09.8.26 1 CHAPTER 3 PRINCIPLES OF EXPERIMENTAL EMBRYOLOGY ZOO3603C Three major research programs in experimental embryology 1. How forces outside the embryo influence its development. 2. How forces within the embryo cause the differentiation of its cells. 3. How the calls order themselves into tissue and organ Sex Determination in Alligators100%100%SexReversal33°C30°CEstrogenTreatment(Estradiol; p,p'-DDD;Dicofol; trans-Nonachlor)Specification Morphogenesis Environment • Environmental sex determination • Adaptation of embryos and larvae to their environment Environmental Developmental Biology Environmental sex determination: Sexual determination in Bonellia viridis Chemical signal-dependent sex determination Female proboscis has the chemical to make a larva to male. In vitro analysis of Bonellia sex determination Larvae were incubated in (1) Control group: pure seawater (2) Experimental group: seawater + proboscis. Environmental sex determination: Sex determination in American alligator Sex Determination in Alligators100%100%SexReversal33°C30°CEstrogenTreatment(Estradiol; p,p'-DDD;Dicofol; trans-Nonachlor) No sex chromosomes  Temperature dependent sex determination (TSD) 33˚C produces male; 30˚C produces female  Chemical signal dependent sex determination Sex reverse can be induced by estrogens or environmental contaminants. 09.8.26 2 Adaptation: Two morphs of Araschnia levana, the European map butterfly Summer Morph Spring Morph Two morphs can be induced by changes in both day light and temperature. Adaptation: Two morphs of Nemoria arizonaria Spring Morph Summer Morph Spring morphs can be induced by feeding them oak leave. Adaptation: Effect of ultraviolet radiation on embryos of the sea urchin Strongylocentrotus droebachiensis • Mycosporine-like protein is a natural sunscreen. • Incubated in seawater without source of mycosporine-like protein. Adaptation: Hatching success rates in three amphibian species in the field 1. UV-B Blocked. 2. UV-B Transactivated 3. No protection Environmental Developmental Biology Developing organisms are adapted to the ecological niches in which they develop.  Environmental sex determination  Seasonal phenotypic changes in caterpillars and butterflies.  The ability of frog eggs exposed to sunlight to repair DNA damage • Autonomous specification • Conditional specification • Stem cells and commitment • Syncytial specification The Developmental Dynamics Of Cell Specification 09.8.26 3 Process of differentiation  Differentiation  Commitment  1st stage: Specification --- still reversible  2nd stage: Determination --- irreversible Autonomous specification Even in isolated culture, the trochoblast cells keep the collect development. Characteristic of most invertebrates. Autonomous specification in the early tunicate embryo Autonomousspecificationintunicateembryo Whole Embryo B4.1 cells Blastomere w/o B4.1 cells Acetylcholinesterase in the progeny of the muscle lineage blastomeres (B4.1) isolated from a tunicate embryo at the 8-cell stage Autonomous specification in tunicate embryo Microsurgery on tunicate eggs forces some of the yellow crescent cytoplasm of the muscle-forming B4.1 blastomeres to enter the b4.2 blastomere pair Autonomousspecification 09.8.26 4 Syncytial specification in the fruit fly Drosophila melanogaster Syncytial specification --- Characteristic of most insect class Bicoid protein is the head morphogen in Drosophila embryos (Part 1) Bicoid protein is the head morphogen in Drosophila embryos (Part 2) Syncytialspecification Conditional specification Characteristic of all vertebrates and few invertebrates. The remaining cells alter their fates so that the role of the missing cells are taken over. Regulative development Spontaneous four separation of cells in the nine-banded armadillo In the early developmental stages of many vertebrates, the separation of the embryonic cells into two parts can create twins. Regulative development 09.8.26 5 Weismann’s germ plasm theory of inheritance The major techniques of experimental embryology  Defect experiment  Destroy a portion of embryo  Isolation experiment  Remove a portion of embryo  Recombination experiment  Replace a portion of embryo by a different region of same embryo.  Transplantation experiment  Replace a portion of embryo by a portion of different embryo. Roux’s attempt to demonstrate mosaic development Defect experiment Q: When is the first cleavage division separated the future right and left? A: At the first cleavage the right-left were determined. Driesch’s demonstration of regulative development Isolation Experiment Q: Regulative development ? A: Yes! Driesch’s pressure-plate experiment for altering the distribution of nuclei (Part 1) Recombination experiment Q: Does the relative position of a blastomere matter? Driesch’s pressure-plate experiment for altering the distribution of nuclei (Part 2) The recombination embryo obtained normal larvae. A: No! The relative position of a blastomere within the whole will probably in a general way determine what shall come from. 09.8.26 6 Modes of cell type specification and their characteristics No embryo uses only one of three. Drosophila embryos use all three. The “French flag” analogy for the operation of a gradient of positional information Morphogen gradients revisited The “French flag” analogy for the operation of a gradient of positional information (Part 1) The “French flag” analogy for the operation of a gradient of positional information (Part 2) Original Transplant It differentiates according to its new position Nodal is a long-range morphogen in zebrafish embryos Nodal: morphogen for the anterior potion of head. inducer for goosecoid. Depending on the concentration of morphogen, cells were differentiate into their appropriate type of cells. Eight cells are maximum distance of Nodal signaling. 09.8.26 7 Negative controls confirm that Nodal is a morphogen Stem cells and commitment General view of stem cell Pluripotent stem cell: can generate any cells Committed stem cell: can give rise to smaller population. Progenitor cells: no longer stem cell The differentiation of stem cell Example: Blood cell formation By surrounding environment • Differential cell affinity • The