Bio 125: Development of Multicellular Systems

Development of Multicellular Systems

From Egg to Early Embryo

• The size of an egg does not determine the complexity of creating a new embryo.
• The fertilized egg (zygote) is a totipotent stem cell, capable of developing into a complete organism and differentiating into any cell or tissue.
• Development relies on surrounding follicle cells.
• The egg contains:
  • DNA: haploid (n) → diploid (2n) during fertilization
  • Yolk: provides food and energy for the embryo
  • Cytoplasm (ooplasm): contains mRNAs and proteins, important for early development events

Maternal Control of Development

• Maternal genes in the cytoplasm control embryonic development until the embryo's genome is activated.
• Maternal phase: early development depends on maternal gene contribution, duration varies among organisms.
• Embryos developing inside (viviparity) have less yolk, while those developing outside (oviparity) have more yolk due to environmental factors.

Mid-Blastula Transition

• Zygotic gene transcription starts, slowing down the cell cycle.
• Asynchrony in cell division and increased cell mobility occur.
• This is controlled by the removal of maternally loaded inhibitors against exponentially increasing DNA amount.

Cleavage Phase

• The zygote undergoes repeated divisions to produce more blastomeres without an increase in embryo size.
• Cell cycle: process of cell replication, skipped G1 and G2 phases in cleavage phase to rapidly divide.
• Cyclins are translated from maternal mRNAs in ooplasm.

Spacial Organization of Early Cleavage

• Some eggs have clear polarity, with an animal-vegetal axis.
• Cleavage patterns differ between species, depending on spindle (microtubules) orientation.
• Mammalian embryos exhibit a rotational cleavage pattern.

Regulation of Cellular Differentiation

• All organisms start as a single cell, dividing to form a mass of cells, generating differences through symmetry breaking.
• Theories of symmetry breaking:
  • Nuclear determinants are differentially distributed among cells.
  • Asymmetric cell division distributes determinants unevenly.
• PAR proteins regulate cellular differentiation, establishing differences between poles of the embryo.

From One to Many: Differential Gene Expression

• Cells acquire different functions through differential gene expression.
• Genomic equivalence: all cells share the same DNA, but cells acquire different phenotypes via differential expression.
• Evidence for genomic equivalence:
  • Lens regeneration in salamanders
  • Cloning of amphibians and mammals

Mechanism of Differential Gene Expression

• Gene expression can be regulated at every step, including transcription, RNA processing, transport, and translation.
• Gene regulatory elements (GREs) and transcription factors (TFs) interact to control gene transcription.
• DNA accessibility is regulated by chromatin conformation, which is modified by epigenetic modifications.

Epigenetic Modifications

• Chromatin conformation is regulated by epigenetic modifications, such as histone modifications and DNA methylation.
• Histone modifications:
  • Acetylation: loosens histone, making DNA accessible.
  • Methylation: can increase or decrease transcription.
• DNA methylation:
  • Adds methyl groups to cytosine, regulating gene expression.
  • CpG islands: enriched in the vicinity of genes, regulating gene expression.

Inheritance to the Next Generation

• Epigenetic modifications are faithfully copied during cell division and inherited to the next generation.
• Provides cellular memory, allowing cells to "know" their status.

Waddington's Landscape

• Metaphor for development and the importance of epigenetics.
• Inclined surface represents a series of choices a cell makes, becoming more committed to its fate.

Determining the Cell Epigenome

• Epigenome: the collection of all epigenetic modifications on the DNA in a single cell.
• Identifying a cell by its epigenome involves:
  1. Tissue dissociation
  2. Single-cell separation
  3. Single-cell encapsulation### Transpose-Accessible Chromatin (ATAC-seq) and Chromatin Immunoprecipitation Sequencing (ChIP-seq)
• ATAC-seq:
  • Determines chromatin accessibility in the genome
  • Uses hyperactive transposase (Tn5) to insert oligonucleotides of known sequence
  • PCR amplification and sequencing
  • Usage of known sequences to determine chromatin organization
  • High frequency of inserted transposons indicates open chromatin
• ChIP-seq:
  • Determines exact epigenetic modifications
  • Chromatin is fragmented, and antibodies recognizing specific epigenetic modifications bind
  • PCR amplification and sequencing
  • Used to study chromatin state and accessibility during development and in different tissues

Study of Chromatin Accessibility in the Mouse

• 1000 ChIP-seq assays and 132 ATAC-seq assays across 72 distinct tissue-stages
• Study shows how chromatin state and accessibility change during development and in different tissues

Differential Gene Expression: GREs and Promoters

• Gene Regulatory Elements (GREs):
  • Are encoded in the genome and turn genes on and off
  • Include enhancers and silencers
  • Can be located near or far from the promoter
  • Interact with specialized proteins: transcription factors (TFs)
• Promoters:
  • Are encoded in the genome and turn genes on and off
  • Located near the gene transcription start site
  • Where the transcription machinery assembles
  • TFs help RNA Polymerase to engage with the promoter and start transcription

Differential Gene Expression: Transcription Factors

• 6-8% of proteins are estimated to work as TFs, regulating gene expression through DNA binding
• Motifs:
  • Most TFs regulate transcription by binding to this defined sequence
  • Are short and degenerate
  • Specific nucleotides at specific positions
• TFs act in a combinatorial way:
  • By collaborating or competing with other TFs, common target genes can be regulated
  • A relatively small number of proteins generates a large diversity of cell types
  • 1 enhancer can contain several different motifs
  • 1 TF has one motif
  • Different combinations of TFs are present in different cells

Mechanism of Enhancer Action

• Enhancers and silencers can be located far away from the promoter they control
• Example: Sonic hedgehog (Shh) promoter
  • Signaling molecule for patterning vertebrate limb depends on an enhancer located 1Mb away
  • Within the enhancer, there are different motifs
  • Point mutations in this enhancer can cause limb defects

Topologically Associated Domains (TADs)

• Enhancers and promoters come to close physical proximity via loop formation
• This leads to the formation of protein complexes that can regulate RNA Polymerase activity
• Genome might be organized in loops and logically associated domains
• TADs:
  • Are interacting genomic regions
  • DNA sequences physically interact more with each other than with sequences outside of the TAD
  • Identification of TADs via Chromosome Conformation Capture (3C)

Chromosome Conformation Capture (3C)

• 3C allows the identification and quantification of DNA sequences that are physically interacting
• Steps:
  1. Formaldehyde treatment: introduces bonds that cross-link protein-DNA interactions
  2. If two loci interact, they will be "frozen" in this conformation
  3. Restriction enzyme treatment: bonded Protein-DNA interactions stay intact
  4. Ligation: in highly diluted DNA
  5. Reverse cross-linking: releases formaldehyde-mediated bonds
  6. Amplification and sequencing: of DNA fragments
  • Loci that are far from each other are ligated together
  • The probability of this happening depends on proximity in the 3D space and frequency of interaction
  • By analyzing hybrid molecules, the quantification of the degree of physical interaction is possible

Insulators

• Form boundaries in 3C diagrams
• Sequences are CCCTC rich
• These consensus sequences are bound by CCCTC-binding factor (CTCF)
• Prevent inappropriate enhancer-promoter interactions
• Mutations cause developmental phenotypes
• Chromosomal rearrangements that disrupt these boundaries
• New enhancer-promoter interactions

Transcriptional Hubs

• Are multiple enhancers and promoters that are co-expressed
• Performed loops: primed and ready to respond to a specific signal
• Signal makes one or more TFs available
• Leads to conformational change
• Allows immediate transcription of all the genes within the loop

Laboratory Experiments

• In vivo enhancer activity assay
• Detection and localization of mRNAs: in situ hybridization
• Single-cell transcriptomics
  • Technique examines the gene expression level of individual cells by simultaneously measuring the mRNA concentration of many genes
  • Helpful if you want to know "all the activity" in the cell

Spatial Patterning of Tissues and Embryos

• Difference between cells and organs/organisms
• Cells are organized into tissues/organs
• Biological function usually emerges in the scale of tissues/organs, not at the individual cell level
• How cells are organized over large scales drives morphological and evolutionary diversity

Organizing Differentiation in Space and Time

• Body axes
  • Animal-vegetal axis: forms future anteroposterior body axis
  • Dorsoventral axis: is orthogonal to the animal-vegetal axis
• Embryo positioning
  • Gastrulation: dynamic organization of the embryo into three germ layers
  • Ectodermal, mesodermal, and endodermal germ layers
• Evidence that differentiation is regulated in space and time
  • Observation: fate mapping and lineage tracing
  • Manipulation: "cut & paste" experiments
  • Impact of position and time on fate
    • Cells normally select a fate appropriate for their location in the embryo
    • Cells are specified if they execute a normal fate when isolated
    • Cells can adapt to a new fate appropriate for the location if moved before determination
    • Fate of late-stage donors is determined

Morphogens and Positional Information

• Exploiting diffusion to pattern tissues
• Morphogens: diffusible chemical substances that drive morphogenesis
• Generating chemical gradients
  • Establish localized source of factor
  • Allow diffusion of factor from source
  • Chemical gradient can provide graded positional information
  • Gradients provide a mechanism to allow positioning to influence differentiation
• Source and sink model
  • Factors deplete/destroy morphogen depletion
  • Ensures morphogen gradient
• From morphogen gradient to domains
  • Morphogen gradient theory offers a mechanism to generate positional information
  • Animal body plans are not organized in continuous gradients
  • Embryos become subdivided into discrete regions/domains

Evidence for Morphogen Thresholds

• Morphogen decreases with distance from the source
• Morphogen provides cells with positional value
• Cells switch on specific target genes that have different morphogen activation thresholds
• Positional information provides a mechanism to go from morphogen source to gradient to tissue pattern
• Multiple morphogen gradients can be combined to form more complex patterns### Heidelberg Screen
• The screen involves two approaches:
  • Knowing where to look: mutated important developmental genes result in strong defects in embryo/larvae
  • Genome-wide approach: isolated many differently patterned mutant larvae, leading to the identification of many genes that caused deletions of body regions when mutated

Bicoid Morphogen

• Bicoid is a maternal-effect gene: the phenotype of an embryo is not determined by its genotype, but by the genotype of its mother
• Bicoid mutant: complete loss of anterior structures (e.g., head) and mirror-image duplication of the most posterior segments
• Bicoid gene encodes transcription factors (TFs) that bind to DNA to regulate transcription and to mRNA to regulate translation
• Maternal Bcd mRNA is localized to the anterior pole of the oocyte, and a single cell contains many nuclei in one cytoplasm (syncytium) during early stages

Spatial Patterning of Tissues and Embryos

• Morphogen gradient: a concentration-dependent gradient of a signaling molecule that induces different gene expressions in different regions of a tissue
• Different morphogens in Drosophila:
  • Hedgehog (hh)
  • Wingless (Wg/Wnt)
  • Decapentaplegic (dpp/BMP)
• Morphogen patterns of activation/repression occur in a concentration-dependent manner
  • Failure of expression: segment polarity and limb defect mutants are often different alleles of the same gene that fail to complement when crossed
  • Pleiotropic: a single gene produces more than one effect in several tissues

Molecular Mechanisms of Morphogen Action

• Morphogen expression and secretion by defined source cells
• Extracellular signal gradients convert to patterns of gene expression
• Wingless (Wg/Wnt) pathway:
  • Ligands: Wnt
  • Receptors: Frizzled, G-protein-coupled receptor (GPCR), and LRP
  • Signal transduction: Dishevelled
  • Transcription factors: B-catenin/Armadillo and TCF/LEF
• Hedgehog (hh) pathway:
  • Ligands: Shh/hh
  • Receptors: Patched (multitransmembrane receptor) and Smoothened (GPCR)
  • Signal transduction: Fused
  • Transcription factors: Gli full-length (GliFL) and Gli Repressor (GliR)

Integrand Models and Molecules

• Competent cells receive positional information from neighboring cells that "organize" them
• Organiser gene expression is induced in floor plate by neighboring notochord
• Shh signaling in cilia: Smoothened receptor must enter cilium to signal
• Receptor localization, signal transduction, and cilia are regulated by Shh signaling

Reaction-Diffusion Systems

• Pauses in the reaction-diffusion system generate complex patterns
• Principles of reaction-diffusion systems:
  • Local increase in diffusible "activator" triggers peak in expression via "auto-activation"
  • Activator triggers expression of its own faster-diffusing inhibitor, which represses activator outside local peak
  • Changing geometry: spots to branch networks

Investigation of Morphogen Activity

• Indirect activity of morphogens: measuring the impact on final embryonic structures
• Direct activity of morphogens: measuring the impact on expression of target genes
• Quantitative light microscopy to measure local concentrations of morphogens