1. BioSci 108 lecture 23 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved Bio 108 - 3/6/2000 Morphogenetic Movements (contd) Contact information –Bruce Blumberg –Office 4216 BioSci II office hours W 3-4 F 12-1 (or by appointment in exceptional cases) –phone 824-8573 –blumberg@uci.edu (preferred contact mode) You are responsible for what I lecture about and what is covered in the assigned reading in Alberts, et al. –Generally speaking, I will stick close to the book as did the other lecturers Today –morphogenetic movements neurulation neural crest migration –cell determination

2. BioSci 108 lecture 23 (Blumberg) page 2 © copyright Bruce Blumberg 2000. All rights reserved Neurulation Last time we finished with gastrulation The next developmental step is when the gastrula ectoderm is induced to form neural tissue by the underlying mesoderm and notochord (Figure 21-10) –neural plate thickens and rolls up into a tube –neural tube pinches off from the rest of the cell sheet –the inducing mesoderm can lead to the formation of an ectopic neural tube if transplanted to a new embryo, much like the organizer transplant –process is driven by changes in cell shape (21-11) cells become columnar apical actin bundles contract and cause curling of neural plate subsequently, dorsal cells of the neural tube pinch off and migrate out through the mesoderm –these neural crest cells will contribute to numerous structures and tissues peripheral nervous system sympathetic ganglia Schwann cells pigment cells –head neural crest will differentiate into cartilage, bone and connective tissue these typically derive from mesoderm in other parts of the body

3. BioSci 108 lecture 23 (Blumberg) page 3 © copyright Bruce Blumberg 2000. All rights reserved Cell adhesion Changes in cell adhesion molecules are important for morphogenetic movements –classic experiments showed that cells can recognize others of the same type (figure 21-14) experiment: –dissociate gastrula cells –allow to reassociate observation: –cells sort out according to tissue type –outer epidermis –central neural tube –mesoderm in between inference: –cells can identify each other –basis of identification is cell surface molecules called cadherins (21-15) Ca-dependent glycoproteins that are differentially expressed in the early embryo cadherins control the formation and dissolution of cell sheets AND provide an anchor for cytoskeleton within the cells recognition of cadherin types and combinations mediates interactions between cells and their neighbors.

4. BioSci 108 lecture 23 (Blumberg) page 4 © copyright Bruce Blumberg 2000. All rights reserved Cells and the extracellular matrix In addition to adhering to each other, cells need to interact with the extracellular matrix –mediated through another class of cell surface proteins called integrins –integrins serve as transmembrane linkers between the ECM and cytoskeleton –How to test the role of this interaction? experiment: –block cells from binding to fibronectin with a synthetic peptide that saturates the binding site observation: –cells do not migrate and appear only loosely adherent conclusion –cell interaction with integrins is very important for migration –Mutations in integrins lead to embryonic patterning defects Drosophila mutation lethal (1) myospheroid leads to failure of muscle attachment –embryos burst at about 24 hours after fertilization when the first muscular movements take place –mutation is in an integrin subunit found in muscles

5. BioSci 108 lecture 23 (Blumberg) page 5 © copyright Bruce Blumberg 2000. All rights reserved Invasion and migration Many types of cells migrate in the early embryo from their point of origin –muscle cells –neural crest –blood cells –primordial germ cells –neurons how to follow migrations? –Mark cells at the beginning of their journey and follow them nontoxic dye heritable genetic marker –quail/chicken chimeras Neural crest follows distinct migratory pathways (21-17) –much of this work was done by Marianne Bronner- Fraser who was previously at UCI –migrating cells extend cellular processes that “test” for interactions with neighboring cells depending on the types of interactions found, the cells go one direction or another cell surface receptors again mediate this process –some types attract or promote adhesion –others inhibit movement and repel connections –Corey Goodman and neural cell migration

6. BioSci 108 lecture 23 (Blumberg) page 6 © copyright Bruce Blumberg 2000. All rights reserved Cell Diversification in the early embryo All of the information needed to form any type of organism is contained in a single cell –the central goal of developmental biology is to understand how what are the molecules? how do they interact? what are the physical processes? Question - does the genome remain intact and totipotent during development or do cells become specialized by genomic modifications? –Answer seems obvious but an important early developmental model was the worm Ascaris. Significant parts of the genome are lost during development and differentiation –experiment: (Figure 21-20) transplant the nucleus from a differentiated cell from adult frog to egg lacking nucleus what is the developmental potential of the resulting embryo? –Observation: normal tadpoles developed that had characteristics of the transplanted nucleus (albino) –conclusion: genome remains intact during development cells differ because they express different genes

7. BioSci 108 lecture 23 (Blumberg) page 7 © copyright Bruce Blumberg 2000. All rights reserved Cell diversification (contd) –Resulting animals are clones, exact copies of the donor animal –Such experiments were previously difficult and not so reproducible –today, the technology has advanced sufficiently that mammals can be readily cloned for $250,000 you can have your pet cloned! it is only a matter of time before someone does this with humans Cytoplasmic determinants –in most plants and animals, the egg is chemically asymmetrical certain components are concentrated in specific regions these localized cytoplasmic determinants vary in importance between species –development was previously characterized in two broad ways in mosaic development, if a blastomere is removed, the structures derived from it fail to form e.g. sea urchin –Inference is that determinants are important in regulative development the embryo develops normally despite blastomere removal –determinants are not important –no animals are completely either way!

8. BioSci 108 lecture 23 (Blumberg) page 8 © copyright Bruce Blumberg 2000. All rights reserved Embryonic induction Induction - the switching of cells from one pathway into another by the influence of an adjacent group of cells –one of the most important processes during early development of virtually all organisms –experiments that demonstrated induction are some of the most famous experiments in biology Organizer transplant (Hans Spemann) –until recently, the only Nobel Prize awarded for Developmental Biology –experiment: transplant the dorsal lip of the blastopore to the ventral side of a host embryo –observation: a complete secondary axis was formed from both host and graft tissue –inference: the transplant was able to organize the host tissue into a secondary axis factor(s) from the graft were able to do this –over the years, many such experiments were done until it became clear that many, many types of molecules could induce secondary axes in salamanders put field into disrepute for many years turns out that salamander is promiscuous for neural induction

9. BioSci 108 lecture 23 (Blumberg) page 9 © copyright Bruce Blumberg 2000. All rights reserved Embryonic induction (contd) Xenopus saved the day –unlike salamander mesoderm and neural tissue could only be induced in Xenopus by a few factors –Pieter Niewukoop performed a classic induction experiment (figure 21-21) isolated animal pole and vegetal pole cells if cultured in isolation, the animal pole cells formed epidermis and the vegetal pole cells stayed as they were. But when put together, mesoderm was formed! This simple mesoderm induction assay was the basis for nearly all of the advances in molecular embryology in recent years –Tiedemanns and Grunz spent many years purifying factors from embryos that could induce mesoderm these turned out to be growth factors in the FGF and TGF-beta families Later, a number of laboratories began to study the molecular nature of the organizer –Viktor Hamburger - Hans Spemann and the Organizer, The Heritage of Experimental Embryology –Prompted by this book, professor Cho and I constructed the first cDNA library from the dorsal lip and identified many factors that were important for its properties (Science 253, 194-196.

10. BioSci 108 lecture 23 (Blumberg) page 10 © copyright Bruce Blumberg 2000. All rights reserved Embryonic induction (contd) Sequential induction is responsible for much embryonic patterning (Fig 21-22) –a series of inductive interactions can generate many kinds of cells, starting from only a few Inductive abilities of cells differs –although all vegetal cells can induce mesoderm, only cells from the dorsal side can induce dorsal mesoderm (organizer) (Fig 21-23) –There are at least three different signals ( and probably many more) responsible for mesoderm induction in the early Xenopus embryo –fertilization -> cortical rotation –cortical rotation -> asymmetrical distribution of dorsal determining factors (several possibilities) dorsal and ventral endoderm –mesoderm and organizer induced by signal from vegetal cells (signals 1 and 2) –organizer dorsalizes adjacent mesoderm (signal 3) mesoderm induction involves a variety of signaling molecules –TGF-beta family (activin, Vg-1, BMP2,4) –Wnt family –FGF family (FGF4) –inhibitors of above chordin, noggin, cerberus

11. BioSci 108 lecture 23 (Blumberg) page 11 © copyright Bruce Blumberg 2000. All rights reserved Actions of inducing molecules One model for how the organizer is induced –cortical rotation leads to activation of Vg-1 on the dorsal side of the embryo –Vg-1 protein in vegetal cells induces the organizer –attractive model but not completely correct many so-called dorsalizing signals are, in fact, inhibitors of ventral mesoderm and ectoderm induction Location (Fig 21-26) –can be intracellular or extracellular –molecules can act at both short and long range nature –secreted molecules –bound to cell surface secreted signals are called morphogens –morphogens are diffusible substances that pattern the embryo –in its strictest definition, a morphogen patterns tissues depending on its concentration model originated with Alan Turing a British mathemetician who almost single-handedly finished off the Germans in WWII –he solved the famous “enigma” code that allowed the Allies to decipher German military transmissions

12. BioSci 108 lecture 23 (Blumberg) page 12 © copyright Bruce Blumberg 2000. All rights reserved Morphogens and gradients –Gradient model was popularized by Child, Huxley and De Beer essential feature is that activity gradients led to the formation of specialized regions of the embryo –Louis Wolpert provided many important theoretical treatments, observed that positional signaling occurs over small distances, -.1-2 mm –Francis Crick provided legitimacy to the model with a quantitative treatment of the activity of diffusible substances how do morphogens work (Fig 21-26) –essential feature is that morphogens diffuse from a source and are degraded or captured by a sink if there was no loss or destruction of the morphogen there would ultimately be no difference in the concentration –cells sense their position in a morphogen gradient and respond according to the concentration of morphogen they encounter ectodermal cells exposed to little or no activin become epidermis low levels of activin induce muscle higher levels induce notochord