

brdu strainings on cancer patients revealed brain regions lighting up found that a set of cells in cortex are always dividing - memory formation-- therefore there has to be a de differentiation process in which the stem cells are going back (glial cells?) or there are stem cells that are set aside

what does cell differentiation result from

differential gene expression! a lot of common genes between regions ie brain and gonad ie neurons express 50% of entire genome but the differentiation results from the differential expression of specific genes

developmental patterns direct life cycle vs indirect life cycle vs parasite life cycle

direct - no larval stage indirect - larval stage + metamorphosis parasites - interactions between organisms

common function of eggs vs common function of sperm exception?

eggs - protection + nutrients sperm - fertilization fruit fly is exception where male fly is making a higher investment (sperm is 1000x longer than human sperm)

most organs contain cell types from several germ layers except

epidermis - cells from the ectoderm only dermis - cells from the mesoderm only

what is changing developmental processes

evolution environment (contaminants)

heterochronies

evolutionary change in rate or timing of development developmental stage vs time allows comparison of different species - rate of development

mammalian stem cells

fertilized egg --> ESC (pluripotent)--> hematopoietic stem cell (HSC - multipotent) --blood cell --> rbc or wbc fertilized egg --> ESC (pluripotent)--> hematopoietic stem cell (HSC - multipotent)--> immune cell --> t cell or b cell ESC and HSC renew themselves

fetal origins of the adult ovarian reserve

fetal ovary germ cell cyst formation cyst breakdown oocyte with primordial follicle (support cell) - 70nm sexual maturation (surge of lutenizing hormone which initiates maturation of the follicle) adult ovary primary follicle secondary follicle antral follicle ovulatory follicle

blastocoel

forms where there are more divisions (animal hemisphere), no space in vegetal bc yolk

blastocoel - where does it form?

forms where there are more divisions (animal hemisphere), no space in vegetal bc yolk

what is involved in regeneration

frogs + salamanders can regenerate regeneration uses several developmental processes in the adult - post embryonic development 1) De-differentation 2) Cell division 3) Migration 4) Induction (external activating factors)

fertilization

fusion of games 2 types of fusion 1) physical - sperm + egg 2) genetic - sperm pronucleus needs to go and find egg pronucleus (pronuclear fusion) to produce zygote - fertilization envelope forms, increase in metabolism directly after fusion

gametogenesis

germ line wake up even if you remove the cells in invertebrates they just regenerate

two types of cleavage

holoblastic - complete cytokinesis (amphibians, mammals, sea urchins) meroblastic - incomplete cytokinesis (birds, fish, insects)

what is morphogenesis and the factors involved

how the cells know where to go morphogens - signals, biological form turing model - oldest model explaining morphogens and form cell-cell interaction - morphogens needed cell adhesion - ca2+, cells need this to communicate cytoskeleton - actin tubulin, allows shape change

HPA axis (hypothalamic-pituitary-adrenal axis)

hypothalamus secretes GnRH (gonadotropin releasing hormone) pituitary secretes the gonadotropins - LH (lutenizing hormone) - FSH (follicule stimulating hormone) steroids: female: estrogen + progesterone male: testosterone

difference between meiosis in the mouse oocyte and meiosis in the sea urchin oocyte

in the sea urchin oocyte you won't see germinal vesicles because it's external fertilization

where can you harvest mammalian stem cells from (2 places)

inner cell mass in the blastocyst-stage embryo primordial germ cells in the fetus

germ cell determination in amniotes

no pole plasm PGCs come from the extra embryonic tissue migrate to the genital ridge where gonads are starting to develop (divide during the migration but then undergo mitotic arrest) there are attraction and repellent cues that guide them genital ridges are 1 in the pic

reproductive cloning (dolly)

nuclear transplantation NUCLEAR DONOR + OOCYTE DONOR 1) collect somatic cells + eggs 2) remove the meiotic spindle from the egg in meiosis II metaphase and enucleate the egg 3) electroporation - cell membrane changes structure + opens up pores so that nucleus/cell goes into egg 4) transplant blastocyst back into female the egg is just a shell, the DNA is from the udder/somatic cell

interphase I

nucleus + nucleolus are visible

what does ploidy value describe

number of homologous/paired chromosomes ie 2n vs n

chromatid

one copy of DNA

difference between oocyte and ovum

oocyte - immature egg before it has a capability of being fertilized oovum - mature egg

oogenesis steps

oogonium --> primary oocyte--> secondary oocyte --> ootid (haploid) --> mature egg there is germinal vesicle breakdown

Randolph and Morgan (1901)

pluripotent stem cell regeneration extensive regeneration capacity of planarian - each piece can regenerate a whole new organism cephalic helps learn more about transdifferentiation (going back to undifferentiated state)

significance of pole plasm

pole plasm is essential for germ cell determination in fruit flies removing pole plasm means no germ cell formation (necessary) transplanting these cells into an anterior portion means gonads will develop in anterior portion -ectopic growth (sufficient)

where do germ cells come from

primordial germ cells (PGCs) - contain all information for features. stem cells - these cells arrest in mitotic development embryonic stem cells (ESC's) give rise to: in embryo - PGCs in adult - PGS's turn into germ cells which turn into gametes

animal examples for mesolecithal cleavage types

radial (displaced) - amphibians

animal examples for each isolecithal cleavage type

radial - echinoderms spiral - molluscs bilateral - tunicates rotational - mammals

radial vs rotational divisions

radial: 1) meridonal (a and b) 2) meridonal (2 additional cleavages) - c and d - e and f rotational: 1) meridonal 2) - 1 meridonal - 1 equatorial

importance of growth

regulates division - # of cells - shape of cells (cytoskeletal) - size of cells changing the SA to volume ratio changes the interaction surface (size + shape of cells)

function of vasa

represses RNA at a translational level inhibits proliferation inhibits cell death necessary for development but not sufficient

paedomorphosis 2 evolutionary processes that allow it to happen

retention of larval physical traits into the adult that has gametes neoteny - somatic, physiological process of development is slowed down (ie. maturation of gametes) progenesis - faster sexual sexual development these two processes are heterochronies

left and right coiled snails

sinistral vs dextral orientation of cells changes after 1st division this change of orientation determines if the cell curves left or right

germ cell development in sea urchins

small micromeres - these cells migrate into cell at division at the tip of the archenteron, give rise to gonadal tissue - migrate to coelomic pouches vasa is a marker of small micromeres

therapeutic cloning

somatic cell nuclear transfer goal is to use patients own genetic material due to compatibility to regenerate organs eventually approach: take nucleus from patient and grow to blastocyst in cell culture

3 fertility issues

sperm activation sperm egg recognition hormonal regulation of oocyte maturation (prior to fertilization)

4 fertility issues

sperm activation sperm egg recognition hormonal regulation of oocyte maturation (prior to fertilization)

what two things do fertility treatments require understanding of

sperm egg interactions hormonal basis of reproduction and development

spermatogenesis order where do mitotic divisions go up to how long does meiosis I last how long does meiosis II last

type A1 spermatogonia --> type a2 spermatogonia --> Type a3 --> type a4 --> intermediate --> type B --> primary spermatocytes --> secondary spermatocytes --> spermatids --> residual bodies --> sperm cells mitotic divisions up to the primary spermatocytes (syncytia, multiple nuclei in the same cytoplasm) meiosis I from primary spermatocyte to secondary spermatocyte meiosis II from secondary spermatocyte to sperm cells

characteristics of stem cells

undifferentiated asymmetry in division (committed cell cannot go back to totipotent state) maintenance of the stem cell pool (one daughter cell out of two becoming committed while the other one is a clone of the original cell - to avoid stem cell depletion) self-renewing - immortal, make clones of themselves

A1 spermatogonia

unipotent stem cells (can only make sperm) self renew no cell division, continuous cytoplasm highly synchronous process

migration of PGCs what plays a major role?

unknown how divide along route migrate through hindgut SCF (stem cell factor) plays a huge rule, no gametes or gonads without SCF

ISH & B-galactosidase relevance what is the name of the gene that inhibits gene expression? name of a mutant?

used to visualize gene expression B galactosidase lights up all genes (blue marker) where there is inhibition of gene expression in the 4 cell stage, those are PGCs/future germ cells PIE- I is inhibitor of gene expression gcl - "germ cell less" is a mutant in drosophila

germ line development in zebrafish

vasa - factor that drives PGC development - expressed in small area + important for RNA transcription and processing - 4 clusters - 2 clusters of somite stage - migrate via SCF

germ cell determination in amphibians where do they come from? what happens after?

vegetal region found out via 1) surgical oblation - removal of germ plasm (essential for development) means there is no development of germ cells - NECESSARY 2) transplantation into something else to see if germ cells develop SUFFICIENT - Migration of germ cells vis cytoskeleton, ECM, actin + microtubules

what is transdifferentiation

when somatic cells turn into germ cells the opposite is when there is early separation of somatic and germ cells