Alberts Johnson Lewis Raff Roberts Walter Molecular Biology
Alberts • Johnson • Lewis • Raff • Roberts • Walter Molecular Biology of the Cell Fifth Edition Chapter 22 Development of Multicellular Organisms Copyright © Garland Science 2008
Development of multicellular organisms § Introduction: general features of animal development § Caenorhabditis elegans: Cell-cell interaction in embryogenesis § Drosophila melanogaster: Pattern formation: Genesis of the body plan
4 essential processes involved in development of multicellular organisms • Morphogens for inducing signaling events • Cell-cell interaction for fate/polarity determination
Functional conservation of developmental genes Homologous proteins functioning interchangeably in the development of mice and flies. § Drosophila Engrailed protein can be substituted for the corresponding gene for the Engrailed-1 protein of the mouse. § Loss of Engrailed-1 in the mouse causes a defect in its brain (the cerebellum fails to develop); the Drosophila protein rescues this deformity. Figure 22 -2 a Molecular Biology of the Cell (© Garland Science 2008)
(Top) Ectopic expression of fly Eyeless in the leg (Bottom) Ectopic expression of squid Eyeless Figure 22 -2 b Molecular Biology of the Cell (© Garland Science 2008)
Basic anatomical features are shared in most animals. § Egg cell divides to form an epithelial sheet § Gastrulation • Ectoderm • Endoderm • Mesoderm between the two layers Figure 22 -3 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008) Initial intucking of the epithelium during sea urchin gastrulation.
Similar basis body plans, radically different body structures Regulatory DNA defines gene expression and development program. Same set of genes Different arrangements of regulatory modules Different states Figure 22 -4 Molecular Biology of the Cell (© Garland Science 2008)
Analysis of development § Description of morphological changes Experimental embryology in 19 th century § Causal mechanisms v Experimental embryologists’ approach Transplantation & track cell (tissue)-cell interaction through division, transformation, migration v Complementary approaches Developmental geneticists Figure 22 -5 Molecular Biology of the Cell (© Garland Science 2008)
Cell-cell interaction studies in large animal embryos (birds/amphibians) Some striking results obtained by experimental embryology. (A)Split of an early amphibian embryo into two parts with a hair loop. (B)An amphibian embryo at a somewhat later stage. Grafting of a group of cells from another embryo at that stage. Figure 22 -6 Molecular Biology of the Cell (© Garland Science 2008)
Developmental decision-making § can occur long before visible differentiation § committed/specified/determined A standard test for cell fate determination Figure 22 -7 Molecular Biology of the Cell (© Garland Science 2008)
Positional values (information) : Position-specific character of cells The early leg-bud cells are already determined as leg, but are not yet irrevocably committed to form a particular part of the leg. Ectopic expression : Eyeless case Figure 22 -8 Molecular Biology of the Cell (© Garland Science 2008)
§ Tbx 4 and Pitx 1: Expressed in the leg bud. § Misexpression of Pitx 1 in the wing bud causes the limb to develop with leg -like characteristics. Figure 22 -9 Molecular Biology of the Cell (© Garland Science 2008)
Inductive signals to create different cell fates. Equivalence group → distinct cell fate Interaction by cell-cell contacts or diffusible molecules. Figure 22 -10 Molecular Biology of the Cell (© Garland Science 2008)
Distinct sister cell fates by asymmetric division : Intrinsic mechanism independent of extracellular signal Asymmetric segregation of cell fate determinant(s) Figure 22 -11 Molecular Biology of the Cell (© Garland Science 2008)
Cell-cell interaction: Genesis of asymmetry through positive feedback. X inhibits X synthesis in adjacent cells Lateral inhibition Figure 22 -12 Molecular Biology of the Cell (© Garland Science 2008)
Positive feedback Symmetry breaking Bistability Memory of the stable states Similar principles in cell fate decisions, regionalization of tissues, asymmetry within a cell, etc
Limited number of signaling pathways : coordinate spatial patterning of embryos : Used repeatedly during development Some inducers are long-range morphogens that function in gradients. Table 22 -1 Molecular Biology of the Cell (© Garland Science 2008)
Sonic hedgehog as a morphogen in chick limb development. Figure 22 -13 Molecular Biology of the Cell (© Garland Science 2008)
Two ways to create a morphogen gradient. (A) By localized production of a morphogen that diffuses away from its source. (B) By localized production of an inhibitor that diffuses away from its source and blocks the action of a uniformly distributed inducer. Figure 22 -14 Molecular Biology of the Cell (© Garland Science 2008)
Patterning by sequential induction. A series of inductive interactions can generate many types of cells, starting from only a few. Figure 22 -16 Molecular Biology of the Cell (© Garland Science 2008)
Morphogen gradient formation § Simple diffusion § Receptor-mediated trap/endocytosis § Excellular matrix-mediated reduction § Cytoneme-mediated transport
S. Brenner § § R. Horvitz J. Sulston An excellent model organism for genetic analysis Small number of cells with fixed cell lineage Self-fertilization in hermaphrodite (female with sperms) Genome sequenced, anatomy reconstructed by EM
Figure 22 -17 Molecular Biology of the Cell (© Garland Science 2008)
Figure 22 -18 Molecular Biology of the Cell (© Garland Science 2008)
Maternal effect genes regulate asymmetric cell division Asymmetric divisions : segregating P granules into the germline founder cell. DNA dye P granule antibody Sperm entry site: future posterior pole Figure 22 -19 Molecular Biology of the Cell (© Garland Science 2008)
Isolation of Maternal Effect Lethal Mutations • Egg laying defective animals were mutagenized, and their progeny were cloned onto single cultures. • F 1 animals with a maternal effect lethal mutation (m/+) produce viable eggs (+/+, m/+, and m/m) that hatch within and consume the parent. • m/m F 2 animals produce only inviable eggs and are therefore not consumed.
To identify maternal effect lethal mutations causing defective early cleavage patterns, 1. Mutant embryos with no catastrophic defects in mitosis or cytokinesis and that 2. Arrest at late stages of cellular proliferation 3. partition-defective: par-1, par-2, par-3, par-4
Abnormal distribution of P granule in par mutants
Asymmetric partitioning in worm embryos and vertebrate epithelia Anterior PAR 3, PAR 6, PKC 3 PAR 1, PAR 2 30
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The pattern of cell divisions in early C. elegans embryo. § Asymmetric P-granule distribution by Par (partitiondefective) proteins. § At the 16 -cell stage, there is just one cell that contains the P granules. § This one cell gives rise to the germ line. Figure 22 -20 Molecular Biology of the Cell (© Garland Science 2008)
Molecular mechanism of cell fate decision § Manipulation of embryo with laser (ablation or position change) § Genetic screen for specific mutants § Mom (more mesoderm) mutations: fails to generate EMS cells. No gut: One gene is wnt, the other is Fz. § Pop (posterior pharynx defective): extra gut cells. § One gene is tcf (downregulated by Wnt). Pop 1 mutants have more Wnt signaling: Both daughters of EMS becomes gut cells.
• Mom (Wnt & Fz): more mesoderm in mom • Pop 1 (more Wnt signaling, more gut cells in pop 1 -) Cell signaling pathways in a four-cell embryo. § P 2 cell sends an inductive signal (Notch) to Abp (ABa cell respond to the same signal, but it is out of contact with P 2. § Meanwhile, a Wnt signal from P 2 cell causes the EMS cell to orient its mitotic spindle and generate MS and E cell (gut founder) due to different exposure to Wnt. Figure 22 -21 Molecular Biology of the Cell (© Garland Science 2008)
Stage-specific pattern of cell divisions
Heterochronic Genes Control the Timing of Development Green: Lin 14+ Premature occurrence of the pattern of cell division and differentiation characteristic of a late larva reiterate the first larval stage patterns Figure 22 -22 Molecular Biology of the Cell (© Garland Science 2008)
Lin 4 antagonizes lin-14 post-transcriptionally. Lin-14 Lin-4
Hormonal control of developmental timing • Metamorphosis in insects and amphibians • Puberty in mammals (Mc. Cune Albright syndrome) ü GOF of lutenizing hormone receptor (LCGR) in precaucious puberty (higher c. AMP & testosterone) ü LOF: delayed puberty
Ced 9 Ced 3/Ced 4 1030 somatic cells 131 cells undergo PCD Apoptotic cell death in C. elegans. §Death depends on Ced 3 and Ced 4 genes in the absence of Ced 9 expression—all in the dying cell itself. §The subsequent engulfment and disposal of the remains depend on other genes in the neighboring cells. Figure 22 -23 Molecular Biology of the Cell (© Garland Science 2008)
Development of multicellular organisms Part II. Drosophila melanogaster
Drosophila and the molecular genetics of pattern formation: genesis of the body plan Figure 22 -24 Molecular Biology of the Cell (© Garland Science 2008)
Figure 22 -25 Molecular Biology of the Cell (© Garland Science 2008)
Segmentation of embryo Syncytial blastoderm Gastrulation, Segmentation, Head involution Figure 22 -26 Molecular Biology of the Cell (© Garland Science 2008)
Relationship between embryo and larval segments Figure 22 -27 Molecular Biology of the Cell (© Garland Science 2008)
Syncytium to cellularized blastoderm Surface view of cellular blastoderm : actin-chromosome stain Figure 22 -28 a Molecular Biology of the Cell (© Garland Science 2008)
Fate map of an embryo at the cellular blastoderm stage. During gastrulation, §the cells along the ventral midline invaginate to form mesoderm. §the cells fated to form the gut invaginate near each end of the embryo. Figure 22 -29 Molecular Biology of the Cell (© Garland Science 2008)
Genetic control of anterior-posterior patterning The domains of the anterior (bicoid), posterior (nanos), and terminal (torso) systems of egg-polarity genes Figure 22 -30 Molecular Biology of the Cell (© Garland Science 2008)
AP and DV patterning in the ovary Follicle cells provide AP and ventral signals. Figure 22 -31 Molecular Biology of the Cell (© Garland Science 2008)
Four egg-polarity gradient systems The receptors Toll and Torso are distributed all over the membrane; the coloring indicates where they become activated by extracellular ligands. Bicoid protein gradient Figure 22 -32 Molecular Biology of the Cell (© Garland Science 2008)
Ik. B NFk. B Nuclear localization of Dorsal and DV gradient
Morphogen gradients patterning DV axis of embryo The concentration gradient of Dorsal protein. Dorsally, it is cytoplasmic. Ventrally, it becomes nuclear via Toll signaling. High Dpp activity: most dorsal tissue Intermediate: dorsal ectoderm Low: neuogenic ectoderm Figure 22 -34 Molecular Biology of the Cell (© Garland Science 2008)
Origin of the mesoderm from cells expressing Twist (b. HLH)-expressing cells move into the interior of the embryo to form mesoderm. The nerve system moves to the ventral region. Dorsal: Dpp is induced Ventral: Sog (short gastrulation), Chordin homolog, Dpp antagonist Figure 22 -35 Molecular Biology of the Cell (© Garland Science 2008)
The vertebrate body plan A dorsoventral inversion of the insect body plan. Dorsal Ventral Drosophila Dpp Sog Figure 22 -36 Molecular Biology of the Cell (© Garland Science 2008) Vertebrates Chordin BMP 4
Anterior-posterior patterning Three classes of segmentation genes The shaded area in green on the normal larva (left) are deleted in the mutant or are replaced by mirror-image duplicates of the unaffected regions. Figure 22 -37 Molecular Biology of the Cell (© Garland Science 2008)
The regulatory hierarchy of egg-polarity Gap, Pair-rule, and Homeotic selector genes Figure 22 -38 Molecular Biology of the Cell (© Garland Science 2008)
Modular organization of the regulatory DNA of the Eve gene Analysis of various eve-lac. Z reporter expression : lac. Z m. RNA in situ-anti-Eve double stain Figure 22 -39 Molecular Biology of the Cell (© Garland Science 2008)
The formation of Ftz and Eve stripes. Ftz (brown) and Eve (gray) are both pairrule genes. Their expression patterns are at first blurred but rapidly resolve into sharply defined stripes. Figure 22 -40 Molecular Biology of the Cell (© Garland Science 2008)
Engrailed, a segment-polarity gene. The Engrailed pattern, once established, is preserved throughout the animal’s life. Figure 22 -41 Molecular Biology of the Cell (© Garland Science 2008)
Expression pattern compared to the chromosomal locations of the genes of the Hox complex. Figure 22 -44 Molecular Biology of the Cell (© Garland Science 2008)
Homeotic transformation Antennapedia Bithorax (haltere to wing)
Ubx: T 3 discs (haltere and 3 rd leg) > T 2 discs (wing and 2 nd leg) - Loss of Ubx in T 3: transformation of T 3 to T 2 (haltere to wing, 3 rd leg to 2 nd leg) Antp : selector for leg identity, repression of antennal fate - Ectopic Antp in the antenna: transform antennae to legs.
The Hox complexes of an insect and a mammal compared and related to body regions Figure 22 -46 Molecular Biology of the Cell (© Garland Science 2008)
Organogenesis & the patterning of appendages
Creation of mutant cells by induced somatic recombination Figure 22 -49 Molecular Biology of the Cell (© Garland Science 2008)
Ectopic overexpression of transgenes by UAS -GAL 4 systme Figure 22 -50 b Molecular Biology of the Cell (© Garland Science 2008)
(Top) Ectopic expression of fly Eyeless in the leg (Bottom) Ectopic expression of squid Eyeless Figure 22 -2 b Molecular Biology of the Cell (© Garland Science 2008)
Figure 22 -51 Molecular Biology of the Cell (© Garland Science 2008)
Wing pouch Gene expression domains in the wing imaginal disc, defining quadrants of the future wing. The wing blade itself derives from the wing pouch. It is divided into four quadrants by the expression of Apterous and Engrailed Figure 22 -53 Molecular Biology of the Cell (© Garland Science 2008)
(A) Compartments in the adult wing. (A) The shapes of marked clones reveal the existence of a compartment boundary. The border of each marked clone is straight where it abuts the boundary. (B) En expression in the posterior compartment Figure 22 -54 a Molecular Biology of the Cell (© Garland Science 2008) (B)
Four Familiar Signaling Pathways Combine to Pattern the Wing Disc: Wingless, Hedgehog, Dpp, and Notch Figure 22 -55 Molecular Biology of the Cell (© Garland Science 2008)
Similar Mechanisms Pattern the Limbs of Vertebrates (A) A wing bud of a chick embryo. At the distal margin of the limb bud a thickened ridge can just be seen—the apical ectodermal ridge. (B) Expression patterns of key signaling proteins and gene regulatory factors in the chick limb bud. Figure 22 -57 Molecular Biology of the Cell (© Garland Science 2008)
Figure 22 -58 Molecular Biology of the Cell (© Garland Science 2008)
Sensory mother cells in the wing imaginal disc. Figure 22 -59 Molecular Biology of the Cell (© Garland Science 2008)
Cell-cell interaction: Genesis of asymmetry through positive feedback. X inhibits X synthesis in adjacent cells Lateral inhibition Figure 22 -12 Molecular Biology of the Cell (© Garland Science 2008)
Lateral Inhibition Singles Out Sensory Mother Cells Within Proneural Clusters SOP Figure 22 -60 a Molecular Biology of the Cell (© Garland Science 2008)
Selection of SOP by lateral inhibition Reduced lateral inhibition - More sensory organs Figure 22 -60 b Molecular Biology of the Cell (© Garland Science 2008)
Basic helix-loop-helix family proteins in neurogenesis Atonal: R 8, chordotonal internal sensory Atonal family Amos: md neuron, olfactory sensilla Math 1: proprioceptive sensory (hearing) Math 5: RGCs in visual system Achaete: external sensory organ Achaete-Scute: redundant with Ac family Mash 1 Mash 2 Neuro. D family Neuro. D 2 Math 3 82
Genetic control of neural development: Proneural (Ac-Sc) & neurogenic genes (Notch-Delta) 83
Use of genetic model systems • Identify developmental genes • Functional analysis in vivo • Reveal functional conservation • New insights for studying higher animals Axial patterning, induction, fate determination, cell death , signaling pathways and more
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