LECTURE COURSE „ Developmental Biology“ Introduction to Developmental Biology

1. LECTURE COURSE „Developmental Biology“ • Introduction to Developmental Biology • II. Basic Principles and Mechanisms of Developmental Biology • III. Stem Cells • IV. Growth Factors and Signal Transduction

2. Em E4-4.5 p.c. + Implantation Em Embryonic axis established ! Ab ICM Ab Primitive ectoderm Primitive endoderm Trophectoderm • Germ cells • gametes • Ectoderm • outer body epithelium • nervous system • skin • mammary glands • Mesoderm • notochord • dorzal mesoderm (myo,sklero,dermato-myotome) • intermedial mesoderm (meta,meso,pro-nefros) • lateral mesoderm (exraembryonal,somatic) • head mesenchyme • Endoderm • foregut • pharynx • lung • liver Polar trophectoderm Extraembryonic ectoderm Chorionic ectoderm Placental trophoblast Mural trophectoderm Ectoplacental cone Trophoblast giant cells Visceral endoderm Parietal endoderm Placenta Parietal yolk sac

3. Em E4-4.5 p.c. + Implantation Em Embryonic axis established !  6.5 p.c. Ab ICM Ab E6.5 - E7.5 Gastrulation- formation of ectoderm, mesoderm, and endoderm Primitive ectoderm Primitive endoderm Trophectoderm • Germ cells • gametes • Ectoderm • outer body epithelium • nervous system • skin • mammary glands • Mesoderm • notochord • dorzal mesoderm (myo,sklero,dermato-myotome) • intermedial mesoderm (meta,meso,pro-nefros) • lateral mesoderm (exraembryonal,somatic) • head mesenchyme • Endoderm • foregut • pharynx • lung • liver Polar trophectoderm Extraembryonic ectoderm Chorionic ectoderm Placental trophoblast Mural trophectoderm Ectoplacental cone Trophoblast giant cells E7.5 - neonatal period Organogenesis-development of tissues and organs Visceral endoderm Parietal endoderm Placenta Parietal yolk sac Embryonic and foetal development in mouse is 21 days !!!

4. Central concept of embryogenesis Based on the understanding the molecular mechanisms, that lead to the differentiation of huge amount of cell lineages that are required to form complex tissues and organs Totipotent cell Pluripotent cells Specialized cells First cleavage Preimplantation developmentGastrulationOrganogenesis -specification of cells/increasing number of cell types increasing variability and complexity of regulatory mechanisms

5. Gastrulation – development of ectoderm, mesoderm, and endoderm E7.5 /Ab E6.5 alantois /Em amnion E7.5 Gastrulation = migration along the embryonic axis that is followed by differentiation

6. Diversification/differentiation of cells during gastrulation is controlled by: Changes in gene expression that are followed by changes in the activity of related proteins (= signalling molecules, growth factors) Mechanism by which the cells are instructed to change gene expression and subsequently activities of signalling molecules during embryogenesis is called EMBRYONIC INDUCTION Sir John Gurdon Wellcome Trust/Cancer Research UK Gurdon Institute

7. Embryonic induction is thought to be initiated by small signalling molecules (morphogens) their expression/synthesis is genetically controlled so that these factors have appropriate biological activity at the correct time and place in the embryo Morphogenetic proteins are produced by well defined and localized groups of cells from the centers that are known as „ORGANIZERS“ Wnt family of proteins FGF family of proteins …and some other proteins (mostly transcription or growth factors) that control the development of more specialized cell lineages (e.g. Oct3/4, Hnf3, Hesx1, Lim1, brachyury, chordin, Sax1, Hoxb1, Flk1, Gata4, Mef2, Bmp, TGF1, Activin, Shh, Tbx6 …) Positional information!!! Morphogen gradient!!!

8. Morphogen gradient Positional information (example of long-range processes) Strategies for the generation of gradients of diffusible substancies (growth factors) mezoderm Limb bud Theory An opposing gradient + extra digits + extra digits Aided transport e.g. Shh and Wnt degradation Parameters affecting cellular responses Sonic hedgehog (Shh), BMP, Wnt, FGF A – normal development B – transplantation of mesodermal cells 1 C – transplantation of mesodermal cells 2

9. Cellular responses to inductive signals of growth factors Competence Community effect The response depends on the presence of a number of molecules that allow a cell to detect, receive, and interpret the signal (A) More cells together - more crosstalk - stronger response If any element is missing, e.g. receptors, transduction molecules or nuclear factors, the cell may lose competence to respond (B, C, D) Example:increasing expression of MyoD gene (development of muscle tissues)

10. Human congenital malformations that result from the aberrant growth factor signalling and thus defects in the formation of ectoderm, mesoderm, and/or endoderm during gastrulation (examples) Sirenomelia (mermaid-like phenotype)  Tsg, Bmp, Chordin Holoprosencephaly  Nodal Cyclopia/Holoprosencephaly  Wnt, Shh Neural tube defects  Blf (bloody fingers) (mostly defects in neuralVan Gogh-like 2 tube closure)Crsh (crash) Scrb1 (circletail) Defects in left-right asymmetry  Bmp, Nodal, Lefty1, Notch …and other defects that are lethal at the beginning of gastrulation and that are caused by abberant signalling of FGFs, Activins, or Cerberus

11. Establishment of cell lineages and development of tissues and organs Molecular principles and examples The specification of particular cell types and generation of tissues and organs requires the accumulation of „molecular information“ in correct spatio-temporal manner Expression pattern of various genes within the mouse embryo (E 12 – 12.5) The development of a specific cell type does not simply involve the expression and activation of genes associated with commitment to a particular lineage, but often also involves the suppression of genes associated with differentiation fates not taken !!! CELL FATE RESULTS FROM INTERPLAY BETWEEN THE ACTIVATION AND SUPPRESSION !!!

12. EXAMPLE: MYOGENESIS I. Spatial organization and influences of different signalling molecules Primitive ectodermmesodermsomite (myotome)skeletal muscles A,B A B Somite Some molecules (BMP a Shh) have dual effects – while promoting the development of a fate in a specific cell population, they suppress other fates Many of signalling molecules can diffuse over a long range (Shh) and resulting gradient might explain the effect that they promote two different fates at different distances The local concentration of specific antagonists (noggin) serves to modulate signals of other molecules (BMP) in space and time Neural tube C C As the development of the cells within the somite proceeds, the signalling becomes more and more complicated (+ FGFs, TGFs, Wnts)

13. EXAMPLE: MYOGENESIS II. Signalling networks acts on the expression of myogenic regulatory factors Myf5 MyoD Primitive ectodermmesodermsomite (myotome)skeletal muscles Ectoderm ? B) The signalling events depicted in (A) result in a complex landscape of signalling molecules over the somite that leads to specification of sclerotome (pink), epaxial myotome (blue), and hypaxial myotome (green). A) Effects of the different signalling molecules on the expression of Myf5 and MyoD in the early somite development

14. EXAMPLE: MYOGENESIS III. Myogenic regulatory factors (TF) progressively specify skeletal muscles Expression and activation of myogenic factors during development – genetic hierarchies that specify cells to become muscles Search for myogenic factors - experimental evidence A-heterokaryon myoblast + non-muscle cell type = muscle cell B-transformation of the fibroblast cell with cDNA from myoblast Margaret Buckingham Pasteur Institute Myoblasts Myotubes Myofibres

15. EXAMPLE: NEUROGENESIS I. DEFAULT MODEL OF NEURAL INDUCTION NEURAL INDUCTION – neural plate is specified from ectoderm Claudio Stern lab University College London chordin noggin follistatin BMP epidermis ectoderm Neural differentiation YES or NO Neural plate Ectodermal cells give rise to neural plate (neural cells) if they receive no signals at all !!! BMP (Bone Morphogenetic Protein) activity directs ectodermal cells to become epidermis. Locally, the activity of BMPs is inhibited by its antagonists (chordin, noggin, follistatin)

16. EXAMPLE: NEUROGENESIS II. Two models (2008) of neural induction - Xenopus (A) and chicken (B) Wnt+ The key regulatory role of BMPs, FGFs a Wnts • FGF signalling cooperates with BMP inhibition to induce a neural (blue) fate by inhibiting Smad1 phosphorylation, • repressing transcription of BMP gene, and inducing expression of the BMP antagonists Chordin (Chd) and Noggin (Nog) • at low levels, FGF seems to induce a neural fate directly. High BMP activity induces epidermis (yellow), while high FGF • signalling cooperating with Nodal-related factors (XNRs) induces mesoderm (red). (B) At the blastocyst stage, medial epiblast cells (prospective neural cells) express FGFs but not Wnts. FGF signalling activates two transduction pathways - repression of BMPs and promotion of neural fate. Lateral epiblast cells (prospective epidermal cells) express both FGFs and Wnts. High Wnt levels block the response of epiblast cells to FGFs, BMPs are expressed and promote epidermal fate and repress neural fate.

17. CONCLUSIONS: Embryonic induction/gastrulation is initiated by the key signalling molecules (morphogens) from specific clusters of cells (organizers). Distribution of morphogens is regulated by a simple mechanisms but the location and timing needs to be precisely controlled The emerging view of organogenesis (e.g. myogenesis or neurogenesis) is of a cascade of sequential events and of cooperation between different signalling pathways, which together allow cells to make not one, but several developmental decisions

18. Basic terminology in this lecture: • Totipotency ability to differentiate into virtually all cell types including • gametes and trophoblast cells • Pluripotency- ability to differentiate into many different cell types • (e.g. cells of inner cell mass or primitive ectodermal cells) • Primitive streak- migrating cells of epiblast (ICM), primitive streak stage = developmental • stage shortly before and during gastrulation process • FGFs- growth factors; fibroblast growth factor (s); family of signalling molecules • BMPs- growth factors; bone morphogenic factor(s)/bone morphogenetic factor(s); • family of signalling molecules • Wnts- growth factors, family of signalling molecules described originally • in Drosophila Melanogaster as Wingless • heterokaryon- cell that results from the fusion of two different nucleated cell types

19. This presentation will be available at: https://is.muni.cz www.med.muni.cz/biologie/ Education materials Medical Biology - lecture course (spring 2008)