Embryology -study of embryos embryo: -juvenile stage of a multicellular organism -contained within egg envelopes or membranes inside or outside

the body of maternal parent -study of development Development: -ontogenetic: individual life history -phylogenetic: evolution -study of ontogenetic development -developmental biology: study of embryonic and other developmental processes Six General Questions in Developmental Biology -Differentiation -Morphogenesis -Growth -Reproduction -Evolution -Environmental integration Phases of Ontogenetic Development -Gametogenesis -Fertilization -Cleavage -Gastrulation -Organogenesis -Growth and histological differentiation CELL DIVISION Stages of Prophase I A. Leptotene -Chromosomes uncoiled and maximally extended -Ends of chromosomes attached at inner surface of nuclear membrane B. Zygotene -Synapsis of homologs takes place -Centrosome duplicates C. Pachytene -Bivalents converted to tetrads -Crossing over occurs -Duplicate centrosomes move apart D. Diplotene -Homologs repel each other but are still connected by chiasma/chiasmata E. Diakinesis -Chromosomes are highly condensed -Duplicate centrosomes reach the opposite poles -Nuclear envelope starts to disappear F. Metaphase I

GAMETOGENESIS Oogenesis Materials stored in the eggs cytoplasm -Proteins and lipids -Ribosomes and tRNA -mRNA -Morphogenetic factors -Protective chemicals Reproductive Endocrinology and Reproductive Cycles Reproductive Hormones -GnRH (hypothalamus) -FSH (anterior pituitary) -LH (anterior pituitary) -Testosterone (gonads) -Estrogen (gonads) -Progesterone (CL, adrenal glands, placenta) -Prolactin (Luteotropic hormone, anterior pituitary) -Oxytocin (posterior pituitary, uterus) -HCG (trophoblast, placenta) -Activins -Inhibins Menstrual Cycle 1. Estrogen -Development of uterine lining -Thinning of cervical mucus -Increase in granulose FSH receptors -Stimulates granulose inhibin secretion -Stimulates LH production 2. Progesterone -Maintenance of vascularized uterine lining -Produced by corpus luteum, then by placenta during pregnancy Estrous Cycle Phases of Estrous Cycle -Follicular phase -Luteal phase Stages of Estrous Cycle 1. Proestrus -Development (enlargement) of follicle -Increase in estrogen levels up to peak -Increase in vascularization of female reproductive tract -Development of endometrial glands 2. Estrus -heat period -Decrease in estrogen levels -Surge in LH -Ovulation within 24-48 hrs -High uterine motility

-Optimal sperm transport -Increase in cervical mucus 3. Metestrus -Very low levels of estrogen -Corpus hemorrhagicum develops -In bovines, ovulation occurs -Uterine contractions decrease -Bleeding may occur, esp. in bovines 4. Diestrus -High progesterone levels -Dip in FSH -Uterine contractions stop -Corpus luteum degenerates if pregnancy does not occur FERTILIZATION -reproduction with genetic recombination (sexual) -Events: -contact and recognition between gametes -regulation of sperm entry into the egg -fusion of pronuclei -activation of egg metabolism Five basic steps in gametic interaction -chemotaxis -acrosomal reaction -binding of sperm to the extraoocytic envelope -penetration of sperm through extraoocytic envelope -plasmalemmal fusion 1. Chemotaxis Resact: in egg jelly of Arbacia punctulata -sperm activator 2. Acrosomal reaction and sperm binding Bindin: egg-binding protein in sea urchins Sperm binding in mammals: zona pellucid glycoproteins ZP3 -primary binding -induction of acrosomal reaction ZP2 -secondary binding Capacitation -makes sperm competent for fertilization -Ca 2+, HCO3 -, albumin -uncapacitated sperm tend to bind temporarily but actively to plasmalemma of cells in the oviductal isthmus Apparent in mammals: -chemotaxis through secretions in different sections of the female reproductive tract

-hyperactivation of sperm Fusion of Genetic Membranes -Fertilization cone formation -Fertilin: in sperm plasmalemma at posterior portion of head Blocks to Polyspermy: Fast block: Depolarization of egg plasmalemma -Slow block: Cortical reactions CLEAVAGE -rapid mitotic divisions -prior to mid-blastula transition: no G1, G2; M and S only -blastomeres with decreasing cytoplasmic to nuclear volume ratio -initiated by MPF activity -large subunit: Cyclin B -small subunit: cdc2 cyclin dependent kinase Activated cdc2 kinase -histones: chromation condensation -nuclear membrane lamin: nuclear membrane degradation -cytoplasmic myosin: formation of mitotic spindle MPF and regulators -stored in egg cytoplasm -maternal genomics/transcriptomics in early cleavage Mid-blastula transition -growth phases (G1 G2) added -synchronicity of division of blastomeres lost -transcription of new mRNA
Process Karyokinesis Cytokinesis Mechanical Agent Mitotic spindle Contractile ring Major protein compo. Tubulin (microtubules) Actin (microfilaments) Location Central cytoplasm Cortical cytoplasm

Factors influencing pattern of cleavage -amount and distribution of yolk in egg cytoplasm -factors that influence the angle and timing of formation of the mitotic spindle Patterns of cleavage I. Holoblastic (complete) A. Isolecithal (evenly distributed yolk) -Radial (echinoderms, amphioxus) -Spiral (annelids, mollusks, flatworms) -Bilateral (tunicates) -Rotational (mammals, nematodes) B. Mesolecithal

-Radial, amphibians II. Meroblastic (incomplete) A. Telolecithal (dense yolk throughout most of cell) 1. Bilateral (cephalods, mollusks) 2. Discoidal (fish, reptiles, birds) B. Centrolecithal (yolk in center of egg) Superficial (most insects) Cell Adhesion in the multicellular embryo Cadherins -Major cell adhesion molecules -Calcium-dependent -Anchored by catenins -P-cadherin (placental cadherin) -N-cadherin (nueral cadherin) -EP-cadherin (C-cadherin): normal movements in gastrulation -Protocadherins: disconnected from cytoskeleton; separation of notochord from rest of mesoderm Gastrulation -highly coordinated rearrangement of blastomeres through morphogenetic movement -formation of embryonic germ layers -endoderm: superficial -mesoderm: intermediate -ectoderm: alimentary Prominent features of physiological changes in gastrulation -morphogenetic movements -decrease in rate and rhythm of cell divisions -insignificant growth -intensified oxidation -more active nuclear activity; involvement of paternal genes -synthesis of new proteins Morphogenetic movements -Invagination: infolding of cell sheet into embryo -Involution: inturning of cell sheet over the basal surface of an outer layer -Ingression: migration of individual cells into the embryo -Delamination: splitting or migration of one sheet into two sheets -Epiboly: the expansion of one cell sheet over other cells Gastrulation involves a combination of the different types of morphogenetic movements

In frog gastrula: invagination, involution, epiboly Functions of the organizer tissue -derivation into the dorsal mesoderm -dorsalization of surrounding mesoderm into lateral plate mesoderm -dorsalization of ectoderm into neural layer -initiation of morphogenetic movements in gastrulation -formation of neural plate into neural tube Proteins expressed in the organizers -Nuclear proteins XLim1, Xnot, Otx2, XFD1, XANF1, Goosecoid, HNF3 betarelated proteins -Secreted proteins Chordin, Dickkopf, ADMP, Frzb, Noggin, Follistatin, Sonic hedgehog, Cerberus FORMATION OF PRIMARY ORGANS RUDIMENTS & AXES -Organogenesis: Neurulation Embryonic germ layers -ectoderm: superficial -mesoderm: intermediate -endoderm: alimentary How the anatomical axes are formed -dorsalization by action of the organizer -ventralization of the rest of the embryo -formation of anterior parts by mesoderm that first migrate inward through the dorsal blastopore lip - formation of posterior parts by mesoderm that migrate through the lateral and ventral lips Two major classes of organizer proteins -BMP4 (bone morphogenesis protein-4) inhibitors -Wnt inhibitors (wingless Int) BMP inhibitors 1. Noggin -induces dorsal ectoderm to form neural tissue -dorsalizes mesoderm cells that could contribute to ventral mesoderm 2. Chordin -induces neural tube formation 3. Nodal-related-3 (Xnr-3) 4. Follistatin 5. Cerberus -Promotes formation of adhesive gland, optic and olfactory placodes 6. Frzb and Dickkopf -Head formation

Regional Specification Homeotic Selector Genes 1) Antennapedia Complex -Specification of head segments Labial (lab) Deformed (dfd) -Identification of thoracic segments Sex combs reduced (scr) Antennapedia (Antp) -Labial palp formation Proboscipedia (Pb) 2) Bithorax Complex -Identification of third thoracic segment Ultrabithorax (ubx) -Identification of abdominal segments Abdominal A (abdA) Abdominal B (AbdB) Characteristics of Homeotic Gene Expression -Patterns of expression are initiated by 1) Gap genes Krppel (Kr) huckendein (hkb) knirps (kni) buttonhead (btd) hunchback (hb) empty spiracles (ems) giant (gt) orthodenticle (otd) tailless (tll) 2) Pair-rule genes hairy (h) odd-paired (odp) even-skipped (eve) odd-skipped (ods) runt (run) sloppy-paired (slp) fushitarazu (ftz) paired (prd) -Homeotic gene expression is dynamic: regulation by expression of other homeotic genes -Transcription patterns are maintained by alteration of chromatin conformation in the genes -Repression by Polycomb proteins -Activation by Trithorax proteins Homeodomain proteins -Transcription factors -Homeodomain -60 aa -Encoded by homeobox (180 bp) -Activate genes that specify the particular properties of each segment -Products of Hom-C expression -Fushitarazu, Caudal, Distal-less, Bicoid

Hox genes -Homologous to Hom-C -Expression specifies anterior-posterior polarity in vertebrates -Four copies located in separate chromosomes per haploid set -Mus musculus: Hoxa, Hoxb, Hoxc, Hoxd -Homo sapiens: HOXA, HOXB, HOXC, HOXD Extraembryonic Membranes Somatopleure-Derived Membranes 1) Amnion -Supplies a hydrated environment for embryogenesis 2) Chorion -Facilitates gas exchange -With endocrine, immune, and nutritive functions in the mammalian placenta Splanchnopleure-Derived Membranes 1) Allantois -Storage of urinary wastes -Mediates gas exchange -A portion (mesodermal layer) fuses with chorion (chorioallantoic membrane in birds) for calcium transport 2) Yolk sac -Mediates nutrition Trophoblast 1) Cytotrophoblast -Initial adhesion to endometrium -Proteolytic enzyme production and secretion for entry into uterine wall and remodeling of maternal blood vessels 2) Syncytiotrophoblast -Digestion of uterine tissue to further implantation -Contact with maternal blood vessels Implantation Cell Adhesion by Trophoblast Cells -E-cadherins -P-cadherins -Integrins : collagen, heparan sulfate glycoproteins, fibronectin, and laminin of uterine wall -Modified glycosyltransferase : carbohydrate residues on uterine glycoproteins Events in the Implantation of the Blastocyst -Direct contact of blastocyst with uterus -Blastocyst capture through extracellular matrix

with collagen, laminin, fibronectin, hyaluronic acid, and heparan sulfate receptors -Digestion of extracellular matrix of uterine tissue by trophoblast proteases (collagenase, stromelysin, plasminogen activator) -Entry of blastocyst into the uterine wall Induction and Competence in Differentiation Induction -Proximate (close range) interaction between two or more different cells or tissues -A group of cells induces changes on another set of cells -Change in morphology -Change in mitotic rate -Change in fate Components of an Inductive Interaction 1) Inducer -Produces a signal or signals that induce changes in the behavior of the other tissue 2) Responder -Changes/differentiates according to signal from Inducer Remarks on Competence -Not all tissues can respond to the inducer; only those with the ability to specific inductive signals. -Competence is actively acquired: competence Factors Pax6 as a Competence Factor Optic vesicles Surface ectoderm Wild-type Wild-type Pax6/Pax6 Wild-type Wild-type Pax6/Pax6 Pax6/Pax6 Pax6/Pax6

Organ Cutaneous structures (hair, feathers, sweat glands, mammary glands) Limb Gut organs (liver, pancreas, salivary glands) Pharyngeal & respiratory assoc. organs (lungs, thymus, thyroid) Kidney

Epithelial component Epidermis (ecto)

Mesenchymal component Dermis (meso)

Epidermis (ecto) Epithelium (endo) Epithelium (endo)

Mesenchyme (meso) Mesenchyme (meso) Mesenchyme (meso)

Tooth

Uretic bud epithelium (meso) Jaw epithelium (ecto)

Mesenchyme (meso) Mesenchyme (neural crest)

Properties of Epithelial-Mesenchymal Interactions -Regional specificity -Genetic specificity -The mesenchyme usually controls organ type specificity within a species. -The responding epithelium controls species specificity. Transmission of Inductive Signals Types of Inductive Interactions Based on Transmission of Signals -Juxtacrine interaction: membrane proteins of inducer cell interact with receptors on adjacent cell surface of responder cell -Paracrine interaction: inducer cell produces proteins that diffuse over a short distance to the responder cell Paracrine factors -Growth and differentiation factors (GDF) -Secreted by inducer into the extracellular space (vs. endocrine = secreted into and carried by blood) -Exhibit homology among different animal taxa Major Families of GDFs -Fibroblast growth factor (FGF) -Hedgehog

Lens induction Yes Yes No No

Modes of Inductive Interaction 1) Instructive interaction -Signal from an inductor is necessary for initiation of new gene expression in the competent responding cell. -e.g. Lens induction 2) Permissive interaction -Potentials are present in responding tissue; only an environment suitable for expression of these potentials is necessary. -e.g. Fibronectin or laminin matrix in tissue Differentiation

-Wingless (Wnt) -TGF- superfamily Fibroblast Growth Factors (FGFs) -Over a dozen in number -e.g. FGF1 (acidic FGF), FGF2 (basic FGF), FGF7 (keratinocyte growth factor) -Variable RNA splicing or initiation codons can produce hundreds of protein isoforms in different tissues -Activate receptor tyrosine kinases (FGF receptors; FGFRs) -Associated with angiogenesis, mesoderm formation, and axon extension -Can substitute for one another, but have separate functions due to differential expression patterns (e.g. FGF2 in angiogenesis, FGF8 in midbrain and limb development) Hedgehog proteins -Induction of particular cell types and creation of boundaries between tissues -At least three homologs Drosophila melanogaster hedgehog gene in vertebrates Sonic hedgehog (shh) Desert hedgehog (dhh) Indian hedgehog (ihh) The Hedgehog Family -Sonic hedgehog -Desert hedgehog: in Sertoli cells -Indian hedgehog: in gut and cartilage cells; important in postnatal bone development Sonic hedgehog -Expressed by notochord -Responsible for -Dorso-ventral patterning of neural tube -Sclerotome development -Formation of left-right axis in chicks -Initiation of anterior-posterior axis in limbs -Induction of regionally specific differentiation of alimentary tube -Induction of feather formation Wnt proteins -Cysteine-rich glycoproteins -At least 15 in vertebrates -wingless (segment polarity gene in D. melanogaster) + integrated (a vertebrate homolog of wingless) -Important in -Establishing polarity of limbs

-Development of urogenital system The TGF- superfamily -With over 30 members -Regulate some of the most important developmental interactions and processes -Includes (but is not limited to) -TGF- -BMP -Activin -Vg1 -Regulation of cell division and formation of extracellular matrix between cells (TGF- 1, 2, 3, and 5) -Critical in control of location and timing of development of ducts of kidneys, lungs, and salivary glands from epithelia -TGF- 1 can be expressed by the placenta and mammary glands Glial-derived neurotrophic factor (GDNF) -Kidney and enteric neuron differentiation Mllerian inhibitory factor -Mammalian sex differentiation The BMP Family -Induction of bone formation -Regulation of cell division and apoptosis, cell migration and differentiation Other Paracrine factors -Epidermal growth factor -Hepatocyte growth factor -Neurotrophins -Stem cell factor -Erythropoietin -Cytokines -Interleukins Urogenital System Development & Sex Differentiation Stages of Kidney Development 1. Pronephros -Initial kidney -Derived from the most anterior nephrotomes -With well-developed nephrostomes (ciliated funnels) -Persist in fish and amphibian larvae -Degenerates in mammals; more posterior portions of the duct may persist (as the Wolffian duct) 2. Mesonephros -Non-functional in excretion in humans and mice -Does not form its own duct -Source of hematopoietic stem cells

-Some tubules persist as the vas deferens and efferent ducts 3. Metanephros -Permanent kidney -Formed from metanephrogenic mesenchyme Epithelial-Mesenchymal Interaction in Metanephros Formation -Formation of the metanephrogenic mesenchyme (WT1 competence) -Secretion of GDNF and HGF by metanephrogenic mesenchyme to induce and direct the ureteric bud -Prevention of mesenchymal apoptosis by FGF2 and BMP7 secretion by the ureteric bud -Ureteric FGF and LIF-induced mesenchyme aggregation and Wnt4 expression (for epithelialization) -Conversion of the aggregated mesenchymal cells into a nephron -Induction of ureteric branching (by GDNF, TGF-1 Activin) -Formation of new nephrons and stromal cells (expressing BF-2, FGF7) from proliferated metanephric stem cells Sex Determination -Chromosomal sex determination -Primary: gonads -Secondary: bodily phenotype -Environmental sex determination -Temperature-dependent -Location-dependent Chromosomal Basis of Sex Differentiation Sex Chromosomes -Mammals: X and Y XX : Female XY : Male -Birds: Z and W ZZ : Male -ZW : Female Fates of the Sex Cords and Ducts GONADS Gonadal type Testis Sex cord Medullary (internal) DUCTS Remaining duct Wolffian Duct Vas deferens, differentiation epididymis, seminal vesicle

Molecular Bases of Early Mammalian Sex Determination SRY/Sry (sex-determining region of the Y chromosome) -223 aa transcription factor with HMG (high mobility group) box -Testis-determining factor -Encoded by SRY/Sry gene (~35 kbp) -Expression in genital ridge cells causes secretion of chemotactic factors for mesonephric cells (presumptive Sertoli cells) SOX9/Sox9 and Sex Reversal SOX9/Sox9 -Transcription factor with HMG box, coded for by SOX9/Sox9 gene (autosomal) -Expressed only in male genital ridge cells (with Sry) -Binds to promoter region of Amh gene -Extra copy of SOX9 genotypic XX develops as phenotypic male -Only one functional copy of SOX9: -Campomelic dysplasia (multiple skeletal and organ system disorders) -Genotypic XY develops as phenotypic female or hermaphrodite Steroidogenic Factor 1 (SF1/Sf1) -Directly or indirectly activated by SRY -Essential in development of bipotential gonad -Expression persists in male genital ridge cells -Active in masculinization of Leydig and Sertoli cells -In collaboration with Sox9, upregulates AMH expression in Sertoli cells -In Leydig cells, activates genes that encode enzymes for testosterone DAX1 and Determination of the Ovary -Encoded by gene located in X chromosome -Expressed in genital ridges shortly after Sry expression -SRY antagonist -Downregulates SF1 expression WNT4/Wnt4 -Expressed in the genital ridge at the bipotential phase -Repressed by SRY Hormonal Basis of Sex Determination

Ovary Cortical (external) Mullerian Oviduct, uterus, cervix, upper port. of vagina

Anti-Mllerian duct hormone (AMH) -560 aa glycoprotein -Causes degeneration of Mllerian duct in male reproductive system development -Synthesized and secreted by Sertoli cells

-Binds to and induces mesenchymal cells surrounding the Mllerian duct to secrete a paracrine factor that leads to apoptosis of cells of the duct epithelium Testosterone -Masculinization of reproductive structures that arise from the Wolffian duct 5-a-Dihydrotestosterone -Masculinization of the urethra, prostate, penis, and scrotum -Converted from testosterone in the urogenital sinus and labioscrotal swellings Estrogen -Essential for complete development of the Mllerian ducts and Wolffian ducts both in males and in females -Needed in males for fertility -Fetal ovarian estrogen Mllerian duct : uterus, oviducts, cervix -Germ plasm region Primordial germ cells -PGCs from extraembryonic mesodermal cells -Yolk sac serves as conduit in mammals Aromatase -Converts testosterone to estrogen