Cellular Mechanisms of Development Chapter 19

Process of development Process of systematic gene-directed changes throughout an organism’s life cycle 4 subprocesses: Cell division Differentiation Pattern formation Morphogenesis

Cell Division Very first process that must occur during embryogenesis After fertilization, the diploid zygote undergoes a period of rapid mitotic divisions In animals, controlled by cyclins and cyclin-dependent kinases (Cdks) Exert control over checkpoints in the cycle of mitosis

Cleavage In animal embryos, period of rapid cell division following fertilization Blastomeres – Enormous mass of the zygote is subdivided into a larger and larger number of smaller and smaller cells Not accompanied by any increase in the overall size of the embryo G1 and G2 phases of cell cycle short or eliminated

Caenorhabditis elegans One of the most completely described models of development Adult worm consists of 959 somatic cells Transparent, so cell division can be followed Researchers have mapped out the lineage of all cells derived from the fertilized egg Fate of each cell is the same in every C. elegans individual

Cell differentiation Human body contains 210 major types of differentiated cells Cell determination – molecular decision to become a particular type of cell Cells become determined prior to differentiation Standard test for determination is to move cell

Determination has a time course Depends on a series of intrinsic or extrinsic, or both events Early tissue will turn into something else but not after a certain time Determination often takes place in stages Cell first becomes partially committed Acquires positional labels that reflect its location in the embryo Tissue determined as leg but not what part of leg

Molecular basis of determination Cells initiate developmental changes by using transcription factors to change patterns of gene expression Once the initial “switch” is thrown, the cell is fully committed to its future developmental path Cells become committed via Differential inheritance of cytoplasmic determinants Cell–cell interactions

Differential inheritance of cytoplasmic determinants Tunicates are marine invertebrates Swimming tadpolelike larval stage Muscles that move the tail develop on either side of the notochord Colored pigment granules asymmetrically localize to tail muscle cell progenitors

Female parent provides egg with mRNA encoded by macho-1 gene Experimentally shifting colored pigment granules causes other cells to become muscle cells Female parent provides egg with mRNA encoded by macho-1 gene Gene product has been shown to be a transcription factor that can activate the expression of several muscle-specific genes

Induction Change in cell fate due to interaction with an adjacent cell Demonstrate the importance of cell–cell interactions in development by separating the cells of an early frog embryo and allowing them to develop independently Ectoderm develops from cells of the animal-pole Endoderm develops from cells of the vegetal-pole No mesoderm develops unless animal-pole cells and vegetal-pole cells are placed next to each other

Another example of inductive cell interactions is the formation of the notochord and mesenchyme in tunicate embryos Muscle, notochord, and mesenchyme all arise from mesodermal cells

Copyright © The McGraw-Hill Companies, Inc Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. FGF signaling Anterior Posterior 32-Cell Stage 64-Cell Stage b. c. Prospective mesodermal cells receive signals from the underlying endodermal precursor cells that lead to the formation of notochord and mesenchyme

Stem cells Cells that are capable of continued division, but can also give rise to differentiated cells Degree of determination Totipotent – cell that can give rise to any tissue in an organism (embryo and extraembryonic membranes) Pluripotent – give rise to all cells in the adult organism’s body Multipotent – give rise to limited number of cells Unipotent – give rise to only a single cell type

Embryonic stem cells (ES cells) Form of pluripotent stem cells Made from mammalian blastocysts ES cells isolated from inner cell mass and grown in culture In mice, have been shown to develop into any type of cell in the tissues of the adult Cannot develop into extraembryonic membranes 1998 – first human ES cells Great promise and controversy

Colony of undifferentiated human embryonic stem cells surrounded by fibroblasts (elongated cells) that serve as a “feeder layer”

Nuclear reprogramming Experiments carried out in the 1950s showed that single cells from fully differentiated tissue of an adult plant could develop into entire, mature plants Cells of an early cleavage stage mammalian embryo are also totipotent Natural twinning Producing multiples artificially for commercial agricultural lines of cattle

Early experiments showed nuclei could be transplanted between cells Cells do not appear to undergo any truly irreversible changes, such as loss of genes More differentiated the cell type, the less successful the nucleus in directing development when transplanted Nuclear reprogramming – nucleus from a differentiated cell undergoes epigenetic changes that must be reversed to allow the nucleus to direct development

Early amphibian work showed that adult nuclei have remarkable developmental potential, but cannot be reprogrammed to be totipotent Nuclear transfer in mammals did not result in reproducible production of cloned animals Did lead to discovery of imprinting

1984 – sheep was cloned using the nucleus from a cell of an early embryo 1996 – Dolly, the first clone generated from a fully differentiated animal cell Used somatic cell nuclear transfer (SCNT) Dolly matured into fertile adult Established beyond all dispute that determination in animals is reversible

Reproductive cloning Uses SCNT to create animal genetically identical to another Efficiency is quite low and other problems Only 3–5% of adult nuclei transferred to donor eggs result in live births Due to lack of genomic imprinting Normal mammalian development depends on precise genomic imprinting Organization of chromatin in adult and embryo very different

Much work has been put into trying to find ways to reprogram adult cells to become pluripotent cells without the use of embryos Different lines of inquiry showed that reprogramming of somatic nuclei was possible 2006 – genes for 4 different transcription factors introduced into fibroblast cells in culture Named induced pluripotent stem cells (iPS cells) Appear to be similar to ES cells in terms of developmental potential, as well as gene expression pattern

Pluripotent stem cells Germ cells Some adult stem cells Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Oocyte Somatic cells Nuclear Transfer Somatic cells Fusion Blastocyst ES cells Defined factors Culture Pluripotent stem cells Germ cells Some adult stem cells Somatic cells

Therapeutic cloning Produce patient-specific lines of embryonic stem cells Artificial embryo created using same process as Dolly (SCNT) Its cells are used as embryonic stem cells for transfer to injured tissue Body readily accepts these cells with no immune rejection May be obsolete with development of iPS cells

Pattern formation All multicellular organisms seem to use positional information to determine the basic pattern of body compartments and overall body Positional information then leads to intrinsic changes in gene activity Cells ultimately adopt a fate appropriate for their location

Polarity – acquisition of axial differences in developing structure Pattern formation can be considered the process of taking a radially symmetrical cell and imposing two perpendicular axes to define the basic body plan Anterior–posterior (A/P, head-to-tail) axis Dorsal–ventral (D/V, back-to-front) axis Polarity – acquisition of axial differences in developing structure

Fruit fly Drosophila melanogaster 2 bodies during development Larva – tubular eating machine Adult – flying sex machine Metamorphosis – passage from one body to next Embryogenesis – process of going from fertilized egg to larva

Development begins before fertilization with construction of egg Specialized nurse cells that help the egg grow move some of their own maternally encoded mRNAs into the maturing oocyte Action of maternal, rather than zygotic, genes determines the initial course of Drosophila development

Syncytial blastoderm – 12 rounds of nuclear division without cytokinesis Nuclei space themselves out Membranes grow forming cellular blastoderm Embryonic folding and primary tissue development soon follow Within a day of fertilization, embryogenesis creates a segmented, tubular body

Anterior/Posterior axis in Drosophila Based on opposing gradients of two different proteins produced by nurse cell mRNAs: Bicoid (anterior) and Nanos (posterior) Morphogens – proteins whose concentration gradients can specify different cell fates 2 other maternal messages, hunchback and caudal, are evenly distributed across the egg Bicoid inhibits translation of caudal mRNA Nanos inhibits translation of hunchback mRNA

Pattern formation in Drosophila along the A/P axis Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Thorax Head Abdomen Pattern formation in Drosophila along the A/P axis Determination of structures is accomplished by the sequential activation of three classes of segmentation genes Create body plan of three fused head segments, three thoracic segments, and eight abdominal segments

Within 3 hr after fertilization, a highly orchestrated cascade of segmentation gene activity transforms the broad gradients of the early embryo into a periodic, segmented structure with A/P and D/V polarity Bicoid Hunchback and other gap genes Pair-rule genes Segment polarity genes

39 A T H Establishing the Polarity of the Embryo Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Establishing the Polarity of the Embryo Setting the Stage for Segmentation Fertilization of the egg triggers the production of Bicoid protein from maternal RN A in the egg. The Bicoid protein diffuses through the egg, forming a gradient. This gradient determines the polarity of the embryo, with the head and thorax developing in the zone of high concentration (green fluorescent dye in antibodies that bind bicoid protein allows visualization of the gradient). About 21/2 hours after fertilization, Bicoid protein turns on a series of brief signals from so-called gap genes. The gap proteins act to divide the embryo into large blocks. In this photo, fluorescent dyes in antibodies that bind to the gap proteins Krüppel (orange) and Hunchback (green) make the blocks visible; the region of overlap is yellow. 500 µm 500 µm Laying Down the Fundamental Regions Forming the Segments About 0.5 hr later , the gap genes switch on the “pair-rule” genes, which are each expressed in seven stripes. This is shown for the pair-rule gene hairy . Some pair-rule genes are only required for even-numbered segments while others are only required for odd numbered segments. The final stage of segmentation occurs when a “segment- polarity” gene called engrailed divides each of the seven regions into halves, producing 14 narrow compartments. Each compartment corresponds to one segment of the future body . There are three head segments (H, bottom right), three thoracic segments (T, upper right), and eight abdominal segments (A, from top right to bottom left). A T H 39 500 µm 500 µm a: © Steve Paddock and Sean Carroll; b-d: © Jim Langeland, Steve Paddock and Sean Carroll

Drosophila mutants with particular segments that seem to have changed identity Mutations in homeotic genes lead to the appearance of perfectly normal body parts in inappropriate places

Homeotic gene complexes of Drosophila Bithorax complex Several homeotic genes map together to 3rd chromosome Control the development of body parts in the rear half of the thorax and all of the abdomen Order of genes corresponds to order of segments Antennapedia complex Governs the anterior end of the fly Order also corresponds to order of segments

a. b. 42 Drosophila HOM Chromosomes Mouse Hox Chromosomes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Drosophila HOM Chromosomes Mouse Hox Chromosomes Drosophila HOM genes Hox 1 Antennapedia complex Bithorax complex Hox 2 lab pb Dfd Scr Antp Ubx abd-A abd-B Hox 3 Hox 4 Head Thorax Abdomen Fruit fly embryo Mouse embryo Fruit fly Mouse a. b. 42

Homeodomain All of the homeotic gene complexes contained a conserved sequence of 180 nucleotides coding for a 60-amino-acid, DNA-binding domain Homeobox DNA that encodes homeodomain Hox gene Homeobox-containing gene that specifies the identity of a body part Function as transcription factors that bind DNA using their homeobox domain Ultimate targets of Hox gene function must be genes that control cell behaviors associated with organ morphogenesis Found in organisms as primitive as cnidarians

Evolutionary split between plant and animal cell lineages occurred about 1.6 BYA Before the appearance of multicellular organisms with defined body plans Multicellularity evolved independently in plants and animals Genetic control of pattern formation in plants is fundamentally different from that of animals Meristems add modules throughout lifetime

Plants have MADS-box genes Homeotic gene family Have homeobox-containing genes Do not possess complexes of Hox genes similar to the regional identity ones in animals Family of transcriptional regulators found in most eukaryotic organisms Plants have many; animals only a few In flowering plants, control transition from vegetative to flowering growth, root development, floral organ identity

Morphogenesis Generation of ordered form and structure Product of changes in cell structure and cell behavior Animals regulate The number, timing, and orientation of cell divisions Cell growth and expansion Changes in cell shape Cell migration (not used in plants) Cell death

Orientation of the mitotic spindle determines the plane of cell division in eukaryotic cells Plane determined by spindle placement Great diversity of cleavage patterns in animal embryos is determined by differences in spindle placement In animals, cell differentiation is often accompanied by profound changes in cell size and shape Neurons, muscle cells

Apoptosis Programmed cell death a part of development Human embryos begin with webbed fingers Necrosis – cells that die due to injury In C. elegans, due to 3 genes Mechanism of apoptosis appears to have been highly conserved during the course of animal evolution C. elegans genes similar to human genes

Cell migration important during many stages of animal development Involves adhesion and loss of adhesion Cell-to-cell interactions are often mediated through cadherins Also involves cell-to-substrate interaction Cell-to-substrate interactions often involve integrin-to-extracellular-matrix (ECM) interactions Extracellular matrix controls extent or route of migration

Cadherins Large gene family with several subfamilies All are transmembrane proteins that share a common motif Cadherin domain 110-amino-acid domain in the extracellular portion of the protein that mediates Ca2+-dependent binding between like cadherins (homophilic binding) Cells with the same cadherins adhere specifically to one another, while not adhering to other cells with different cadherins

Integrins Attached to actin filaments of the cytoskeleton and protrude out from the cell surface in pairs, like two hands “Hands” grasp a specific component of the matrix Actions Provides anchor Can initiate changes in cell Alter growth of cytoskeleton Activate gene expression Gastrulation depends on fibronectin–integrin interactions