BB1725 Lecture 9 Differentiation and development (2) PDF

BB175 Biology of the Cell Lecture 9: Cell Differentiation and development Dr Joseph Hetmanski [email protected] Today’s Lecture The lecture today will cover: Development of an embryo The fate of cells during development By the end of the lecture you should be able to e...

BB175 Biology of the Cell Lecture 9: Cell Differentiation and development Dr Joseph Hetmanski [email protected] Today’s Lecture The lecture today will cover: Development of an embryo The fate of cells during development By the end of the lecture you should be able to explain: Why certain model organisms are used The early stages of development How cell fate is determined Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 2 Somatic cells, gametes and zygotes Each somatic cell (everything except sperm/egg) nucleus contains essentially the same DNA in a defined number of chromosomes (Karyotype - varies between species) The number of chromosomes in each Gamete (sperm or egg) haploid cell is half the number in each somatic diploid cell At fertilisation, the egg and sperm fuse to give rise to the Zygote (diploid cell) Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 3 Human Karyotype Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 4 Multicellularity Single fertilised egg cell (zygote) becomes billions of cells in the adult A human body contains more than 210 different cell types (germ-line and soma) How do the differences in cell type arise? Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 5 Model Organisms To study the development of embryos in the lab we use model organism (because we can’t use human embryos freely) The model organism must be Produce a lot of eggs and embryos Easy to work with Transparent Large Develop outside the body Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 6 C. elegans - Caenorhabditis elegans Transparent nematode About 1 mm in length Lifespan, 2 to 3 weeks Generation time - 3 to 4 days Adult hermaphrodite - 959 somatic cells Male C. elegans - 1031 cells Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 7 Fruit Fly - Drosophila melanogaster Easy to work with in the lab Fast life cycle - 5 days Embryos and larva easy to work with Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 8 Zebrafish - Danio rerio Easy to work with in the lab Eggs and embryos easy to work with Rapid development Larval stage transparent Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 9 Xenopus laevis - African clawed frog Why use Xenopus? Easy to handle and keep in the lab Produce eggs all year round Eggs laid in water Development of the embryo is easy to observe Quick development Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 10 Xenopus laevis - African clawed frog Why use Xenopus? Easy to handle and keep in the lab Produce eggs all year round Eggs laid in water Development of the embryo is easy to observe Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 11 Asymmetry of oocyte Frog development - a model system Frog eggs are radially asymmetrical Pigmented top half - Animal pole White bottom half - Vegetal pole – less ‘active’ cytoplasm and more yolk Frogs have many planes of asymmetry: Left/Right Anterior/Posterior (i.e. top/bottom) Figure 21–13A The frog egg and its asymmetries Molecular Biology of the Cell - Seventh Edition - Dorsal/Ventral (i.e. back/front) Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 12 Asymmetry of oocyte The oocyte is asymmetric: Figure 21–13B The frog egg and its asymmetries Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 13 Generation of polarity The Dorsal/Ventral (i.e. back/front) axis is established at the point of fertilisation! Single sperm enters egg to cause fertilisation The point opposite the sperm Figure 21–13B The frog egg and its asymmetries Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. entry point will become the Dorsal (back) side of the frog Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 14 Dorsal ventral axis - Frogs Sperm can bind anywhere on the surface of the oocyte Requires binding to receptors Density of receptors is highest just above the equator Single sperm entry point - SEP Point opposite the SEP will Figure 21–13B The frog egg and its asymmetries form the dorsal (back) surface Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. of the organism Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 15 Dorsal ventral axis Sperm enters oocyte and migrates towards oocyte pronucleus (in the middle) Egg cortex - a pigmented layer just below the surface - rotates 30° Gives rise to the “grey crescent” Region around the grey Figure 21–13B The frog egg and its asymmetries Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. crescent is destined to become the dorsal surface (back) Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 16 Grey crescent Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 17 Development of the embryo Figure 21–3A The early stages of development, as exemplified by a frog Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. From single cell to blastula to gastrula? How is the process controlled? Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 18 From one cell to blastula From one cell to blastula: Consists of about 4000 basically ‘identical’ cells After this stage gene expression really starts Cell divide synchronously Takes around 7 hours to reach the final stage https://youtu.be/fUpc93T12bk Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 19 Development of the embryo Figure 21–3A The early stages of development, as exemplified by a frog Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. From single cell to blastula to gastrula? How is the process controlled? Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 20 Blastula to gastrula Gastrulation: when cells rearrange and differentiate to form tissue layers Ectoderm: the outermost layer of cells or tissue of an embryo, which include the epidermis and nerve tissue Mesoderm: the middle layer of cells or tissues of an embryo, which include Ecto – Outer cartilage, muscles, and bone Meso – Middle Endoderm: the innermost layer of cells or Endo - Inner tissue of an embryo, which include the lining of the gut and many internal organs Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 21 Role of mRNA localisation VegT mRNA localised: After fertilisation, VegT mRNA produce VegT protein VegT protein - transcription regulator - located in vegetal Figure 21–13B The frog egg and its asymmetries Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 pole W.W. Norton & Company, Inc. VegT protein activates genes that code for mesoderm and endoderm Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 22 Fate Why does that cell always become muscle? Because it is pre- programmed to become that cell type? Because of its position? Figure 21–27 Blastula fate map in a frog embryo Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 23 Becoming Different How can we determine fate? Inject particular blastula cell (blastomere) with a “vital” dye Look for stained cells in fully developed animal Figure 21–27 Blastula fate map in a frog embryo Molecular Biology of the Cell - Seventh Edition - Copyright © Create a “fate map” 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 24 Fate - experiment 1 Select blastomere Inject marker Allow animal to develop Identify location of ‘stained’ cell Same blastomere, same Figure 21–27 Blastula fate map in a frog embryo final tissue Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 25 Fate - experiment 2 Select same blastomere as in experiment 1 Inject marker Move the cell in the blastula Identify location of ‘stained’ cell in adult Figure 21–27 Blastula fate map in a frog embryo Molecular Biology of the Cell - Seventh Edition - Copyright © animal 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 26 Fate - experiment 2 - result ‘Stain’ is not in the same tissue as in experiment 1 ‘Stain’ now located in different tissue Tissue is different to experiment 1 Figure 21–27 Blastula fate map in a frog embryo Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 27 Fate - conclusion Final fate of blastomere is determined by position in the blastula! Figure 21–27 Blastula fate map in a frog embryo Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 28 Blastula Fate Map Position of the cells in the blastula can be mapped to their final fate in the adult frog Cell fate depends on position Figure 21–27 Blastula fate map in a frog embryo Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 29 Development robustness Cells are a ‘blank canvas’ until later in development, therefore it doesn’t matter if cells move to the wrong location early They’ll still become the ‘correct’ cell type later after being influenced by neighbours This is an example of self generated positive feedback Therefore early development is robust Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 Human vs xenopus development comparison Xenopus Human How good is the xenopus as a model for human development? What differences can you see? Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 Human vs xenopus development comparison Similarities Differences Start with zygote Much more complicated and Early doubling – ‘cleavage’ multilayered in humans Earlier breaking out of constrained Constrained area at first area First differentiation based on Initial differentiation into inner cells location and trophoblasts Endoderm, mesoderm, ectoderm Endoderm, mesoderm and formation crucial ectoderm form later, as embryo is growing in size Many more steps after endoderm, ectoderm, mesoderm formation Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 Developmental defects https://journals.biologists.com/dev/arti cle/122/12/4119/39143/Xenopus-VegT- RNA-is-localized-to-the-vegetal https://www.sciencedirect.com/ science/article/pii/S0092867400 815925#aep-abstract-id15 In xenopus, miss expression of vegT (which controls formation of mesoderm and endoderm) leads to: supressed head formation Mislocalised mesoderm position and growth abnormalities In humans, in general the earlier the development defect, the great the damage Embryonic lethality Abnormal limb growth Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 Fate Moved cell comes under the influence of new neighbouring cells and their protein secretion ‘Induction’ of tissue-determining transcription factors Figure 21–5 Regulatory DNA defines the gene expression patterns in development Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 34 Fate Potential to become different cell types Totipotent – can become all cell types E.g. Early embryonic stem cells Pluripotent – can become most cell types E.g. Later embryonic stem cells (inner cell mass) Multipotent – can become several cell types E.g. fibroblasts Figure 21–4 The lineage from blastomere to a terminally differentiated cell type Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 35 Fixed Fate Later in development, fate becomes fixed Potential becomes reduced Cell becomes “terminally differentiated” Figure 22–2 The defining characteristics of a stem cell Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 36 Differentiation All cells express common proteins Housekeeping - e.g. ATP synthase Differentiated cells express specific proteins: Epithelial cells - Keratin Red Blood Cells - Haemoglobin Skin fibroblasts - Collagen Figure 21–4 The lineage from blastomere to a terminally differentiated cell type Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 37 Methods to reprogram adult cells The process of differentiation can be reversed One differentiated cell type can be transformed into another via an induced pluripotent (iPS) cell Figure 7–39 A combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 38 Methods to reprogram adult cells Which The process of breed will differentiation can be be born? reversed As revealed by: “Dolly the sheep” Pluripotent stem cells Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 39 Weismann’s Central Dogma S S S S S G G G G G The Weismann Barrier - information is only inherited via the germ line (G) - however, the somatic cells (S) can impact the germ line through methylation of the germ cell DNA – ‘epigenetics’ Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 40 Weismann’s Central Dogma Protein Protein Protein Protein Protein DNA DNA DNA DNA DNA A Modern Interpretation of the Weissman’s Central Dogma - instead of germ line (G) and somatic cells (S) we can think of DNA and protein, where epigenetic regulation can ‘alter’ DNA (not the code) Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 41 Summary Oocytes have polarity - true from insects to mammals Development and cell fate is determined by position in the blastula This is key for self generated development robustness Cells terminally differentiate, but can be ‘re- programmed’ Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 42 Suggested Reading Explore the material for this week on Brightspace Molecular Biology of the Cell by Alberts et al. 7th Ed. - Chapters 7, 21 and 22 Brunel University London – BB1725 Biology of the Cell – Lecture 9 – 2024/25 43 Questions?