ANAT3231 Cell Biology: Development Dr. Steve Palmer

Transcription

1 ANAT3231 Cell Biology: Development Dr. Steve Palmer Honours projects: 1. Characterizing genes involved in the neurocognitive/behavioural disorder Williams-Beuren syndrome 2. Identification of genes involved in the specification of muscle fibre types

2 Textbook/Online References Essential Cell Biology Chapter19 Tissues p Molecular Biology of the Cell (3rd ed) Chapter 19 Cellular Mechanisms of Development p Online References UNSW Embryology Molecular Biology of the Cell online Molecular Cell Biology online. 4th ed. Lodish etal

3 This lecture is about how the embryo makes use of cellular mechanisms (described during this term) to construct itself. You should understand and learn: The meaning of the terms cell lineage, embryonic patterning, morphogenesis, cell commitment, determination, differentiation. The utility of model organisms in research on developmental mechanisms The conceptual importance of somatic cell nuclear transfer (cloning) experiments Where embryonic stem cells come from What induced pluripotent stem (ips) cells are and what are the therapeutic benefits of making them The concept of lineage restriction The concept of how lineage restriction is controlled by the expression of DNA-binding transcription factors The concept of transcription factor patterning in time and space How transcription factor expression can be controlled by signaling pathways How the cytoskeleton of the cell can generate cell movements How apoptosis can be used as a morphogenetic mechanism.

4 Development involves many different cell biology mechanisms Including: Cell proliferation: careful control of cell division is needed to ensure that tissues achieve their correct size at the right time and in the right place. Lineage commitment: specialization of cells involves activation and repression of many structural genes e.g. blood cells globin heart cells actin and myosin Patterning: by restricted temporal and spatial expression of DNA-binding proteins e.g. the homeobox (Hox) genes. Induction and cell signaling: Short range by cell-cell contact e.g. delta/notch Long range by diffusible morphogen e.g. sonic hedgehog Cell migration and shape change cells (or parts of cells e.g. neurons) need to move through other tissues to reach the right location e.g. germ cells and limb myoblasts. Shape of tissues is defined by reorganization of the internal cytoskeleton, making and breaking cell-cell contacts and laying down of extracellular matrix components. Coordinated movement of cell layers can create structures (e.g. formation of the neural tube) and permit large-scale morphogenetic movements e.g. gastrulation and neurulation in the mouse. Morphogenesis by selective apoptosis shapes can be created by the formation of temporary structures that are later removed by coordinated apoptosis e.g. formation of digits

5 Features of Williams-Beuren Syndrome Physical Supravalvular aortic stenosis (SVAS) Craniofacial dysmorphology Growth retardation Hypotonia Infantile hypercalcaemia Hoarse voice Dental abnormalities Strabismus Behavioural Reduced social anxiety Leading to inappropriate approach behaviour Increased non-social anxiety phobias, strong emotional reactions, sleep disturbances Cognitive Reduced IQ (mean 57) Visuospatial construction deficit Delayed motor and language milestones

6 Williams-Beuren syndrome is caused by a hemizygous microdeletion of chromosome 7 Chr7 Chr7

7 Aim 1: Identify the gene that causes the genetic disease Aim 2: Determine the cellular mechanism that is affected OMIM # WILLIAMS-BEUREN SYNDROME; WBS Q. How can we work out how these genetic mutations lead to the observed developmental abnormalities?

8 Genes and mechanisms of development can be highly conserved throughout evolution Example 1: Mutation of the mouse Engrailed gene leads to a failure of normal cerebellum development in the mouse brain. If a Drosophila fruit fly gene with a similar DNA sequence is substituted, cerebellum development seems normal. Example 2: By mis-expression of the Drosophila Eyeless gene in a fly s leg it is possible to induce an extra eye (TOP). If a gene with similar sequence from a squid is used (Pax6) the same thing happens (BOTTOM). Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

9 Therefore we can use animal models to study the development function of genes Caenorhabditis elegans: nematode worm, external development in egg, transparent, fate of every cell (about 1000) mapped, short life-cycle, rapid development, very cheap and easy to keep, easy to manipulate gene expression (feed them DNA), genome sequenced to completion, very poor relevance to human development, Drosophila melanogaster: fruitfly, external development in egg, excellent for genetics, short life-cycle, rapid development, easy and cheap to keep, genome sequenced to completion, poor relevance to human development. Xenopus laevis: African clawed frog, external development in egg, easy and moderately inexpensive to keep, short life-cycle, rapid development, very poor for genetics, genome not sequenced due to tetraploidy, very good for grafting studies and fate mapping, better relevance to human development.

10 Animal models continued: Gallus gallus: Chicken, external development in egg, easy and cheap to produce (eggs), relatively rapid development, very poor for genetics, genome sequence incomplete, very good for grafting studies (chick/quail grafting) and fate mapping, good relevance to human development. Danio rerio: Zebrafish, external development in egg, easy and moderately inexpensive to keep, short life-cycle, rapid development, good for transient genetic manipulation, translucent embryo, genome sequenced to completion, good relevance to human development, good all-round model. Mus domesticus: Mouse, internal development in uterus (virtually impossible for grafting studies), very expensive to keep, 20 day gestation, 8 weeks before sexually mature, difficult and expensive genetic manipulation, genome sequenced to completion, excellent relevance to human development,

11 A glossary of developmental terms: Cell lineage a linear sequence of cell fate that traces progressive states of differentiation. Like the ancestry of the cell e.g. liver cells are derived from the endodermal cell lineage. Embryonic patterning the underlying mechanism by which a shapeless ball of cells is provided with the information required to develop into its appropriate anatomical form and structure. Cell commitment (specification) the process by which a cell becomes dedicated to becoming some other more mature cell type due to its position in the embryo or as a result of its cell lineage: may be reversible if exposed to a different environment e.g. grafted into another location. Cell determination the process by which a cell becomes irreversibly locked into a particular cell fate: precedes differentiation. However, the cell shows no outward signs of what they are destined to be. Differentiation - The process by which a less specialized cell undergoes a phenotypic transformation into a more specialized cell type: usually irreversible. Morphogenesis The overall process by which the embryo resolves itself into a mature shape

12 The nucleii of somatic cells contain the same chromosomal DNA content and have the potential to make a clone. Somatic cell nuclear transfer in frogs and sheep John Gurdon Dev Biol : Adult frogs derived from the nuclei of single somatic cells. Dolly the sheep Nucleus from a mammary cell derived from a 6 year old ewe Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

13 Cell division: The early embryology of the mouse: fertilized egg to blastocyst. Early cleavage stages 2,4,6,8,16 cells - all cells are totipotent Compaction Formation of the blastocoel cavity Differentiation of two cell types - outer trophectoderm and an inner cell mass Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

14 Embryonic stem cells Embryonic stem cells are derived from the inner cell mass. They are pluripotent i.e. they can contribute to all of the primary germ layers of the embryo (endoderm, mesoderm and ectoderm) and the germ cells. express a number of genes that maintain the pluripotent state including DNA-binding transcription factors encoded by the genes Oct4 and Sox2. Inactivation of one of these genes leads to a loss of pluripotency. Can be grown in culture indefinitely, will divide continuously without differentiating further, can be genetically modified by DNA transfection e.g. to make knockouts. Main utility as a research tool for genetic manipulation Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

15 Induced pluripotent stem cells (ips cells) ips cells Retroviral transduction of Oct4, Sox2, Klf4 leads to induced pluripotent stem cells (ips) 13.5dpc mouse embryo Differentiated mouse embryonic fibroblasts If human ips cells can be made from adult somatic cells e.g. from peripheral blood or fibroblasts, it would avoid the problems of: Graft cell rejection without using immunosuppresants Ethical problems of using human embryonic stem cells

16 Q: How do all of the different cell types arise? A: lineage restriction AB (hypodermis, neurons, muscle, others) MS (neurons, muscle, somatic gonad, others) E (intestine) C (hypodermis, neurons, muscle) D (muscle) P4 (germ line) Edward T. Kipreos Nature Reviews Molecular Cell Biology 6, (October 2005) The early cell division stages of Caenorhabditis elegans embryo and the lineage restriction of the cells

17 Lineage restriction Imagine a series of balls rolling down a set of hills and valleys, representing every cell in a developing organism But whatever the immediate operations of the genes turn out to be, they most certainly belong to the category of developmental processes and thus belong to the province of embryology. Waddington, C. H., 1956, Principles of Embryology

18 Gene regulation differential activation or repression in each cell type g=gonad m=mesonephros tc=testis cords o=ovary A small region of a microarray (DNA chip) slide containing 625 samples. The relative amount of green and red colour is accurately read by a laser scanning device.

19 Chromosomal sex determination in humans: an example of a developmental cascade that is controlled by the expression of a single DNA-binding transcription factor protein SRY SRY NO SRY Image from Developmental Biology 6 th Edition Scott Gilbert. Limited content available at NCBI Bookshelf

20 Some major transcription factor families and subfamilies critical for development Family Representative transcription factors Some functions Homeodomain: Hox Hoxa-1, Hoxb-2, etc. Axis formation POU Pit-1, Unc-86, Oct-2 Pituitary development; neural fate LIM Lim-1, Forkhead Head development Pax Pax1, 2, 3, etc. Neural specification; eye development Basic helix-loop-helix (bhlh) MyoD, achaete, daughterless Muscle and nerve specification; Drosophila sex determination Basic leucine zipper (bzip) C/EBP, AP1 Liver differentiation; fat cell specification Zinc finger: Standard WT1, Krüppel, Engrailed Kidney, gonad, and macrophage development; Drosophila segmentation Nuclear hormone receptors Glucocorticoid receptor, estrogen receptor, testosterone receptor, retinoic acid receptors Secondary sex determination; craniofacial development; limb development Sry-Sox Sry, SoxD, Sox2 Bend DNA; mammalian primary sex deter- mination; ectoderm differentiation

21 Gene regulation at the molecular level: transcription factor complexes bind to genes via recognition sequences in the DNA Transcription factor protein General TATA binding protein (TBP) Specific Hox bhlh Sox5 Tcf/lef Example DNA Recognition sequence TAATATAT TAAT CANNTG AACAAT AGATCAAAGG Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

22 Developmental transcription factor proteins are expressed in restricted spatial patterns e.g. the Hox proteins mrna in-situ hybridization Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

23 Time of expression of developmental transcription factor proteins can generate patterns e.g. Hox proteins and development of the limb Hoxd-9 and Hoxd-10 are expressed in the newly formed limb bud. Cells become committed to form all of the tissues surrounding the humerus. Nested expression of Hoxd genes such that Hoxd-9 through Hoxd-13 are expressed in the posterior of the limb bud, while only Hoxd-9 is expressed both anteriorly and posteriorly. Cells become committed to the tissue around the radius and ulna. Inversion of Hox gene expression. Hoxd-13 and Hoxa-13 are expressed anteriorly and posteriorly, while Hoxd-10 through Hoxd-12 and Hoxa-12 are expressed posteriorly. Cells become committed to metacarpal and digit formation. Image from Developmental Biology 6 th Edition Scott Gilbert. Limited content available at NCBI Bookshelf

24 What controls the spatial and temporal patterns of transcription factor expression? Other transcription factors: Regulatory gene networks Cell signaling: Cell-cell contact e.g. Delta/Notch Diffusible morphogens e.g. Sonic Hedgehog

25 Signaling by cell-cell contact lateral inhibition via delta/notch (A) The basic mechanism shown with just two cells that begin by expressing both delta and notch on the cell surface. Binding of the delta ligand to the notch receptor leads to cleavage of the notch protein which moves into the nucleus. A series of events leads to reduced expression of the delta gene and increased expression of the notch gene. Thus, competitive interactions become polarized. (B) Delta notch signaling in a multicellular context leads to patterning within a homogeneous layer of cells Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

26 The result of switching off lateral inhibition during the singling-out of sensory mother cells. The thorax of a Drosophila fruitfly containing a mutant patch of cells in which the gene Delta has been partially inactivated. The reduction of lateral inhibition has caused almost all the cells in the mutant patch to develop as neuronal sensory mother cells, producing a great excess of sensory bristles. Notch signaling Notch signaling P. Heitzler and P. Simpson (1991) The choice of cell fate in the epidermis of Drosophila. Cell 64: Epidermis Neuroblast (bristle)

27 Cell signaling via diffusible morphogens e.g. Sonic Hedgehog in the chick limb (A) Expression of the Sonic hedgehog gene in a 4-day chick embryo, shown by mrna in situ hybridization dorsal view. Expression is in the midline and at the posterior border of the wing buds. (B) Normal wing development from the wing bud. (C) A graft of tissue from the region that expresses sonic hedgehog, which is called the zone of polarizing activity (ZPA) into a second site on the anterior margin leads to development of a mirror image wing. Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

28 Production of the inducer from a point source generates a diffusion gradient across a field of cells. If sufficient receptors are activated in the receiving cells to generate a threshold intracellular signal then cell fate is switched. Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

29 The 3-dimensional organization of the vertebrate limb bud uses a number of spatial cues A wing bud of a chick embryo at 4 days of incubation. The scanning electron micrograph shows a dorsal view. At the distal margin of the limb bud a thickened ridge can just be seen The apical ectodermal ridge (AER). Expression patterns of key signaling proteins and DNA-binding transcription factors in the chick limb bud. Sonic hedgehog FGF4 FGF8 Wnt7a BMP2 diffusible morphogen diffusible morphogen diffusible morphogen diffusible morphogen diffusible morphogen Notch En1 Lmx1 cell-cell signaling DNA-binding transcription factor DNA-binding transcription factor Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

30 Importance of the cytoskeleton in cell migration and movement migration of individual cells through other cell populations e.g. neural crest cells and primordial germ cells Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

31 Cell migration and movement - migration of parts of cells through other cell populations e.g. growth cone of developing axons Images adapted from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

32 Cell migration and movement mass movements of cell populations relative to others to reorganize shape, e.g. formation of the neural tube Image from Developmental Biology 6 th Edition Scott Gilbert. Limited content available at NCBI Bookshelf

33 Cell migration and movement mass movements of cell populations relative to others to reorganize shape, e.g. gastrulation Gastrulation in a sea urchin Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

34 Video of gastrulation and neurulation in a frog embryo

35 Morphogenesis by apoptosis of temporary developmental structures e.g. tissue between the digits in the mouse paw Fluorescent green marker indicates cells undergoing apoptosis Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

36 Extracellular matrices and development Endochondrial ossification in long bones cartilage forms first (green) then osteoblasts and blood vessels invade. Intramembranous ossification in skull. Does not require a cartilaginous framework mesenchymal cells differentiate into osteoblasts within the membrane overlying the brain. The basal lamina and Sertoli cell (testis) differentiation (A) grown on plastic (B) grown on basal lamina extracellular matrix. Elastin in the aorta low power and high power image Images from Molecular Biology of the Cell (4 th Ed) Alberts et al. Online content available at

37 So what cell mechanisms could be affected during development in Williams-Beuren syndrome patients? Craniofacial abnormalities Signaling -Sonic hedgehog? Neural crest migration? Aortic stenosis lack of elastin Teeth odontoblasts neural crest derived migration? Cell division? Gene regulation? Gene expression? Patterning? Signaling? Cell movement and shape? Apoptosis? Extracellular matrices? Neurons Signaling, gene regulation, cell movement, apoptosis, gene expression? Brain shows widest diversity of gene expression compared with all other tissues.