Experimental Embryology PDF

EXPERIMENTAL EMBRYOLOGY Introduction  Descriptive and evolutionary embryology both had roots in anatomy  However, by end of 19th century, physiology made inroads into embryological research  The questions of "what?" became questions of "how?"  It was felt that embryology…  s...

EXPERIMENTAL EMBRYOLOGY Introduction  Descriptive and evolutionary embryology both had roots in anatomy  However, by end of 19th century, physiology made inroads into embryological research  The questions of "what?" became questions of "how?"  It was felt that embryology…  should not merely study anatomy and evolution, but  should answer the question, "How does an egg become an adult?" Introduction  This new program was called Entwicklungsmechanik, often translated as "developmental mechanics" or “causal embryology”  Its goals were to find the molecules and processes that caused the visible changes in embryos  Embryologists focused on studying mechanisms of organ formation (morphogenesis) and differentiation  Experimentation were done to supplement the observations in the study of embryos Introduction  Experimental embryology focusses on three of the major research programs  How forces outside the embryo influence its development? Environmental Developmental Biology  How forces within the embryo cause the differentiation of its cells? Developmental Mechanics of Cell Specification  How the cells order themselves into tissues and organs? Morphogenesis and Cell Adhesion Environmental Developmental Biology  The developing embryo is not isolated from its environment  In several species, environmental cues are a fundamental part of the organism's life cycle  Removing or altering these environmental parameters can alter development Environmental Sex Determination  Sex determination in an echiuroid worm: Bonellia  Baltzer (1914) showed that sex of Bonellia viridis depended on where the Bonellia larva settled  Female Bonellia worm Marine, rock-dwelling, about 10 cm long The proboscis can extend over a meter in length  Male Bonellia worm Only 1– 3 mm long Resides within the uterus of the female, fertilizing her eggs Environmental Sex Determination  Sex determination in an echiuroid worm: Bonellia  Baltzer showed that If a Bonellia larva settles on the seafloor, it becomes a female Instead, if a larva land on a female's proboscis it becomes male Proboscis apparently emits chemical signals that attract the larva Larva enters female's mouth, migrates into her uterus, and differentiates into a male  Baltzer (1914) and Leutert (1974) duplicated this phenomenon in laboratory Incubated larvae in either absence or presence of adult females Environmental Sex Determination  Sex determination in a vertebrate: Alligator  Sex of alligators, crocodiles, and many other reptiles depends not on chromosomes, but on temperature  Ferguson and Joanen (1982) Studied sex determination of Mississippi alligator both in lab and field Sex is determined by temperature of egg during 2nd and 3rd weeks of incubation Eggs incubated at  30C produce female alligators Eggs incubated  34°C produce males alligators Adaptation of Embryos and Larvae to their Environments  Norms of reaction  August Weismann (1875) noted that… Butterflies that hatched during different seasons were colored differently This season-dependent coloration could be mimicked by incubating larvae at different temperatures  Phenotypic variations caused by environmental differences are often called morphs Adaptation of Embryos and Larvae to their Environments  Norms of reaction  E.g., European map butterfly, Araschnia levana Has two seasonal phenotypes The spring morph is bright orange with black spots The summer morph is mostly black with a white band Change from spring to summer morph is controlled by changes in both day length and temperature during larval period When researchers experimentally mimic spring conditions, summer caterpillars can give rise to "spring" butterflies Adaptation of Embryos and Larvae to their Environments  Norms of reaction  E.g., moth Nemoria arizonaria It has a fairly typical insect life cycle Eggs hatch in spring, and caterpillars feed on young oak flowers (catkins) These larvae metamorphose in late spring, mate in summer, and produce another brood of caterpillars on oak trees These caterpillars eat oak leaves, metamorphose, and mate Their eggs overwinter to start the cycle again next spring Adaptation of Embryos and Larvae to their Environments  Norms of reaction  E.g., moth Nemoria arizonaria Caterpillars hatching in spring look nothing like their progeny hatching in summer In spring, they eat oak catkins (flowers) and resemble oak catkin In summer, after all catkins are gone, they feed on oak twigs/leaves and resemble a twig Adaptation of Embryos and Larvae to their Environments  Norms of reaction  E.g., moth Nemoria arizonaria What controls this difference? Greene (1989) conducted reciprocal feeding experiments Converted spring caterpillars into summer morphs by feeding them oak leaves But reciprocal experiment did not turn summer morphs into catkin like caterpillars Conclusion:  Catkin form is "default state“  Something induces the twig like morphology  That something is probably a tannin that is concentrated in oak leaves as they mature Adaptation of Embryos and Larvae to their Environments  Norms of reaction  Thus, what gets inherited is not a deterministic genotype  It is a genotype that encodes a potential range of phenotypes  The environment can select the phenotype that is adaptive for that season or habitat  This continuous range of phenotypes expressed by a single genotype across a range of environmental conditions is called the “reaction norm” The Developmental Mechanics of Cell Specification  The development of specialized cell types is called differentiation  The changes associated with differentiation are preceded by a process involving commitment of the cell to a certain fate  At this point:  The cell does not appear phenotypically different from its uncommitted state  However, its developmental fate has become restricted The Developmental Mechanics of Cell Specification  The process of commitment can be divided into two stages:  The first stage is a labile phase called “specification” The fate of a cell or a tissue is said to be specified It is capable of differentiating autonomously when placed in a neutral environment such as petri dish or test tube (The environment is neutral with respect to the developmental pathway) At this stage, the commitment is still capable of being reversed The Developmental Mechanics of Cell Specification  The process of commitment can be divided into two stages:  The second stage of commitment is “determination” The fate of a cell or tissue is said to be determined It is capable of differentiating autonomously even when placed into another region of the embryo It is able to differentiate according to its original fate even under these different circumstances The commitment is irreversible The Developmental Mechanics of Cell Specification  Three basic modes of commitment  Autonomous specification Specification by differential acquisition of certain cytoplasmic molecules present in the egg  Conditional specification Specification by interactions between cells. Relative positions are important.  Syncytial specification Specification of body regions by interactions between cytoplasmic regions within a syncytial (multinucleated) cell The Developmental Mechanics of Cell Specification  Autonomous specification  A particular blastomere removed from an embryo early in development will produce same cells it would have made in the embryo  The embryo from which the cell is taken will lack the cells that would have been produced by the missing blastomere The Developmental Mechanics of Cell Specification  Autonomous specification  Autonomous specification gives rise to a mosaic pattern of development  Morphogenetic determinants Are placed in different regions of egg cytoplasm Are apportioned to different cells as the embryo divides Specify the cell type  E.g., molluscs, annelids, and tunicates The Developmental Mechanics of Cell Specification  Autonomous specification  Laurent Chabry (1887) Experimented on tunicate embryos Tried to produce malformations by isolating specific blastomeres from cleaving embryo He discovered that each blastomere was responsible for producing a particular set of larval tissues In the absence of particular blastomeres, larva lacked only structures normally formed by those cells The Developmental Mechanics of Cell Specification  Autonomous specification  Whittaker (1973) Stained blastomeres in the 8-cell tunicate embryo for presence of enzyme acetylcholinesterase found only in muscle tissue When the two cells specific for producing tail muscle tissue (B4.1) were removed, they produced muscle tissue that stained positively for acetylcholinesterase When he transferred some of the yellow crescent cytoplasm of B4.1 blastomere into ectoderm-forming b4.2, it generated muscle cells as well as its normal ectodermal progeny The Developmental Mechanics of Cell Specification  Conditional specification  Each cell originally has ability to become many different cell types  Its interactions with other cells restricts fate of one or both of participants  Thus, fate of a cell depends upon conditions in which the cell finds itself (conditional specification)  If a blastomere is removed from early embryo using conditional specification... Remaining embryonic cells alter their fates to take over roles of missing cells The Developmental Mechanics of Cell Specification  Conditional specification  This ability of embryonic cells to change their fates to compensate for missing parts is called regulation  Isolated blastomere can also give rise to a wide variety of cell types It sometimes generates cell types that the cell would normally not have made if it were part of embryo The Developmental Mechanics of Cell Specification  Conditional specification  Research leading to discovery of conditional specification began with testing of hypothesis claiming that there was no such thing  August Weismann (1883) proposed first testable model of cell specification  The Germ Plasm Theory Chromosomes carried inherited potentials of the new organism Not all determinants on chromosomes entered every cell of embryo The Developmental Mechanics of Cell Specification  Conditional specification  The Germ Plasm Theory Chromosomes divided such that different chromosomal determinants entered different cells Certain cells retained "blood-forming" determinants while others retained "muscle-forming" determinants Only cells destined to become gametes (germ cells) retained all determinants The Developmental Mechanics of Cell Specification  Conditional specification  Testing of this hypothesis pioneered three of the four major techniques involved in experimental embryology: The defect experiment A portion of embryo is destroyed and development of impaired embryo is observed The isolation experiment A portion of embryo is removed and development of partial embryo and isolated part are observed The Developmental Mechanics of Cell Specification  Conditional specification  Testing of this hypothesis pioneered three of the four major techniques involved in experimental embryology: The recombination experiment Development of embryo after replacing an original part with a part from a different region of embryo is observed The transplantation experiment One portion of embryo is replaced by a portion from a different embryo The Developmental Mechanics of Cell Specification  Conditional specification  Wilhelm Roux (1888) Took 2- and 4-cell frog embryos and destroyed some cells of each embryo with a hot needle He obtained half-blastulae These developed into half neurulae having a complete right or left side, with one neural fold, one ear pit, and so on The Developmental Mechanics of Cell Specification  Conditional specification  Wilhelm Roux (1888) His results were just as Weismann’s Germ Plasm Theory had predicted He therefore concluded that Frog embryo was a mosaic of self-differentiating parts It was likely that each cell received a specific set of determinants and differentiated accordingly The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1892) Performed isolation experiments He separated sea urchin blastomeres from each other by vigorous shaking Observations Each blastomere from a 2-cell embryo developed into a complete larva When blastomeres from 4- and 8-cell embryos were separated, some of them produced entire larvae The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1892) The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1892) The results were drastically different from predictions of Weismann or Roux Rather than self-differentiating into its future embryonic part, each isolated blastomere regulated its development so as to produce a complete organism Thus, these experiments provided first experimentally observable instance of regulative development The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1893) He performed recombination experiment to confirm regulative development in sea urchin embryo In sea urchin eggs First two cleavage planes are meridional, passing through both the animal and vegetal poles Third division is equatorial, dividing the embryo into four upper and four lower cells The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1893) He changed the direction of third cleavage by gently compressing early embryos between two glass plates This caused the third division to be meridional like preceding two After he released pressure, the fourth division was equatorial The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1893) This procedure reshuffled the nuclei A nucleus that normally would be in the region destined to form endoderm was now in the presumptive ectoderm region Some nuclei that would normally have produced dorsal structures were now in ventral cells He obtained normal larvae from these embryos The Developmental Mechanics of Cell Specification  Conditional specification  Hans Driesch (1893) If segregation of nuclear determinants had occurred (as proposed by Weismann and Roux), the resulting embryo should have been strangely disordered He concluded that The relative position of a blastomere within the whole embryo will determine what shall come from it The fate of a nucleus depended solely on its location in the embryo The Developmental Mechanics of Cell Specification  Conditional specification  J. F. McClendon (1910) Highlighted difference between isolation and defect experiments and importance of interactions among blastomeres He showed that isolated frog blastomeres behave just like separated sea urchin cells The mosaic-like development of first two frog blastomeres in Roux's study was an artifact of the defect experiment Something in or on the dead blastomere still informed the live cells that it existed The Developmental Mechanics of Cell Specification  Conditional specification  The influence of neighboring cells Jon Henry et al (1989) Pairs of cells isolated from animal cap of a 16-cell sea urchin embryo, can give rise to both ectodermal and mesodermal components However, their capacity to form mesoderm is severely restricted if they are aggregated with other animal cap pairs Thus, presence of neighbor cells, even of same kind, restricts potencies of both partners The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients Cell fates can also be specified by specific amounts of soluble molecules secreted at a distance from the target cells Such a soluble molecule is called a morphogen A morphogen may specify more than one cell type by forming a concentration gradient The concept of morphogen gradients had been used to model another phenomenon of regulative development: Regeneration The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients: Regeneration Allman (1864) When hydras and planarian flatworms were cut in half, the head half would regenerate a tail from the wound site, while the tail half would regenerate a head He hypothesized that this phenomenon indicated a polarity in the organization of the hydra The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients: Regeneration Thomas Hunt Morgan (1905, 1906) Polarity indicated an important principle in development Pointed out that: If both head and tail were cut off a flatworm  A head would regenerate from former anterior end  A tail would regenerate from former posterior end Never the reverse If medial segment were sufficiently small, the regenerating portions would be abnormal The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients: Regeneration Thomas Hunt Morgan (1905, 1906) Postulated that A gradient of anterior-producing materials concentrated in head region The middle segment would be told what to regenerate at both ends by the concentration gradient of these materials If the piece were too small, the gradient would not be sensed within the segment It is possible that there are actually two gradients in flatworm  One to instruct formation of head and one to instruct the production of tail The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients Makoto Asashima et al (1994) In the frog Xenopus, animal cap of embryo normally becomes ectoderm It responds differently to different concentrations of activin produced in the vegetal hemisphere It can be induced to form mesoderm if transplanted into other regions within the embryo The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients Makoto Asashima et al (1994) With no exposure, animal cap blastomeres form an epidermis-like mass of cells If exposed to small amounts of activin, they form ventral mesodermal tissue blood and connective tissue With progressively higher concentrations of activin, they develop into other types of mesodermal cells: muscles, notochord cells, and heart cells The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients John Gurdon et al (1994,1995) Animal cap cells respond to activin by changing expression of particular genes They placed activin-releasing beads or control beads into "sandwiches" of Xenopus animal cap cells The Developmental Mechanics of Cell Specification  Conditional specification  Morphogen gradients John Gurdon et al (1994,1995) Little or no activin: No expression of genes associated with mesodermal tissues. Cap cells differentiated into ectoderm Higher concentrations: Genes such as Brachyury turned on  Cells become mesoderm Still higher concentrations: Expression of genes such as goosecoid, associated with the most dorsal mesodermal structure, the notochord The Developmental Mechanics of Cell Specification  Conditional specification  Morphogenetic fields A group of cells whose position and fate are specified with respect to same set of boundaries General fate of a morphogenetic field is determined A particular field of cells will give rise to its particular organ (forelimb, eye, heart, etc.) even when transplanted to a different part of embryo However, individual cells within the field are not committed, can regulate their fates to make up for missing cells in the field The Developmental Mechanics of Cell Specification  Conditional specification  Morphogenetic fields One of the first morphogenetic fields identified was the limb field Mesodermal cells that give rise to a vertebrate limb can be identified by… Removing certain groups of cells and observing that no limb develops in their absence Transplanting these cells and observing that they form a limb in this new place Marking groups of cells with markers and observing that their descendants partake in limb development The Developmental Mechanics of Cell Specification  Conditional specification  Morphogenetic fields The morphogenetic field is referred to as a "field of organization" or a "cellular ecosystem“ There are webs of interactions among cells in different regions of a morphogenetic field Molecular connections among various cells of such fields are being studied E.g., vertebrates: limb, eye, and heart fields E.g., insects: imaginal discs that form eyes, antennae, legs, wings, and balancers The Developmental Mechanics of Cell Specification  Syncytial specification  Interactions occur not between cells, but between parts of one cell  In early embryos of several insects, cell division is not complete  Rather, nuclei divide within the egg cytoplasm creating many nuclei in the large egg cell  A cytoplasm that contains many nuclei is called a syncytium  The egg cytoplasm, however, is not uniform: Anterior of egg cytoplasm being different from posterior The Developmental Mechanics of Cell Specification  Syncytial specification  In Drosophila egg… The anterior-most portion contains mRNA encoding protein called Bicoid The posterior-most portion contains mRNA encoding protein called Nanos After fertilization, these mRNAs are translated into respective proteins The Developmental Mechanics of Cell Specification  Syncytial specification  In Drosophila egg… The concentration of Bicoid protein is highest in the anterior and declines toward the posterior The concentration of Nanos protein is highest in the posterior and declines as it diffuses anteriorly The Developmental Mechanics of Cell Specification  Syncytial specification  In Drosophila The long axis of egg is spanned by two opposing gradients They form a coordinate system based on their ratios Each region of embryo will be distinguished by a different ratio of the two proteins As the nuclei divide, they enter a different regions of egg cytoplasm The Developmental Mechanics of Cell Specification  Syncytial specification  In Drosophila The nuclei will be instructed by these ratios based on their position along anterior-posterior axis High Bicoid and little Nanos: Activate genes necessary for producing head Slightly less Bicoid with small amount of Nanos: Activate genes that generate thorax Little/no Bicoid and plenty of Nanos: Activate genes for abdominal structures The Developmental Mechanics of Cell Specification  No embryo uses only one of the autonomous, conditional, or syncytial mechanisms to specify its cells  Autonomous specification is seen even in a "regulative embryo“, e.g., sea urchin  Nervous system and some musculature of "autonomously developing" tunicate come from regulative interactions between its cells  Drosophila use all three modes of specification to commit their cells to particular fates Morphogenesis and Cell Adhesion  A body is more than a collection of randomly distributed cell types  Development does not involve differentiation of cells only  It also involves organization of cells into multicellular arrangements such as tissues and organs  A tissue/organ has an intricate and precise arrangement of many types of cells  How can matter organize itself so as to create a complex structure such as a limb or an eye? Morphogenesis and Cell Adhesion  There are five major questions on morphogenesis: 1. How are tissues formed from populations of cells? 2. How are organs constructed from tissues? 3. How do organs form in particular locations, and how do migrating cells reach their destinations? 4. How do organs and their cells grow, and how is their growth coordinated throughout development? 5. How do organs achieve polarity? Morphogenesis and Cell Adhesion  How are tissues formed from populations of cells?  E.g., How do neural retina cells stick to other neural retina cells and not become integrated into the pigmented retina or iris cells next to them? How are various cell types within retina (three distinct layers of photoreceptors, bipolar neurons, and ganglion cells) arranged so that retina is functional? Morphogenesis and Cell Adhesion  How are organs constructed from tissues?  E.g., Retina of eye forms at a precise distance behind cornea and lens Retina would be useless if it developed behind a bone or in middle of kidney Moreover, neurons from retina must enter brain to innervate regions of brain cortex that analyze visual information All these connections must be precisely ordered Morphogenesis and Cell Adhesion  How do organs form in particular locations, and how do migrating cells reach their destinations?  E.g., Eyes develop only in the head and nowhere else What stops an eye from forming in some other area of body? Some cells for instance, precursors of our pigment cells, germ cells, and blood cells must travel long distances to reach their final destinations How are cells instructed to travel along certain routes in our embryonic bodies? How are they told to stop once they reach their appropriate destinations? Morphogenesis and Cell Adhesion  How do organs and their cells grow, and how is their growth coordinated throughout development?  E.g., Cells of all tissues in eye must grow in a coordinated fashion if one is to see Some cells, including most neurons, do not divide after birth In contrast, in intestine, new cells are regenerated each day If intestine generated more cells than it sloughed off, it could produce tumors If it produced fewer cells than it sloughed off, it would soon become nonfunctional What controls rate of mitosis? Morphogenesis and Cell Adhesion  How do organs achieve polarity?  E.g., In fingers, there is organized collection of tissues: bone, cartilage, muscle, fat, dermis, epidermis, blood, and neurons Forearm has same collection of tissues, but arranged very differently. Similarly, in different parts of arm How is it that same cell types can be arranged in different ways in different parts of same structure? Morphogenesis and Cell Adhesion  All these questions concern aspects of cell behavior  There are two major types of cell arrangements in the embryo  Epithelial cells: Tightly connected to one another in sheets or tubes  Mesenchymal cells: Unconnected to one another and operate as independent units  Morphogenesis occurs via a limited set of variations in cellular processes within these two types of cell arrangements Morphogenesis and Cell Adhesion  The variations in cellular processes include: 1. Direction and number of cell divisions 2. Cell shape changes 3. Cell movement 4. Cell growth 5. Cell death; and 6. Changes in composition of cell membrane or secreted products Morphogenesis and Cell Adhesion  Differential cell affinity  Properties of cell surface play a major role in morphogenesis  Each type of cell has a different set of proteins in its surfaces  Some of these differences are responsible for forming structure of tissues and organs during development Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Took amphibian embryos that just had neural tube formation Prepared single-cell suspensions from each of the three germ layers Dissociated them into single cells by placing in alkaline solutions Combined two or more single-cell suspensions in various ways Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 1: Cells adhered to one another, forming aggregates Re-aggregated cells became spatially segregated Instead of the two cell types remaining mixed, each cell type sorted into its own region When epidermal and mesodermal cells were brought together to form a mixed aggregate  Epidermal cells moved to periphery of the aggregate  Mesodermal cells moved to inside of the aggregate  In no case recombined cells remained randomly mixed  In most cases, one tissue type completely enveloped the other Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 1: Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 2: Final positions of re-aggregated cells reflected their embryonic positions When epidermal cells were combined with mesodermal cells… Mesoderm migrated centrally w.r.t. epidermis, adhering to inner epidermal surface Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 2: When mesodermal cells were combined with endodermal cells… Mesoderm migrated centrally w.r.t. gut or endoderm Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 2: However, when the three germ layers were mixed together… Endoderm separated from ectoderm and mesoderm Then, it was enveloped by ectoderm and mesoderm Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 2: When epidermal cells were combined with neural plate cells… The presumptive epidermal cells migrated to periphery as before Neural plate cells migrated inward, forming a structure similar to neural tube Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 2: When axial mesoderm (notochord) cells were added to presumptive epidermal and presumptive neural cells, it formed… An external epidermal layer A centrally located neural tissue, and A layer of mesodermal tissue between them Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 2: Final configuration Somehow, cells were able to sort into proper embryonic positions Ectoderm was on the periphery Endoderm was internal Mesoderm lied in region between them Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Interpretation: The findings demonstrated selective affinity Inner surface of ectoderm has a positive affinity for mesodermal cells and a negative affinity for endoderm Mesoderm has positive affinities for both ectodermal and endodermal cells Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Boucaut (1974) also observed selective affinities Injected individual cells from specific germ layers into body cavity of amphibian gastrulae These cells migrated back to their appropriate germ layer Endodermal cells were found in host endoderm Ectodermal cells were found only in host ectoderm Thus, selective affinity is important for imparting positional information to embryonic cells Morphogenesis and Cell Adhesion  Differential cell affinity  Townes and Holtfreter (1955) Observation 3: Selective affinities change during development Embryonic cells do not retain a single stable relationship with other cell types For development to occur, cells must interact differently with other cell populations at specific times Such changes in cell affinity are extremely important in the processes of morphogenesis Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Cells do not sort randomly  Instead, they actively move to create tissue organization  What forces direct cell movement during morphogenesis?  Malcolm Steinberg (1964) Proposed “differential adhesion hypothesis” Explained patterns of cell sorting based on thermodynamic principles Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Malcolm Steinberg (1964) He mixed cells derived from trypsinized embryonic tissues Showed that: Certain cell types always migrated centrally when combined with some cell types But they migrated peripherally when combined with other cells Studied interactions between pigmented retina cells and neural retina cells by mixing them Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Malcolm Steinberg (1964) Observation: A. Initially, they formed aggregates of randomly arranged cells B. After several hours, the pigmented retina cells were no longer on the periphery of the aggregates C. After 2 days, two distinct layers were seen Pigmented retina cells lying internal to the neural retina cells Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Malcolm Steinberg (1970) Conclusion: Such interactions form a hierarchy If final position of cell type A is internal to cell type B If final position of cell type B is internal to cell type C Then final position of cell type A will always be internal to cell type C Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Malcolm Steinberg (1970) Conclusion: E.g., Pigmented retina cells migrate internally to neural retina cells Heart cells migrate internally to pigmented retina cells Therefore, heart cells migrate internally to neural retina cells Hypothesized that: Cells interact so as to form an aggregate with smallest interfacial free energy In other words, cells rearrange themselves into most thermodynamically stable pattern Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Malcolm Steinberg (1970) E.g., If cell types A and B have different strengths of adhesion If strength of A-A connections > strength of A-B or B-B connections  Sorting will occur with A cells becoming central If strength of A-A connections ≤ strength of A-B connections  Aggregate will remain as a random mix of cells If strength of A-A connections is >>> strength of A-B connections (Cells A and B have no adhesivity)  A cells and B cells will form separate aggregates Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Malcolm Steinberg (1970) According to this hypothesis The early embryo can be viewed as existing in an equilibrium state It stays in equilibrium until some change in gene activity changes cell surface molecules The movements that occur as a result tries to restore the cells to a new equilibrium configuration Morphogenesis and Cell Adhesion  The thermodynamic model of cell interactions  Foty et al (1996) Demonstrated that for sorting to occur, all that is needed is the cell types to differ in strengths of adhesion Cell types with greater surface cohesion sorted within those cells that had less surface tension Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  Studies have shown that boundaries between tissues can be created by Different cell types having different types of cell adhesion molecules, and Different cell types having different amounts of cell adhesion molecules  Several classes of molecules mediate cell adhesion  Major cell adhesion molecules are the cadherins Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  Cadherins Calcium-dependent adhesion molecules Critical for establishing and maintaining intercellular connections Crucial for spatial segregation of cell types, and Necessary for the organization of animal form Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  Cadherins interact with other cadherins on adjacent cells  They are anchored into cell by a complex of proteins called catenins  The cadherin-catenin complex forms classical adherens junctions that connect epithelial cells together  Moreover, since catenins bind to actin cytoskeleton of cell, they integrate epithelial cells together into a mechanical unit Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  In vertebrate embryos, several major cadherin classes have been identified: E-cadherin (epithelial cadherin) Expressed on all early mammalian embryonic cells, even at the 1-cell stage Later, this molecule is restricted to epithelial tissues of embryos and adults P-cadherin (placental cadherin) Expressed primarily on trophoblast cells (placental cells of embryo) and uterine wall epithelium P-cadherin facilitates connection (implantation) of embryo to uterus Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  In vertebrate embryos, several major cadherin classes have been identified: N-cadherin (neural cadherin) Prominent in neural plate, neural tube, and mesodermal tissues Essential for neural tube formation and neural crest cell migration In mesoderm, aids in cell-cell adhesion during somite formation EP-cadherin (Ectodermal Placode Cadherin) or C-cadherin (Classical Cadherin) Expressed primarily in embryonic tissues, especially blastomeres of Xenopus blastula Mediates adhesion during early cleavage, gastrulation, and tissue sorting Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  In vertebrate embryos, several major cadherin classes have been identified: Other types of cadherins R-Cadherin (Retinal Cadherin) VE-Cadherin (Vascular Endothelial Cadherin) K-Cadherin (Kidney Cadherin) Desmosomal Cadherins Protocadherins (non-classical cadherins) Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  Cadherins join cells together by binding to same type of cadherin on another cell Cells with E-cadherin will stick best to other cells with E-cadherin, and Cells with E-cadherin will sort out from cells containing N-cadherin Similarly, cells expressing N-cadherin readily sort out from N-cadherin-negative cells  This pattern is called homophilic binding Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  Cells sorting Can occur according to amount and types of cadherins on their cell surfaces Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  These adhesion patterns have important consequences in embryo  In gastrula of frog Xenopus Neural tube expresses N-cadherin, while epidermis expresses E-cadherin Normally, these two tissues separate from each other such that neural tube is inside the body and epidermis covers the body Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  If epidermis is experimentally manipulated to remove its E-cadherin Epidermal epithelium cannot hold together  If epidermis is made to express N-cadherin, or neural cells are made to lose it Neural tube will not separate from epidermis Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  The amount of cadherin can also mediate formation of embryonic structures  Differences in degree of cell adhesion is critical in development of embryo  E.g., in Drosophila ovary The developing egg, or oocyte, is at the most posterior side of egg chamber/follicle The oocyte's nurse cells are found more anteriorly This pattern reflects distribution of E-cadherin in these cells Oocyte and posterior follicle cells express E-cadherin at far higher levels than other cells Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  E.g., in Drosophila ovary When E-cadherin was experimentally removed from the oocyte and nurse cells (or from the follicle cells) The position of the oocyte became random Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  During development, cadherins work with other adhesion systems  In mammals, when embryo passes through uterus, for development to continue, embryo must adhere to uterus and embed itself in uterine wall  That is why the first differentiation event in mammalian development distinguishes trophoblast cells from inner cell mass Morphogenesis and Cell Adhesion  Cadherins and cell adhesion  Trophoblast cells have several adhesion molecules to adhere to uterine wall Both E-cadherins and P-cadherins recognize similar cadherins on uterine cells They have receptors (integrins) for collagen and heparan sulfate glycoproteins of uterine wall They also have a modified glycosyltransferase enzyme on their membrane that binds to specific carbohydrate residues on uterine glycoproteins