Zoo 470 unit 2 Learning Objectives

Explain the basic components required for pronuclear migration

• sperm brings in a centriole associated with the centriole in the flagellum.

• guided by the sperm aster

• dynein moves female pronucleus along MT

Describe ICSI and how it is being used in the clinic and lab

Intracytoplasmic Sperm Injection; bypasses normal sperm/egg union, fixes poor sperm mobility/lack of functional flagellum

Explain what cleavage accomplishes for the embryo

• Multicellularity

• regionalization (different areas of the embryo can become different)

• new cell-cell interactions

• cell movement

• partitioning of localize determinants to different cells

Explain how cleavage in early embryos differs from that in somatic cells in adult animals, including whether it involves cell growth

• cleavage in early embryos does not involve cell growth(reductive cleavage) resulting in smaller cells compared to somatic cells

• cleavage much faster in embryonic cells than somatic

Explain how differences in the cell cycle in rapidly dividing cells in early embryos vs somatic cells

• two gap phases in cell cycle essentially absent in early embryos

Recall how cyclins and MPF regulate cell division, and extend this principle to cleavage

• anaphase promoting complex(APC) leads to cyclin degradation, but then cyclins are resynthesized at each cell division for MPF to be activated

• mitotic cyclin (cyclin B) synthesis precedes entry into mitosis

Describe how astral microtubules are thought to influence positioning of the cleavage furrow

• radiate to the cortex and influence where the cleavage furrow/contractile ring forms

• cortex contains activated myosin and actin

• move astral MT with glass bead and see where cleavage furrow forms

Provide evidence that Rho regulates assembly of the actomyosin network of the contractile ring

• when rho is activated it is associated with GTP and leads to changes in actin cytoskeleton/ actin recruitment

• rho activation necessary for cytokinesis

describe why providing additional membrane surface area is an important challenge faced by early embryos

requires extra membrane to enclose which takes efficient synthesis of lipid membranes

Explain how yolk content and phylogeny correlate with different cleavage patterns in early embryos, and how this affects placement of the blastocoel

• yolk content: higher yolk leads to incomplete divisions

• phylogeny: grouping animals by their mode of development

  • protosome: mouth first

  • dueterostome: mouth second

Explain how cleavage works in conjunction with localized determinants to influence fates of cells in early embryos

• partitioning the cytoplasm into blastomeres so that they can inherit different regulatory molecules

Cite examples of each major type of cleavage presented in the textbook

• radial Isolecithal: amphibians

• spiral isolecithal: annelids

• bilateral isolecithal: tunicates

• rotational isolecithal: mammals

• radial mesolecithal: amphibians

• bilateral telolecithal: birds

• Discodial telolecithal: fish, reptiles, birds

Describe the central dogma of molecular biology, and explain in a general sense how the various steps in processing of gene products provide opportunities for regulation of gene expression

• DNA →RNA via transcription, RNA→ Protein via translation

• transcriptional factors bind to DNA sequences to turn them on/off,

• alternative splicing removes introns to provide distinct proteins

explain why chromatin remodeling is necessary for efficient transcription, and how specific histone and DNA modifications lead to change in the status of chromatin for two different types of promoter

• chromatin remodeling needed for transcriptional machinery to bind

• HCP: usually open, but can be shut down by histone modifications

• LCP: associated with more mature, differentiated cell types, selectively activated by transcription factors, DNA methylation can shut down LCP

• Methylation blocks TF binding or stabilize nucleosomes via histone deacetylases/histone methyltransferases, reducing access to transcriptional machinery

Outline basic steps in eukaryotic transcription, and list the basic elements of a transcription unit(gene).

• transcriptional regulation: regulation of the likelihood that RNA pol II and friends will bind to a promoter

• promoter (initiation site), structural gene, terminator

• TFs bind to DNA within promoter region, then RNA pol II, forming the transcription initiation complex

Explain what maternal-to-zygotic transition(MZT) is, and what its main features are

• degradation of maternal transcripts and new transcription of zygotic transcripts

• occurs at different times in different species

• not be regulated by common pathways in all animals

Explain in basic terms how transcriptional regulators interact with DNA

• TFs bind gene promoters and organize the transcriptional machinery

Explain how enhancers and their corresponding transcriptional regulatory proteins can regulate the efficiency of transcription of a gene

• other TFs bind to enhancers to either activate or repress

• enhancers found far away but 3d conformation of DNA allows them to regulate Pol II binding to a promoter through a 3d complex.

• bring themselves closer to promotor and recruit RNA Pol II and regulatory TFs

Provide at least 3 examples of how overexpression of transcriptional regulators can force cells to adopt new fates

1. MyoD I: transforms non muscle cells → muscle cells

2. Pax6/eyeless: expressing eyeless in atennal or leg discs produces fully formed eyes in those tissues

3. yamanaka factors: expression of these reprograms them into iPSCs

Explain the combinatorial effects of transcriptional activators and repressors interacting with enhancer elements fine-tunes level of transcription of developmentally regulated genes

determines the total amount of transcription that occurs for a particular gene in a differentiated cell

Explain the basis for reporter constructs and CHIPseq and why they are useful

• fuse the regulatory DNA associated with a gene to a reporter

• examples are GFP and LacZ

• If it’s an essential gene, we can’t destroy the ability of the normal gene to be expressed. Reporters allow us to study regulation in transgenic animals without affecting the normal (endogenous) gene.

• ChIPseq: allows specific DNA sequences that are bound by specific transcription factor proteins

• allows pinpointing which sequences are enhancers and other regulatory elements

Explain how alternative splicing can result in different protein products from the same nuclear RNA and provide an example that indicates it is functionally important in embryos.

• different patterns of splicing (removing introns) or keeping exons allows one gene to produce multiple distinct protein products.

• Dscam→produces over 38k protein products that allow for neurons to distinguish themselves from neighboring cells

Explain what domain of a mRNA regulates its spatial localization, and provide specific examples of such localization

• 3’UTR regulates localization of mRNAs

• oskar and bicoid mRNAs attach to microtubule motors via their 3’UTR; kinesin→posterior, dynein→anterior

Be able to explain how translational regulators can regulate translation by mediating interactions between the 3’ UTR and 5’ ends of a mRNA, an provide examples

-proteins can bridge 3’ end to 5’ end physically looping the mRNA, block of promote access of initiation factors to the 5’cap, or control polyA tail length

ex. bicoid represses caudal translation. Bicoid binds to caudal 3’ UTR which allows for a protein to bind the 5’ end of the caudal mRNA and prevents translatioin of caudal mRNA

Explain how microRNAs regulate stability and translation of mRNAs

• precursors to miRNAs are processed by Drosha

• double stranded miRNAs are processed by Dicer into short double stranded RNAs

• duplexes recruit RNA-induced sliencing complex (RISC) which targets matchng mRNA

  • if match inexact, translation blocked

  • if match exact mRNA destroyed

• have tissue specific expression to regulate mRNAs in specific tissues even if mRNAs are expressed everywhere

Explain how ubiquitylation of proteins leads to their destruction, and provide an example relevant to developmental biology

• ubiquitins, small protein sequences, are added to proteins that are destined for destruction

• molecular machine that recycles fragment of proteins is called the proteasome

• ex. Wnt signaling

Explain how analysis of the pseudoautosomal region led to the hypothesis of a “testis determining factor” controlling sex determination in male mammals

region that has sufficient homology to allow crossovers between the ends of the X and Y chromosome

• the TDF region can be carried along with the primary pseudoautosomal region

• leads to unsuaual x chromosome or deficient y chrosome during recombination

• sufficient to confer maleness (male parts)

Describe evidence that Sry is sufficient and necessary to confer maleness in mammals

make transgenic xx mouse and analyze phenotypes/or make knockout xy and analyze phenotypes

• Sry and one other gene needed to generate viable offspring

• sry→ sox9 which is not on y chromosome but sufficient to testis development

Describe the basic aspects of gonadal differentiation in male and female humans from an indifferent gonad

males: sex cords→ seminiferous tubules

females: surface epithelium→surface of ovary

Describe which structures arise from the Mullerian and Wolffian duct during gonadal differentiation

wolfian duct: males, develops testes

mullerian duct; females, develops ovaries

Describe which cells/structures produce important hormones during gonadal differentiation

• XY→Sry→Sox9→testis, leydig cells, sertoli cells→ testosterone and dht, anti-mullerian duct hormone

• XX→Wnt4 expression→Ovary→Thecal(testosterone), granulosa cells→aromatase(converts testosterone to estrogen)

provide evidence that Sox9 is important for testis differentiation

• Sry produces Sox9(downstream effector)

• X O mice can’t be male→but adding Sox9 and other gene results in functional spermatids and males

how does AIS develop

Androgen insenstivity syndrome: results from mutations in androgen(testosterone, dht) receptors

Provide evidence that Wnt4 is important for ovary differentiation, and how Sox9 expression is prevented in ovaries

• Wnt4 is necessary for normal ovaries to promote female and prevent male type differentiation in females

• FoxL2 keeps Sox9 off in ovaries 

  • Removing FoxL2 leads to Sox9 expression and trans-differentiation of granulosa cells 

Explain how defective testosterone signaling leads to defects in secondary sex characteristics in mammals

Androgen insenstivity syndrome: results from mutations in androgen(testosterone, dht) receptors, so external genetilia do not masculinize and secondary characteristics do not devleop

Describe the basic components of germ plasm

• material associated with mitochondria(nuage)

• RNA helicases(enzymes that alter RNA structure)

Describe the basic function of germ granules in the C. elegans embryo

• form RNA processing centers in germ cells that associate with nuclear envelopes

• localize in posterior prior to first cleavage

• NOT membrane bound; liquid-liquid phase condensates

pole plasm in drosphila

• germ plasm equivalent

• use cytoplasm transplantation to see if pole plasm is sufficient to allow flies that would normally be sterile to make germ cells(it is)

provide evidence that oskar localization is sufficient to recruit germ plasm in drosophila

• make engineered form of oskar that encodes oskar+ 3’UTR of bicoid. bicoid mRNA is normally at the anterior, so the gene, if expressed in mothers, will cause some oskar to be at the anterior

• pole cells with grow in both posteior(normal) AND anterior(not normal) therefore is sufficient

Describe evidence that vertebrae germ cells use a chemokine-based chemotactic mechanism to migrate to the genital ridge, citing evidence from mice and zebrafish

• primordial germ cells migrate into the genital ridge

• done by SDF-I(chemokine) and CXCR4(receptor on PGCs)

• Adding excess SDF-I perturbs germ cell migration in mice

• SDF-1 or CXCR4 knockdown perturbs germ cell

  migration in zebrafish

Describe the basic events that occur during axis specification in all triploblastic animals.

• establish polarity in egg

• activation of signaling pathways

• segregation of germ layers

Describe the basic types of morphogenetic movements that shape animal embryos, and be able to recognize examples of each.

invagination: inward bending (formation of digestive tube)

epiboly: spreading (ectoderm in frogs)

involution: rolling (lip of blastopore)

ingression: detachment of single cells (mesemchyme)

delamination: splitting of one to two (hypoblast in bords)

convergent extension: direced rearrangment (germ band in flies)

Explain how the reproducible cell lineage of C. elegans aids the analysis of cell fate, and identify which cells make endoderm and mesoderm in the C. elegans embryo.

• you can trace every cell back to its origin→which blastomere produced which tissue, when a fate decision occured, which ealier signals influenced that decision

Explain what the initial cue is that polarizes the one-cell zygote

Explain how PAR protein distributions change after fertilization, and provide evidence that they are important for blastomere identity in the early embryo.

Identify which cytoskeletal system is largely responsible for the reorganization of the one-cell zygote

Explain the role of MEX proteins in creating two cells of distinct fate in the two-cell zygote

Explain how SKN-1 acts as a crucial protein for EMS (and hence MS) differentiation, and how PIE-1 restricts SKN-1 activity to a specific blastomere.

Explain how signaling from P2 induces EMS to produce a daughter cell that produces endoderm, cite experimental evidence for such signaling, and identify the signaling pathway responsible.

Predict the effects of placing P2 and EMS blastomeres of specific genetic constitutions in contact (e.g., P2 cells lacking MOM-2/Wnt, EMS lacking MOM-5/Frizzled, etc.).

Describe the morphogenetic movement that internalizes endodermal precursors during gastrulation, and what cytoskeletal system is probably responsible

Describe the morphogenetic movement that covers the embryo in epidermis during ventral enclosure, and what cell-cell adhesion system is required.