Developmental Biology

• Different model organisms are useful for studying different aspects of developmental biology.
  • Nematode worms: Signal transduction pathways, genetic control of development, and cell identity decisions.
  • Drosophila flies: RNA and protein localization, genetic control of development, segmentation, and cell identity.
  • Xenopus frogs: RNA and protein localization, cell movements, cell cycle, and cell identity.
  • Zebrafish: RNA and protein localization, cell movements, cell cycle and cell identity, and genetic control of development.
  • Chicken: Organ formation and cell death.
  • Mouse: Organ formation and genetic control of organ formation.
• There is a positive correlation between size (number of cells) and time for a given developmental stage.

Processes Before Fertilization

• Many developmental processes begin before fertilization.
• Axis formation establishes a coordinate system.
• Maternal genes, proteins, and organelles accumulate.
• Maternal determinants are partitioned to give rise to different cell types.
• A determinant is a substance (RNA, protein, organelle, etc.) localized to a part of a cell that controls its differentiation.

Cell Fate After Fertilization

• After fertilization, cell identities become restricted.
• Asymmetric distribution dictates that daughter cells have different fates due to differing amounts of a determinant.
• In proliferative mode, a cell (A) produces daughter cells with the same fate (A).
• In stem cell mode, a stem cell (A) can produce more stem cells and cells with a certain fate (B).
• In diversifying mode, cell (A) gives rise to cells with one of two fates (B, C).
• Orientation of cell division leads to different fates.

Induction

• Induction: one cell (inducing) interacts with another cell (responding), causing the responding cell to change its identity.
• Cells signal through diffusion, direct contact, or gap junctions.
• Induction occurs at various times and between different tissues.
• After initial patterns are set, cells communicate to modify differences or similarities.
• Cell response is limited by manner and timing.

Morphogenesis

• Groups of cells become organized through morphogenesis, which involves:
  • Cell adhesion.
  • Cell migration (localization to region).
  • Interactions with ECM.
  • Cell shape changes.
• Morphogens form a concentration gradient that cells use to determine position and fate.

Developmental Mechanisms

• Developmental mechanisms are generally redundant due to evolutionary and developmental reasons.
• Determinate development: normal division patterns are stereotyped
• Indeterminate development: normal division patterns are not stereotyped
• Determination (commitment): a cell's potential becomes autonomously and heritably restricted, allowing it to express only one fate (restricted) or a smaller set of possible fates (pluripotent).

Experimental Determination of Commitment

• Commitment can only be assayed experimentally.
• Transplant a cell from region B to region A: if it becomes cell type A, it is not committed; if it becomes cell type B, it is committed.

Theories of Development

• Weismann’s mosaic development theory: the egg is a mosaic of localized determinants, and early divisions make daughter cells different due to unequal distribution of determinants.
• Roux’s experiment: killing one cell at the two-cell stage results in a half-formed frog embryo, suggesting early cleavages determine cell fates (mosaic development).
• Drisch’s experiment: removing one cell at the two-cell stage results in a normal (but small) sea urchin, suggesting the ability to restore normal development (regulation).
• Regulative development involves cell interactions and signaling between cells (indeterminate).

Anterior-Posterior Axis in Frogs

• Why study frogs?
  • Easy to maintain in lab.
  • Eggs are large and easy to obtain.
  • Good embryological preparation.
  • Easy to manipulate.

Frog Embryology

• Oocyte grows without dividing for months before fertilization.
• Cleavage occurs.
• Gastrulation forms germ layers.
• Organogenesis completes the body plan.
• Embryos can be holoblastic or meroblastic at the first cleavage.
• Holoblastic: no material passed; asymmetry must occur before this stage.
• Meroblastic: material and organelles can pass
• Frogs are holoblastic, and the first three cleavages set up asymmetry, correlating with future body axes.
  • First cleavage: right/left.
  • Second cleavage: dorsal/ventral.
  • Third cleavage: anterior/posterior (cytoplasmically asymmetric).

Methods to Determine If Cleavages Segregate Different Fates

• Ablations: Roux's and Drisch's experiments on cell ablation at the two-cell stage.
• Fate mapping: cells are marked and observed after development.
• Blastomere isolation: remove two cells and culture independently to observe if they produce the same or different .
• Transplantation: cells are moved to see if they keep initial fate or take on a new one.

Frog Specification Map

• Anterior side forms ectoderm and mesoderm; the posterior side forms mesoderm and endoderm.
• Separated anterior cells form ectoderm only, while posterior cells form endoderm only.
• The animal-vegetal axis is determined maternally before fertilization.
• The nucleus is in the highly pigmented animal half.
• Maternal mRNAs concentrate at the vegetal pole.
• Vg1 maternal mRNA is synthesized early and localized to the vegetal pole, functioning as an early signal for mesoderm induction.
• Vg1 mRNA levels remain constant across the early oocyte.
• In the late oocyte, Vg1 concentrates at the vegetal pole due to localization by microtubules and anchoring by microfilaments.
• Vg1 is released from the cytoskeleton and diffuses locally.

Vegetally Localized Maternal RNAs

• Vg1
• Wnt11
• VegT

METRO Pathway

• Some maternal mRNAs follow the METRO pathway—localized to vegetal cortex in order to transport cytoplasmic messenger transport.

Dorsal-Ventral Axis in Xenopus

• Sperm entry induces events that define the dorsal-ventral axis, with the dorsal side forming opposite the sperm entry point.
• Sperm entry point (SEP) occurs in the animal hemisphere.
• Sperm entry causes the cortex to loosen from the denser cytoplasm, enabling independent movement.
• Cortical rotation occurs.
• The grey crescent forms opposite the SEP, becoming the Spemann Organizer.
• An array of sub-cortical microtubules orients with the + ends away from SEP.
• Sperm provides a centrosome, forming parallel microtubules.
• Microtubules are inhibited by UV light, effects are transient.
• This forms parallel array of “train track” microtubules in the direction of rotation in the vegetal hemisphere only
• Direction of cortical rotation is more important than SEP. DAI or Dorso-Anterior Index is a measure of dorsal axis development and is useful for experiments that disrupt dorsal development.
• The higher the DAI, the more normal development of dorso-anterior characteristics.
• DAI scale:
  • 0: Gut piece, lacks all dorsal structures.
  • 1: Gut piece, somites.
  • 2: Gut piece, somites, otic vesicle.
  • 3: Gut piece, somites, otic vesicle, eyes.
  • 4: Gut piece, somites, otic vesicle, eyes, cement gland.
  • 5: Normal embryo.
• The amount of cortical rotation correlates with the DAI scale of development.

Relocation of Dorsalizing Factors

• Relocation of dorsalizing factors during cortical rotation confers the dorso-ventral axis.
• Vegetal dorsal cell transplantation rescues UV-irradiated embryos (Nieuwkoop center).
• Dsh protein (Disheveled) and Wnt11 mRNA dorsalizing factors relocated from the vegetal cortex stabilize β-catenin on the dorsal side, which is needed for Nieuwkoop formation, inducing the Spemann Organizer.

Mid-Blastula Transition (MBT)

• Early development depends on maternally supplied mRNA, proteins, and organelles.
• During cell cycles 1-12:
  • Abbreviated cell cycle (no G1 or G2).
  • No RNA transcription; development depends on maternal genes and products.
  • Cell division without growth produces synchronous cell divisions and DNA synthesis.

Characteristics of MBT

• No zygotic RNA synthesis, no active migrations.
• MBT is marked by new RNA synthesis, cell migrations, and non-synchronous cell divisions.
• After cell cycle 12, cells may begin desynchronizing, acquiring G1/G2 phases and beginning transcription.
• Signal for entering that MBT:
  • ratio of DNA to cytoplasm.

Proposed Model for Determining Signal

• • MBT is due to titration of a cytoplasmic factor that binds DNA.
• MBT onset can be altered by changing nuclear content or cell volume.
• Increasing the DNA:cytoplasm ratio (through polyspermy or DNA injection) causes premature transcriptional activity.
• As the egg cleaves, cytoplasmic volume stays the same while DNA increases, reducing the amount of repressor relative to DNA until a threshold is reached.

Mitotic Domains

• Mitotic domains is the expression of cell cycle genes.
• MBT occurs at cycle 14 in Drosophila.
• Cycles 1-13: divisions.
• Cycle 14-16: zygotic transcription, varied G2 phases.
• Mitotic domains are discrete groups of cells that enter M phase in synchrony with each other but not with surrounding cells.
• A single mitotic domain may include discrete clusters of cells.

Important Cell Cycle Proteins

• Zygotic expression of cell cycle proteins are controlled spatially and temporally after MBT.
• String: cdc25 homolog that controls mitosis; string mutants have no mitotic divisions after M13.
  • Loss of String, lack of mitotic divisions
  • String expression corresponds to cell cycle of mitotic domains.
• Cyclin E: controls G1 to S phase; cyclin E mutants arrest after cycle 16.
  • Overexpression of cyclinE leads to cells entering immediately.

Mesoderm Identity and Mitotic Activity

• Snail and twist are mesoderm-specific repressors.
• Snail represses ectodermal genes.
• Twist activates mesodermal genes.
• Snail and twist mutants lack mesoderm, which is preceded by altered mitotic behaviors of cells.
• String expression in mesoderm is disrupted, leading to the loss of mesoderm activity.

Mapping the Fates of Cells

• Fate maps can be elucidated at an early cleavage stage in frogs, but late in zebrafish.

Mapping Methods

• In frogs, intracellular markers are used.
  • Not harmful to cell
  • Labels only intended cell
  • Is passed on faithfully to progeny
  • Reasonable long-lived
• In zebrafish, cells are injected with intracellular fluorescent tracer dye
• Other methods of fate mapping:
  • Direct observation.
  • Genetic and physical mosaics (transplantation).
  • Extracellular labeling and membrane markers.
  • Introduced genetic markers.
  • Lineage mapping defines what pluripotent cells will be come

Fate maps (Zebrafish)

• Anterior: epidermis.
• Posterior: endoderm.
• Middle: mesoderm.
• Brain and notochord: dorsal.
• Blood and muscle: ventro-posterior.
• Fate maps look fairly similar across many different species.
• Fate map vs specification map:
  • Fate map: what cell regions will become in an intact embryo.
  • Specification map: what different cell regions will become in isolation.

Gastrulation

• Gastrulation involves cell rearrangements.
• Epiboly: movement of cells around the yolk cell.
• Radial intercalation: thinning of cell layer.
• During gastrulation, cell movements get cells inside to form germ layers.
  • Involution: turning in of cells.
  • Blastopore lip: curved edge of blastopore opening around the yolk plug.
• Other ways to "get cells inside":
  • Invagination: bending inward of a sheet of cells.
  • Ingression: individual cells pop inside a deeper layer.
  • Delamination: cell in a layer divide in the same direction inside the layer.
  • Cavitation: formation of a hollow lumen within cells
• Convergent extension: lengthening cells in one direction while narrowing in another to elongate the embryonic axis
• Medial-lateral intercalations: convergence of cells reducing medial-lateral thickness and lengthening anterior-posterior axis
• New cellular interactions due to 3D arrangement of axes
• Cell movements occur in synchrony. Germ Layers and Resulting Biological Structures
• Ectoderm: epidermis, nervous system, hair.
• Mesoderm: muscle, skeleton, kidneys, gonads, blood, connective tissue.
• Endoderm: liver, guts, lungs, pancreas, bladder.
• Embryonic axes are modified, going from spherical egg to elongated body plan.

Mesoderm Induction

• Organizers are key signaling centers.

Specification of Mesoderm

• Mesoderm becomes patterned along the dorsal-ventral axis:
  • Blood.
  • Lateral plate mesoderm.
  • Somites.
  • Notochord (Spemann organizer).
• Spemann organizer: future notochord mesoderm, patterns remaining mesoderm and induces the nervous system.
• Spemann organizer homologs are a common feature in vertebrate embryos.
• Nieuwkoop center: induces Spemann organizer and dorsal mesoderm.

Signal Hypothesis

• Four signal hypothesis: four signals occur at key points of development:
  • Vegetal signal (Vg1, Activin): general mesoderm formation in marginal zone.
  • Spemann/Nieuwkoop signal (Wnt11, Dsh, β-catenin, Noggin): dorsal mesoderm
  • Ventral mesoderm signal (BMP4): blood, kidneys, and lateral mesoderm.
  • Posterior mesoderm signal (FGF): somite formation, muscle development, and axial elongation.

Mesoderm Inducing Factors (MIF)

• MIF must be biologically active in a test system, must be expressed at the right time and specific inhibition must prevent in vivo action.
• Animal cap assay for mesoderm induction: animal cap forms skin only; endoderm forms gut only; animal cap and endoderm attached form skin, gut, and muscles (mesoderm!).
• Fibroblast Growth Factor (FGF) induces ventrolateral mesoderm but not dorsal mesoderm.

Vg1:

• induces dorsal mesoderm in animal caps; varied levels induce different mesodermal genes.

Activin:

• gradient effects on mesoderm formation (high levels induce dorsal mesoderm, intermediate levels induce lateral mesoderm, and low levels induce ventral mesoderm).
• Noggin and chordin—not needed for mesoderm formation, but used for patterning of preexisting mesoderm for dorsalization
• BMP—not needed for mesoderm formation, but used for patterning of preexisting mesoderm for ventralization
• Vg1, FGF, and somewhat activin—induce mesoderm

Cell Interactions Can Lead to Induction

• Signals from Nieuwkoop center induce overlaying mesoderm to become dorsal mesoderm and Organizer.
• Signals from the Spemann organizer induce dorsalization of mesoderm
• Cell spread through fibronectin due to 3d arrangements
• Integrins are receptors that mediate cell attachments to ECM.

Disruption of Integrins

• Blocking integrin-fibronectin interactions halts gastrulation movements by inhibiting migration of mesodermal cells.
• If epiboly occurs but involution movement is blocked, extra SA has nowhere to go and wrinkles to compensate

Neural Induction

• The Spemann organizer gives rise to the notochord, while the dorsal ectoderm gives rise to the CNS.
• Dorsal mesoderm induces the nervous system by signaling molecules.

Neural Tissue and Induction

• Grafting ventral ectoderm at the dorsal ectoderm forms neural tissue.
• Organizer molecules include chordin, noggin, and follistatin (which antagonize ventral signaling).

Neural Tube Formation

• In a late gastrula, the neural tube (from ectoderm) is directly above the chord.
• Neural inducing factor: endogenous molecule that must be present at the right time.
• Follistatin: Suggests that activin in neural development is an inhibitor.
• Noggin induces anterior neural markers without formation of mesoderm in animal caps.
• Anterior-posterior patterning: signals released vertically from the mesoderm.
• Planar signaling passes through the ectoderm, originating at posterior layers.

Neurula Graft Assay

• Neurula anterior mesoderm grafted into early gastrula induces a head with eyes and forebrain.
• Neurula posterior mesoderm grafted into early gastrula induces trunk and tail.
• Signals from underlying layers pattern ectoderm and organizer regions
• Formation of tissue starts at the neural tube
• Nodal structure forms floorplate, epidermal structure forms roof plate
• BMP is used for dorsalization

Role of Sonic Hedgehog

• Sonic Hedgehog (shh) mediates local cell to cell interactions
• Neural tube pattern is defined by Shh
• BMP is reused to define dorsalization

Limb Development

• Limb outgrowth represents a new developmental set of axes.
• New axis—proximal-distal axis where proximal means close to body and distal means far from body
• Dorsal represents back of hand while ventral represents the palm

Zone of Polarizing Activity (ZPA):

• Mesenschymal cells proximal to progress zone

Apical Ectodermal Ridge (AER):

• Most distal cells, distal of the Progress Zone Progress Zone- actively growing cells responsible for limb length
• Mesenchymal mesoderm: area of differentiation, proximal to progress zone
• AER supports the outgrowth pattern of limbs
• Cells closer to truncation differentiate more easily

Limb Development Requirements

AER is permissive, but will form. Removing AER, then re-adding, will still form

• AER maintains proliferation and prevents differentiation of progress zone cells.
• The AER is a source of FGF.
• ZPA polarizes and orients, mirror image if switched

High/Low Conc leads to the formation of posterior/anterior structure

Key limb signals

Shh : morphogen in ZPA Hox: Maintains AER to facilitate limb growth

AER and Zona interact to allow limb development

• Positive feeback mechanism
• FGF is produced, leads to SHH release, which regulates AER

Hox Genes

Proximal-distal: Hox 9: Sacpula Hox 10: Humerus Hox 11: Ulna/Radius Hox 12: Metacarpals Hox 13: Digits

This quiz explores the fundamental principles of developmental biology, including model organisms like Drosophila and C. elegans, and processes such as cell death, axis formation, and stem cell behavior. It contains material about induction and morphogenesis. Key concepts cover genetic control and cellular interactions during development.