SEMBRYOLOGY Its Scope, History, and Special Fields

Historical Background

Galen learned about structure of relatively advanced fetuses minute dimensions resisted serious analysis development of microscope allowed study of early stages

de Graaf described ovarian follicles

Hamm and Leeuwenhoek first saw human sperm

Spermists vs Ovists spermists: sperm contained the new individual in miniature and was nourished in ovum ovists: ovum contained a minute body, stimulated to grow by seminal fluid Bonnet: discovered parthogenetically developed insect eggs (supported ovists)

Spallanzani and Wolf Laid to rest spermist/ovist view (preformation) Spallanzani: demonstrated that both male and female sex products are necessary for initiation of development Wolf: Epigenesis, embryonic development occurs through progressive remodeling and growth

von Baer von Baers law: the more general basic features of any animal group appear earlier in development than do the special features that are peculiar to different members of the group Germ layer theory: demonstrated existence of germ layers in embryos Significance could not be grasped until cellular basis of animal structure was known

Schleiden and Schwann Formulation of cell theory

Embryology Zygote has dual origin from two gametes: spermatozoa and ovum Fertilization is starting point of life history of individual (ontogeny) Period starting with fertilization until metamorphosis, hatching, or birth

Weismann Germ cell theory Made distinction between soma and germ-cell line Germ-cell line was all-important for perpetuation of species; soma was primarily vehicle for protecting and perpetuating germ plasm

Special Fields in Embryology

Descriptive Embryology Basic structural pattern of the embryonic body Serial sections, 3D wax plate reconstructions

Comparative Embryology Provided insight for concept that ontogeny recapitulates phylogeny Recognition of different modes of development Adoption of model organisms

Experimental Embryology Understand causative factors in development by posing hypotheses and testing them by manipulation of embryos Roux Experiment of 2 cell embryo Destroyed one blastomere Each cell is capable of giving rise to complete individual Provided proof of untenability of the preformationist doctrine

Chemical Embryology Provided description about chemical and physiological events in embryo Interaction between components and how basic body pattern is laid down

Teratology Concerned with study of malformations Identify and eliminate genetic and environmental factors that cause congenital defects

Reproductive Biology Related to problems of conception and contraception Emphasis on gametogenesis, endocrinology, transport of gametes, fertilization, embryonic development

Developmental Biology Embryonic development + postnatal development and processes Focus on processes and concepts, rather than specific morphological structures Plant and animal systems included

Embryology in Contemporary Society

Test tube baby In vitro fertilization and embryo transfer

The Cell and its Environment

Intracellular Synthesis and its Regulation Regulatory mechanisms restrict or permit synthesis of specific proteins and other macromolecules DNA (transcription) mRNA with introns and exons (mRNA processing) definitive mRNA(1, formation of structural proteins/enzymes) ribosome linkage (2, secretory proteins) mRNA-rER complex Golgi complex membrane vesicles May be mediated by receptor molecules located at cell surface activated by binding of a ligand, causing stimulation of signal transduction.

Cell Surface Junctional complexes Desmosomes bind epithelial cells focal points for attachment of fibrillar intracellular proteins Gap junctions Mediates communication and exchange of small molecules Tight junctions On surface of many epithelia Bind adjacent cells together, forming impermeable barrier Prevent mingling of membrane proteins on either side of junction Cell adhesion Experiment: disaggregated and reaggregation of sponges Ca++ mediated adhesion Glycoproteins E-cadherin (epithelium) N-cadherin (nerves, mesoderm) P-cadherin (placenta) Heterophilic binding between complementary saccharides Occurs during mammalian fertilization, when head of spermatozoa encounters zona pellucida

Extracellular Matrix Cells embedded on extracellular matrix Collagen (glycoprotein with glycine) Basic unit is tropocollagen (I, II, III, IV, V, X) Attachment glycoproteins involved in attaching cells to other components of extracellular matrix Fibronectin (I, III, V) Chondronectin (II, X) Laminin (IV) Glycosaminoglycans Example: hyaluronic acid in raising fertilization membrane of the egg

Fundamental Processes and Concepts in Development

Cell Division and the Cell Cycle Postmitotic cells further division does not occur Maturation-promoting factor (MPF) Cdc2 Present throughout cell cycle Activation by cyclin and dephosphorylation Activation of MPF exerts mitotic effects on cells Cyclin Produced during G1 Broken down after mitosis

Gene Activation Derepression of heterochromatin or repression of euchromatin Derepression of general genes from zygote to blastula Derepression of tissue specific genes from gastrula to organogenesis

Restriction and Determination Restriction: the reduction of developmental options permitted to a cell At gastrulation, one stage of restriction has occurred (endo, meso, ectoderm) Part of ectoderm thickens and undergoes Neurulation Determination restriction has proceeded to a point where a group of cells becomes committed to single developmental fate final step in process of restriction Inductions (tissue interactions) precede determination (and restriction)

Induction Form of embryonic signal calling Effect of one embryonic tissue on another, so that the developmental course of responding tissue is changed from what it would have been in absence of inductor First major inductive event: induction of mesoderm in cleavage Primary induction: induction of nervous system during and shortly after gastrulation Secondary induction: nervous system induces other structures Permissive induction: inductive signal required to bring about development of structure Instructive induction: responding tissue has options of forming more than one type of tissue, depending on inductive stimulus

Differentiation Restriction and determination signify progressive limitation of developmental capacities Differentiation: actual morphological or functional expression of a particular cell or group of cells; the process where cell is specializedMorphogenesis Processes that mold external and internal configuration of embryo Pattern formation: laying down of morphological blueprint Morphogenesis: actual realization of plans Homeotic genes: determine the regional characteristics of each segment (14 segments)

Intercellular Communication Intercellular communication takes place in localized gap junctions

Cell Movements Individual cells commonly migrate by means of amoeboid movements Unique: in avian embryos, primordial germ cells move from wall of yolk sac into the bloodstream and are carried via blood to the gonads Amoeboid movement examples: Ectoderm: migration of cells away from neural crest Mesoderm: spreading out of mesodermal cells Endoderm: migration of primary germ cells from yolk sac to gonads in mammals Movement as sheet seen in epithelial cells

Cell Death (Apoptosis) Examples: Tail and opercular resorption Separation of digits May be hormonally controlled Male and female genital ducts

The Clonal Mode of Development Clones: group of cells arising from single precursor

Regulation and Regeneration Regulation restoration of missing material occurring before differentiation of structure basis for development of identical twins subdivision of inner cell mass Regeneration Differentiation of structures already occurredGrowth Differential growth: all parts of the embryo do not grow at the same rate Determinate growth: body grows to certain point that is characteristic of species and sex (mammals) Indeterminate growth: ancestral vertebrates (fish) Recapitulation Biogenetic law of Muller and Haeckel Ontogeny is an abbreviated recapitulation of phylogeny

REPRODUCTIVE ORGANS AND THE SEXUAL CYCLE

Reproductive Organs

Female Reproductive Organs Paired gonads (ovaries) located in pelvic cavity Each ovary lies close to funnel-like opening (ostium tubae) at end of a uterine (fallopian) tube Around abdominal orifice of tube is fimbriae Uterus: thick, vascular, smooth muscle, caudally continuous with the cervix, will project into vagina Vagina: organ of copulation and birth canal

Male Reproductive Organs Testes suspended in scrotum (with a countercurrent heat-exchange system), with lower temperature Spermatozoa produced in seminiferous tubules, then to tubuli recti, rete testis, ductuli efferentes, epididymis, ductus deferens Spermatozoa stored in epididymis and ductus deferens

Sexual Cycle in Mammals

Estrous Cycle in Mammals Sexual cycle Estrus (prepared for reproduction accompanied by ovulation and sexual desire) Postestrum (regression of preparations) Diestrum (period of rest) Proestrum (period of active preparatory changes) Light is a critical factor: higher than certain threshold causes hypophysis to become active and produce enough FSH Light Nervous transmission FSH Follicular growth Estrogen in ovarian follicle estrogen in blood stream mating behavior, estrous uterus, estrous vagina

Primate Menstrual Cycle Sexual cycle is characterized by menstruation Commences at menarche until menopause Three phases Menses Proliferative (follicular) Secretory (luteal) Menstruation initiated by reduction of blood flow into superficial uterine blood vessels, resulting in deterioration and extravasation of blood into tissue, bringing with it the necrotic superficial tissue

Hormonal Regulation of the Female Sexual Cycle Levels of hormonal control 1: Hypothalamus GnRH (stimulates LH and FSH in hypophysis) PIH (inhibits prolactin release by hypophysis) 2: Hypophysis FSH (stimulate follicle cells to produce estrogen) LH (male: stimulate Leydig to produce testosterone; female: stimulate follicle to produce progesterone) Prolactin (promotion of lactation) 3: Ovaries (hormones secreted into blood and placental tissues) Estradiol Progesterone Testosterone: precursor of estrogen biosynthesis; induces atresia Inhibin: inhibits FSH secretion 4: Ovarian steroid hormones into body tissues Ovarian follicle maturation brought by rise in pituitary FSH FSH and LH stimulate follicle estrogen production high estradiol production in ovarian follicle causes LH and FSH peak LH peak as final stimulus for follicular maturation ovulation follicle transforms into corpus lutem (actions of LH) corpus luteum secretes estradiol and progesterone increased ovarian hormone levels and inhibin cause feedback inhibition inhibition results in low levels of LH and FSH later regression of follicle comes decrease in gonadotropin levels stimulation of GnRH and gonadotropins

Hormonal Regulation of Reproduction in the Male Testosterone: secreted by Leydig cells (stimulated by LH) Sertoli cells take up FSH synthesis of ABP

GAMETOGENESIS

Gametogenesis Germ plasm gametes + cells that give rise to them Gametogenesis: germ plasm is converted to specialized sex cells capable of uniting at fertilization and producing a new being Four phases Origin of germ cells and migration to gonads Multiplication of germ cells in the gonads (mitosis) Reduction of number of chromosomes (meiosis) Maturation and differentiation of gametes

The Origin of Primordial Germ Cells and Their Migration to the Gonads Primordial germ cells of mammals, reptiles, birds arise in epiblast take up temporary residence in extraembryonic tissue before returning Birds: in germinal crescent Mammals: posterior wall of yolk sac (near allantois) Amphibians: vegetal pole cytoplasm Note: PGCs do not produce gonads, they produce gametes PGCs in extragonadal sites may develop into teratomas

Proliferation of Germ Cells by Mitosis Mitotically active germ cells: oogonia, spermatogonia Settling in the gonads induce a proliferative phase Mammalian oogonia proliferative phase Mitosis brings numbers to about 7 million at 5th month of pregnancy Atresia causes sharp decline Primary oocytes formed (suspended at prophase I)Meiosis Genetic recombination occurs by Random distribution of chromosomes to daughter cells Crossing over (2n, 4c) (Me I) (1n, 2c) (Me II) (1n, 1c)

Spermatogenesis and Oogenesis Compared 4 functional spermatozoa vs 1 viable ovum Arrests in meiosis Spermatogenesis: none Oogonesis meiotic arrest First arrest at diplotene prophase I in primary oocyte Broken by hormonal changes Arrested again at metaphase II Broken by fertilization (sea urchins complete meiosis II at once, no second arrest)

Spermatogenesis Transition from mitotically active PGCs to mature spermatozoa Three phases Mitotic multiplication (spermatocytogenesis) Meiosis (spermatidogenesis) Maturation and differentiation (spermiogenesis) Spermatogonia Type A: stem cell population Ad: long term reserves Ap: mitotically active, give rise to B Type B: committed to finish spermatogenesis Spermatocytes Preleptotene spermatocytes Primary spermatocytes (Me I) secondary spermatocytes (Me II) haploid spermatids Differentiation of spermatid to spermatozoa Golgi complex forms proacrosomal granules, into acrosome Centrioles as point of anchorage for developing flagellum Intercellular bridges: facilitate synchronous differentiation and division of sperm-producing cells Sertoli cells: FSH target Sertoli cells Synthesis of ABP (to maintain high testosterone levels) Maintain blood-testis barrier Create environment for differentiation of sperm cells Facilitate release of mature spermatozoa Degradation of residual bodies Blood-testis barrier (held by tight junctions) responsible for preventing bodys immune system from destroying mature sperm cells (antigenically different from body)

Gene Expression during Spermatogenesis Posttranscriptional control

Sperm Maturation Sperm coated with glycoprotein which must be removed in female reproductive tract before fertilization can occur (activation, capacitation) Seminal fluid provides external energy source (causes nonmotile sperm to become motile)

Oogenesis

Oogenesis in Amphibians Mitotic phase of oogenesis does not come to early halt New crop of eggs each year, 3 years for maturation Follicular epithelium, theca, ovarian epithelium

Development of the Amphibian Egg Three phases Previtellogenesis (before yolk deposition) Vitellogenesis (period of yolk deposition) Maturation (released from meiotic block by progesterone) Previtellogenic phase Period upto early diplotene phase of meiosis Lampbrush chromosomes: spread out configuration forming loops Loops are where RNA synthesis occurs Large numbers of nucleoli for specific gene amplification (ribosomes, rRNAs) Vitellogenic phase Principally concerned with yolk formation Lipochondria stores lipids Glycogen granules stores carbs Yolk platelets stores proteins Yolk protein produced by liver cells under estrogen influence Gonadotropin release from hypothalamus to oocyte estrogen secretion from oocyte into liver secretion of vitellogenin from liver to oocyte Yolk precursor vitellogenin (phospholipoprotein) Incorporated into oocyte by micropinocytosis Represented by phosvitin and lipovitellin (packed in crystalline form to form yolk platelets) Yolk formation was thought to be function of Balbiani body (yolk nucleus) Pigment granules concentrate at animal hemisphere Maturation phase Hormonally induced release of egg from first meiotic block Breakdown of germinal vesicle Completion of first meiotic division Formation of first polar body Begins with secretion of progesterone (stimulated by gonadotropin) Causes breakdown of germinal vesicle, meiotic maturation Meiosis arrested again at Metaphase II by CSF (cytostatic factor)

Oogenesis in Birds Yolk is a single cell (the ovum) Gradually accumulates in cytoplasm of ovum before it is liberated from ovary All other noncellular secretions (egg white, shell membrane, shell) are contributed as ovum passes reproductive tract Yolk still produced by the liver and transported via blood to the follicular cells surrounding ovum (as in amphibians) 50% water, 33% fat, 16% protein, 1% carb Water NaCl, Ca salts (bone formation) Proteins lipovitellin (binds w/ lipids), phosvitin (binds w/ phosphorus) Protuberance containing ovum is ovarian follicle With zona radiata (irregular striated plasma membrane due to microvilli), for increase in membrane surface, enhancing metabolic interchanges Compared with mammalian ovum: No yolk in mammals, just liquor folliculi Both have two layered CT theca Yolk release albumen secretion shell membranes (isthmus) shell (uterus)

Oogenesis in Mammals Primary oocyte (so called as it is undergoing meiosis I) + flat follicular cells = primordial follicle Phase I: pool of primordial follicles developing into primary follicles (flat cuboidal) Meiotic arrest follows (diplotene meiosis I) Both oocytes and follicular (granulosa cells) develop microvilli, connected by gap junctions (allow high MW molecules to pass through) Zona pellucida beings to develop Phase II: growth of oocyte and granulosa covering (under influence of gonadotropic hormones) Overall growth of follicular covering mediated by FSH receptors Secondary follicle when antrum is identifiable LH receptors develop, allowing production of testosterone by theca Transported into granulosa cells, wherein granulosa converts testosterone to estrogen by aromatase Phase III: further follicular growth and selection of one follicle (highest receptivity to FSH) which will undergo ovulation Begins late in follicular phase of menstrual cycle After LH surge, angiogenesis occurs, causing estradiol to spill out into blood Before ovulation, ovum is released from first meiotic block (diplotene), allowing meiosis I to finish After that, follicle is now ready to respond to preovulatory FSH and LH surge, and release itself (wherein it is now at metaphase II, second block) Ovulation Increased antral fluid pressure within follicle causes bursting of follicular wall Weakening of follicular wall by lytic enzyme (stimulated by LH) Corpus luteum Stratum granulosum and theca interna involved in corpus luteum formation Endocrine organ, secreting progesterone and estrogen Granulosa cells swell and develop to secrete high levels of hormones Formation of corpus lutem require continuous presence of LH from pituitary regression happens with decreased sensitivity to LH receptors corpus lutem of pregnancy maintained by chorionic gonadotropin (secreted by embryo) Corpus lutem produces large amounts of progesterone and estrogen Progesterone prepares lining of uterus for implantation

Accessory Coverings of Eggs

Covering of the Sea Urchin Egg Inner to outer Plasma membrane Vitelline envelope (composed of glycoproteins, contain species-specific receptors for spermatozoa) Jelly coat (polysaccharides, glycoproteins, hydrates and expands when eggs are shed)

The Membranes Surrounding the Amphibian Egg Plasma membrane: forms microvilli Follicular cells: form macrovilli Narrow space between oocyte and follicular epithelium becomes filled with noncellular basement membrane: vitelline envelope (equivalent of zona pellucida in mammals) Gap junctions join the villous processes At ovulation, perivitelline space forms between vitelline envelope and plasma membrane Coated with jelly coat as it goes through oviduct (same function as sea urchins)

Formation of the Accessory Coverings of Bird Eggs At ovulation, ovum surrounded by inner vitelline membrane Remainder of accessory coverings secreted about ovum during passage toward cloaca Outer vitelline membrane laid down when it is in oviduct adjacent to ovary Albumen laid down in upper oviduct Rotation twists albumen into spiral strands at two ends of yolk: chalazae Serve to suspend yolk in albumen Egg white Ovalbumin and lysozyme by estrogen Avidin secreted by goblet cells by progesterone Shell membrane added farther along oviduct Shell secreted as egg passes through shell gland (at uterus)

The Coverings of Mammalian Eggs Noncellular zona pellucida (mostly synthesized by oocyte) ZP-1 ZP-2 ZP-3 acts as sperm receptor and plays a role in inducing acrosome reaction Corona radiate still surrounds mammalian ovum (may continue to secrete steroids and prostaglandins)

FERTILIZATION Initial contact between egg and sperm Entry of sperm cell into egg Prevention of polyspermy by egg Metabolic activation of egg Completion of meiosis by egg Formation and fusion of male and female pronuclei

The Sea Urchin

Gamete Release and Transport 100 billion spermatozoa and 4 billion eggs

Sperm Penetration of the Egg in Invertebrates and the Acrosome Reaction When spermatozoa encounter egg, former undergoes changes In presence of egg cells, spermatozoa will cluster and increase motility Direct contact with jelly coat increases motility and stimulates acrosome reaction Speract is responsible for increased motility and activated respiration that occur when sperm contacts with jelly coat Increase in permeability of plasma causes influx of Na and Ca, efflux of H+ Raises intracellular pH, stimulating flagellar activity Contact w/ jelly coat stimulates acrosome reaction Begins with breakdown and subsequent fusion of outer acrosomal membrane and plasma membrane Polymerization of G-actin to F-actin (forming acrosomal process) Tip of process is covered with bindin, mediating sperm binding to surface of eggs Spermatozoa digest through vitelline membrane by lysins

Binding of Sperm to the Egg Sperm receptor molecule (on microvilli of egg) Intracellular: remains constant among species Extracellular: differs accdg to species After sperm-egg fusion, fertilization cone forms by microvilli engulfing sperm head

Blocks to Polyspermy Fertilization of egg by more than one sperm Fast block Membrane event, set in place within 2-3 seconds, and lasts for 60 seconds Acrosomal process and plasma membrane fusion causes depolarization of plasma membrane (by influx of Na+) From -70mV to +10mV Positive potential does not permit fusion of other spermatozoa to plasma membrane Slow block Mobilization of Ca from within egg first released at site of sperm entry wave of released Ca initiates cortical reaction (rupture of cortical granules and release of these contents into perivitelline space) cortical granules move to inner surface of plasma membrane, fuse with it, and open up contain sulfated mucopolysaccharides (GAGs), which have high water affinity, causing swelling and forcing vitelline envelope away from plasma membrane (raising the fertilization membrane) Fertilization membrane: name given to vitelline envelope after changes by cortical reaction Hydrated mucopolysaccharides form hyaline layer (between plasma and fertilization membrane) As fertilization membrane is elevated, an enzyme alters it, causing attached sperm to drop off Final step: release of ovoperoxidase from cortical granules (for breakdown of H2O2, results in hardening of fertilization membrane) H2O2 released by egg during cortical reaction (spermicidal) Polyspermy is normal in urodele amphibians and birds

Metabolic Activation of the Egg Other events that prepare egg for fusion of genetic material: Increased oxygen consumption Activation of NAD kinase (facilitate biosynthesis of new membrane lipids) Another influx of Na+ (with efflux of H) causing increased intracellular pH Increased pH leads to increased protein synthesis and initiation of DNA synthesis

Penetration of the Spermatozoon into the Egg and Fusion of the Genetic Material Sperm nucleus begins to interact with egg cytoplasm, and chromatin relaxes As chromatin dispersion nears completion, new membrane forms around what can now be called a male pronucleus Sperm centrioles persist, and provide basis for formation of sperm aster, important in getting male and female pronuclei together Pronuclear fusion occurs at center of egg After fusion, chromosomes replicate in preparation for cleavage

Mammalian Fertilization

Sperm Transport in the Female Reproductive Tract of Mammals Barriers to fertilization: Natural acidity of vagina (bacteriostatic) Seminal fluid acts as buffer (raises vaginal pH) Seminal fluid may cause contractions in upper vagina, helping propel spermatozoa Orgasms cause uterine contractions Entrance to uterine tubes (ovulation can sometimes only occur on one tube) Positive rheotactic response (face an oncoming current generated by uterine ciliary movement) Capacitation: removal of glycoprotein covering spermatozoa, enabling better penetration of egg

Egg Transport Ciliary currents and smooth muscle contractions transport egg into uterine tube Corona radiata adds mass faster movement down tube

Union of Gametes Mammals: occurs in upper part of uterine tubes Spermatozoa must penetrate corona radiata cells and then zona pellucida before contact with plasma membrane Zona pellucida: molecules on sperm head bind with species-specific sperm receptors (consist of exposed part of ZP-3) Further contact with other core regions stimulate acrosome reaction (capacitation is prerequisite, so that lytic enzymes are released to facilitate passage of sperm through zona pellucida) Acrosome reaction: Localized fusion and breakdown of outer acrosomal membrane and plasma membrane Acrosin bound to inner acrosomal membrane digests through zona pellucida Fertilization cone bulged out when sperm makes contact with egg

Development and Fusion of Pronuclei With sperm entry, block to second meiotic division is lifted, and second polar body is released Breakdown of sperm nuclear membrane and decondensation of chromatin and protamine replacement with histones New pronuclear membrane forms around decondensed material DNA synthesis occurs as male and female pronuclei migrate toward each other (as opposed to sea urchins, where chromosomes condense to prepare for metaphase, and DNA synthesis occurs after fusion of pronuclei)Parthenogenesis Activation of unfertilized eggs and development into viable individuals Mammals: all female because females are homogametic (XX) Birds, reptiles: males and females (heterogametic females)

Sex Determination Occurs at fertilization, determined by Mammal sperm Bird/reptile egg

Establishment of Polarity in the Embryo 3 polar axes: Craniocaudal (anteroposterior) Dorsoventral Mediolateral

Establishment of Polarity in Amphibians Primary polarity of egg by animal and vegetal poles Denser pigment concentration at animal pole Nucleus located near animal pole Gradient of increasing density of ribosomes and glycogen granules towards animal pole Size and concentration of yolk platelets increase toward vegetal pole Region of animal pole = head Region of vegetal pole = tail Anterocaudal axis Fertilization is next establishment of polar axes 2 major reorganizations General convergence of cytoplasm beneath the thin cortical region toward the sperm entry point 30 degree shift between subcortical cytoplasm and overlying cortex These changes cause reduction in density of pigment granules in the region of the animal hemisphere along equatorial zone opposite to sperm entry point Reduced pigmentation called gray crescent Midpoint of gray crescent is middorsal point of body (determines dorsoventral axis of future embryo) Determination of two axes determines third Even before cleavage, the three axes are established and secondary polarization is completed

Establishment of Polarity in Birds Cleaving embryo represented by flat disk of cells on yolk surface: cells on outer surface become dorsal part, those closest to yolk become ventral Cells are shed from part of blastoderm uppermost (with respect to gravity). The area from which these cells fall become the caudal end of embryo

CLEAVAGE AND FORMATION OF THE BLASTULA Cleavage: waves of cell division following one another almost without pause Many of changes and differences in cleavage patterns among embryos of various species are related to the amount of yolk present in egg Cleaving embryo develops a central cavity (blastocoel) and enters blastula stage

The Cell during Cleavage Cleavage division consists of karyokinesis followed by cytokinesis Cleavage furrow first forms in the region of cortex nearest to mitotic spindle, and then moves around cell Position of cleavage furrow gets fixed or established at anaphase Asters (composed of microtubules) interact with cell cortex to stimulate cleavage furrow formation Asters are the effective agent in initiating cleavage furrow formation (not the mitotic spindle) Mitotic spindle asters cell cortex

Distribution of Yolk and its Effect on Cleavage Blastomeres: cells that arise from cleavage Holoblastic cleavage: characterized by complete division of cells Oligolecithal eggs produce blastomeres of equal size Mesolecithal eggs displace nucleus towards animal pole Net result is appearance of later and larger blastomeres at vegetal pole Meroblastic cleavage: newly formed plasma membrane does not separate inner borders of dividing cells from the underlying yolk Telolecithal eggs: displaces the embryo-forming cytoplasm into a small disk on one edge of ovum

Cleavage and Formation of the Blastula

Amphioxus Radial holoblastic equal cleavage, microlecithal eggs Meridional meridional equatorial meridional equatorial

Sea Urchins Early cleavage only consists of S and M phase, with alternating periods of cyclin synthesis and degradation G1 and G2 phases evident only later in cleavage Microlecithal Meridional meridional equatorial Lower tier undergoes unequal equatorial division Micromeres (becomes primary mesenchyme) Macromeres Upper tier undergoes equal meridional division, forming 8 mesomeres During entire cleavage, embryo is enclosed in fertilization membrane and closely associated with hyaline layer Blastomeres later form motile cilia, penetrating hyaline layer and secreting hatching enzyme into perivitelline space, digesting the fertilization membrane End product is mesenchyme blastula

Amphibians Meridional (begins at animal pole, bisects gray crescent) meridional equatorial double meridional double equatorial Amphibian blastocoel Formed from specialization of cleavage furrow of animal hemisphere Filled with Na+ ions, then water enters to maintain ionic balance, causing expansion Amphibian blastula has three main regions Region around animal pole, including cells forming roof of blastocoel Future ectodermal germ layer Region around vegetal pole, including large cells in interior Future endodermal cells Marginal ring of cells in the subequatorial region of the embryo, including gray crescent Embryonic mesoderm Nieuwkoop discovered basis for mesodermal induction Experiment 1: directly apposed sheet of cells from animal hemisphere above blastocoel to a yolk mass from vegetal hemisphere Inductive influence from yolk mass caused animal pole cells to form mesodermal structures Therefore, blastocoel may function to restrict interaction between future endodermal and ectodermal cells Experiment 2: isolated pieces of ectoderm induced to form mesoderm by transforming growth factor-beta Source of mesodermal induction (Nieuwkoop center or dorsalizing center or mesodermal inducing center), resides in a number of vegetal endodermal cells located in prospective dorsal midline Nieuwkoop center Stimulates formation of mesoderm Establishes dorsal properties of induced mesoderm Dorsal induced mesoderm is direct forerunner of what is called the Spemann organizer (dorsal lip of blastopore), the dominant organizing region of amphibian embryo during gastrulation

Birds Mitotic spindles align themselves so subsequent cleavage furrows form at right angles to the preceding one (first 3) Fourth cleavage furrow is a circumferential one Blastomeres formed by first few divisions are dorsally bound by plasma membrane but basal surfaces are open to underlying yolk Further cleavage of blastoderm results in radial extension of embryo 32-cell embryo shows cleavage parallel to surface, establishing several strata of superficial cells At around 100 cells, blastoderm is underlain by a subgerminal cavity pH of subgerminal cavity 6.5, while albumen: 9.5, leading to establishment of transepithelial potential of 25mV. Electrical gradient determines dorsoventral axis of blastoderm. Shedding of cells begins from undersurface of area of blastoderm that is farthest away from source of gravity (this area becomes caudal end of embryo) Area pellucida: central portion of blastoderm thinned out by shedding of cells Area opaca: region where blastoderm cells still abut directly onto yolk Primary hypoblast: aggregates of cells shed from lower surface of blastoderm by a process of polyingression/delamination First occurs at posterior end of embryo Separated from epiblast by a blastocoel Polarity and location of primary hypoblast determine location and direction of future primitive streak by inductive interaction EXTRAembryonic endoderm Comparison with amphibians Two layered blastoderm is compared to a flattened blastula (epiblast: animal hemisphere:: hypoblast: vegetal) Both hypoblast and vegetal pole have ability to induce formation of mesoderm in epiblast and animal pole by inductive interaction

Mammals Equal holoblastic cleavage of an isolecithal egg Persistence of traits characteristic to large-yolked embryos (later in development) Second cleavage division may not occur simultaneously in both blastomeres Mitotic spindle of one blastomere may rotate 90 degrees, causing a rotational pattern of cleavage (as opposed to radial in echinoderms, Amphioxus) 16-cell stage still is contained in zona pellucida Morula: internal secretion of fluid by blastomeres leads to formation of blastocoel, or blastocyst cavity Similar to amphibian (Na-K-ATPase system brings in Na, and then water) Blastocyst has two populations of cells Trophoblast: cells that constitute outer wall of blastocyst maternal X chromosomes are preferentially expressed assumed epithelial properties (tight junctions, microfilaments) forms large part of placenta Inner cell mass: joined together by gap junctions, retains ability to reaggregate if separated Comparison with birds Embryos of mammals form a layer of cells beneath inner cell mass, called primitive endoderm equivalent to primary hypoblast of avian embryos Note that primitive endoderm does not contribute to embryo proper, as primary hypoblast does not too

GASTRULATION AND THE FORMATION OF THE GERM LAYERS

Gastrulation as a Process Well-ordered rearrangements of cells in embryo Morphogenetic movements result in reorganization Rearrangement of blastula to a stage characterized by presence of three germ layersTable 6-1Type of movementDescriptionExample

InvaginationInpocketing of sheet of cellsArchenteron formation in Amphioxus

EvaginationOutpocketing of sheet of cellsExogastrulation

InvolutionRolling around a corner of an expanding outer layer of cells and spreading over an internal surfaceCell movements through the amphibian blastopore

EpibolySpreading of a cell sheetSpreading of outer cells towards amphibian blastopore

IngressionSinking of individual cells from a surface into an areaPrimary mesenchyme formation in sea urchin embryos

Polyingression (delamination)Separation of second sheet from an original single sheetFormation of primary hypoblast of avian embryos

Ameboid motionMigration of cells as single individuals through their own motilityMigration of neural crest cells

Two main strategies for gastrulation: Carry out gastrulation movements within context of a sphere (Amphioxus, amphibians) Elaboration of three germ layers as two-dimensional sheets upon one sector of an enormous sphere (birds, reptiles, even mammals) Blastopore: opening from outside into the archenteron

Gastrulation in Sea Urchin Embryos Separation of primary mesenchyme from vegetal plate of blastula signifies start of gastrulation Primary mesenchymal cells develop filopodia, moving along the basal lamina until forming a ring-like structure near base of invaginating archenteron Three stages of formation of archenteron Invagination of cells at vegetal pole Presence of secondary mesenchyme (will later make contact with animal pole by filopodia) As tip of archenteron makes contact with animal pole, secondary mesenchyme undergoes final determination (can no longer dedifferentiate into primary mesenchyme cells) Pluteus larva

Gastrulation in Amphibian Embryos Cortical rotation, stimulated by sperm fusion, leads to generation of an early organizing center (Nieuwkoop center) in dorsal cells of vegetal hemisphere Again, major activity of vegetal organizing center is mesodermal induction As embryo enters late blastula, organizing activity shifts to a more superficial dorsal location Under genetic influence, cells of dorsal marginal region migrate resulting in formation of blastopore Upper margin of blastoporal groove is known as dorsal lip of blastopore and will later become the major organizing region (Spemann organizer) Initial formation of blastoporal groove is related to a change in cell shape in this area; cells elongate inwardly, bottle cells, associated with inward pulling movement, results in formation of blastoporal groove. Prospective endoderm: around ventral margins of blastopore and extending to ventral part of embryo, and lines archenteron Chordamesoderm: passes over dorsal lip of blastopore and gives rise to notochord and cephalic mesoderm Cell movement in dorsal lip of blastopore: Surface cells are underlain by a deeper marginal zone consisting of several layers of cells Movement associated with convergence and extension phenomena Account for overall elongation of embryo Early gastrulation: cells of deep layer of marginal zone interdigitate by radial intercalation to form single layer Late gastrulation: mediolateral intercalation, where lateral cells insert processes between cells in medial part of layer and later become interposed between them Involuted cells from marginal zone will form mesoderm of embryo In surface layers, epiboly happens by cell division, flattening, and spreading. As these surface cells cross dorsal lip, they become endodermal lining of archenteron. Ectoderm: neural ectoderm and general cutaneous ectoderm At end of gastrulation, all future endoderm and mesoderm lie within Spemann and Mangold: demonstrated that dorsal lip of blastopore possesses ability to organize future development Early, it initiates convergent extension movements Late, it exerts dorsalizing effect on lateroventral region of marginal zone Organizer signals neighboring cells of animal cap to form neural plateGastrulation in Birds Review: Blastula as two-layered structure (epiblast and hypoblast, with blastocoel in between) Embryo proper occupies area pellucida and surrounded by area opaca Kollers sickle (thin sickle-shaped mass of cells) at posterior end of embryo, where a secondary hypoblast pushes anteriorly, folding primary hypoblast ahead of it Distribution of primordial germ cells along anterior border of blastoderm due to compression of primary hypoblast (PGCs are found in primary hypoblast) Gastrulation begins with condensation of cells in posterior part of epiblast, gradually assuming cephalocaudal elongation, eventually forming primitive streak Appearance of primitive streak is result of inductive interaction of epiblast with hypoblast Primitive streak now contains: Primitive groove: central furrow Primitive ridges: thickened margins Hensens node: cephalic end of streak Begins to regress after 18hrs, with corresponding elongation of notochord above it (head process) Embryonic germ layers are formed by migration of cells in epiblast towards primitive streak, and subsequent ingression (not delamination) to form middle and lower germ layers (embryonic mesoderm and endoderm) First cells to pass are the future embryonic endodermal cells They displace primary hypoblast outward and cephalad toward area opaca Mesodermal cells migrating through: Hensens node: notochord Cranial part of primitive streak: embryonic mesoderm Caudalmost part of streak: extraembryonic mesoderm

Comparison of Avian and Amphibian Development Blastula Bird: epiblast contains endodermal and mesodermal germ layers Amphibian: surface layers contain endoderm and mesodermal parts Induction Effect of hypoblast on epiblast Mesoderm induction by vegetal yolk Blastopore Pre-primitive streak thickened area of chick blastoderm Blastopore in amphibians Cell migration: Cells that constitute endodermal layer migrate to interior first, and mesodermal layers follow later

Origin of the Germ Layers in Mammals Review: Mammalian blastocyst segregated into embryo forming inner cell mass and trophoblastic cells Hypoblast: first cells to segregate out from ICM Forms extraembryonic endoderm, similar to birds Forms lining of yolk sac Remainder of ICM is now called epiblast Contains future ectodermal cells Also contains cells that will migrate through primitive streak and become definitive endodermal and mesodermal germ layers Remaining ICM now called embryonic disk One margin of disk becomes thickened, becomes caudal part of embryo From caudal thickening, cephalad expansion of cells results in primitive streak

Origin of the Germ Layers in Rodents Early hypoblast is called primitive endoderm in mouse Cells from this layer spread out beneath trophoblast (trophectoderm) to form endodermal layer of parietal yolk sac Parietal endoderm cells create basement membrane called Reicherts membrane Polar trophectoderm: overlying ICM can undergo mitosis, daughter cells become mural Mural trophectoderm: surrounding blastocyst cavity Mitosis results in polyploidy giant cells ICM undergoes transformation different from other mammals Protrudes deeply into blastocyst cavity (like a tongue-like lobe) Cavity forms within the lobe (proamnion), and cells surrounding it are primitive ectoderm (or epiblast) Called an inverted egg cylinder

NEURULATION AND THE FORMATION OF AXIAL STRUCTURES

Primary (Neural) Induction

Neurulation in Amphibians

Formation of the Neural Tube

The Neural Crest

The Mesoderm of the Early Embryo

Secretion of Extracellular Materials in the Early Embryo

The Formation and Differentiation of Somitomeres and Somites