Model organisms in development Cell and embryology

A few have been studied extensively; each has advantages and disadvantages. Model Systems

Xenopus laevis: development is independent (in vitro), easy catch and observation but poor genetics. Model organisms: vertebrates (frog, mouse, zebrafish) Chick: available, surgical manipulation and in vitro culture but poor Model organisms: invertebrates (sea urchin, Drosophila, nematode) genetics. Mouse: surgical manipulation, good genetics, transgenic model, Identifying development genes mammalian but development is in utero . Drosophila: great genetics, great development (recent Nobel Prize to Textbook: Wolpert L, Beddington R, Jessell T, Lawrence P, Meyerowitz E, Smith J. (2007) Principles of Development. 3th ed. London: Oxford university press. Lewis, Nusslein-Volhard & Wiechaus). C. elegans: has less than 1000 cells and is transparent. Gilbert SF. (2003) Development Biology. 7th ed. Sunderland: Sinaure Sea Urchin : in vitro Associates Inc. 1 Arabidopsis thaliana: flowering plant. 2

Summary of the main patterns of cleavage

Lecithal

3 4

1 Model organisms: vertebrates

All vertebrate embryos undergo a similar pattern of development. 1) fertilization 2) Cleavage (cell number ↑, but total mass X) Fig. 2. 1 3) blastulation (blastcoel formation and three germ layers) 4) gastrulation (where ectoderm covers embryo, endoderm and The skeleton of a mouse embryo illustrates the mesoderm are inside), A-P axis (body plan), notochord vertebrate body plan formation, embryo affected by yolk in egg. In mammalian, yolk to small but have extra-embryonic structure of placenta for nutrition. 5) Phylotypic stage, at which they all more or less resemble each other an show the specific features of notochord, somites and neural tube. Fig. 2.2

5 6

The phylotypic stage Xenopus laevis: egg At the end of gastrulation all embryos appear to be similar (the (Amphibians) phylotypic stage). Structures that are common to the phylotypic stage of the Advantage: easy observation, fertilized, catch (sperm, egg), low infection vertebrates are: 1) the notochord (an early mesoderm structure along A/P axis), The egg is composed of an animal and a vegetal Animal 2) the somites (blocks of mesoderm on either side of notochord region, bo th covere d by v ite lline mem brane (ge l which form the muscles of the trunk & limbs), coat). Fig.2.4 3) the neural tube - ectoderm above notochord forms a tube (brain Meiosis is stopped at 1st division with apparent 1 polar and spinal cord). body (the 2nd polar body comes after fertilization). Box 2A Extra- After fertilization, the cortex (the layer below plasma embryo membrane) rotates to determine future dorsal region nic at a position opposite to the site of sperm entry . vegetal Vertebrate embryo tissue to through a phylotypic state, but differences in form before Fig. 2.3 gastrulation 7 8

2 Box 2A

Cleavage of a frog egg.

9 10

Early developmental stages of Xenopus laevis Xenopus laevis : fertilization and early growth

1. one sperm enters animal region (grow to embryo, plant pore to yolk) morula Blastula 2. completes meiosis 3. egg and sperm nuclei fuse 4. v ite lline mem brane lifts 5. yolk rotates down (15 minutes) 6. cortical rotation occurs (60 minutes). 囊胚 7. 1st cleavage occurs (90 mins) Animal / Vegetal (A/V) 8. Every 20 mins, one cleavage 2.5 hpfp 3.5 hpfp 5 hpfp 10 hpfp 9. 2nd cleavage (110 mins) A/V 90 degrees to 1st 10. 3rd cleavage (130 mins) equatorial (4 small animal and 4 large blastocoel - vegetal= 8 , it is blastomeres). 11. Continued cleavage → blastomeres ↓, cells at vegetal region hpf: hours post-fertilization large than those at the animal region.

11 12

3 Xenopus laevis: blastulation

The blastula (after 12 divisions) has radial symmetry. The marginal zone will become mesoderm and endoderm. Marginal zone, the belt of tissue around the equator , plays a crucial part in future development. Internalization of the mesoderm and endoderm starts at the blastopore.

Fig 2.3 Life cycle of the frog Xenopus laevis. In blastula stage, it is in the form of a hollow sphere with radial symmetry 13 14

Types of cell movement during gastrulation Xenopus laevis: gastrulation Gastrulation step: 1. Mesoderm and endoderm converge and begin to move inwards at dorsal lip of the blastopore. 2. Mesoderm and endoderm extend in along A/P axis. 3. Ectoderm spreads to cover embryo (epiboly). 4. Dorsal endoderm separates mesoderm from the space between the yolk cells, the archenteron (future gut). Do not forget, mesoderm come from ectoderm 5. Lateral mesoderm spread to cover inside of archenteron. 6. dorsal mesoderm is beneath dorsal ectoderm 7. mesoderm spread to cover gut 8. epiboly - ectoderm covers embryo 9. yolk cells are internalized (food source), dorsal mesoderm develops into a) notochord (rod along dorsal midline) and b) somites (segmented blocks of mesoderm along notochord). Invagination Blastopore Involution ↓ Ingression Archenteron ↓ Delamination Large Eiboly: ectoderm covers embryo ↓ Blastocoel ↓ Close ↓ 15 gut 16

4 Xenopus laevis: Neurulation

• Neuralation or neural tube formation: 1) The neural plate is the ectoderm located above notochord and somites. 2) The edge of the neural plate forms neural folds which rise towards midline. 3) The folds fuse to form neural tube. 4) The neural tube sinks below epidermis. • The anterior neural tube becomes brain. Mid and posterior neural tube becomes spinal cord.

Gastrulation → neurulation → neural plate → fold → tube

notochord Neural crest cell Anterior posterior Autonomic nerves ↓ ↓ 17 Brain spinal cord18

Fig. 2.7 Neurulation in amphibian Xenopus laevis: Somites The somites formation, after neurulation The dorsal part of somites have ready begun to differentiate into dermatome (future dermis). The rest of each somite becomes vertebrae and trunk muscles (and limbs). Lateral plate mesoderm becomes heart, kidney, gonads and gut muscles. VlVentral meso dbderm becomes bldblood-fiiforming tissues. Also at this stage, the endoderm gives rise to the lining of the gut, liver & lungs. Brain andspinal Fig. 2.8 A cross-section through a stage 22 Xenopus embryo just after gastrulation and neurlation are completed

Notochord begins to form in the midline Neural plate develops neural folds 19 20

5 The major lineages of the mesoderm Xenopus laevis: tail bud stage

• After gastrulation comes the early tail bud stage In the anterior embryo: a) the brain is divided, b) eyes and ears form, c) 3 branchial arches form (anterior arch later becomes the jaw.

In the posterior embryo, the tail is formed last from dorsal lip of blastopore by extension of notochord, somites and neural tube.

Circulatory body cavity Fig. 2. 9 The ear ly ta ilbu d system stage of Xenopus embryo Scler Myo tome Cartilage skeletal dermis

21 22

Schematic representation of neural crest formation Xenopus laevis : neural crest cells (in chick embryo)

Neural folds meet and adhere Neural crest cells come from the edges of the neural folds after neural tube fusion. Neural crest cells can form from the dorsal side of the Cells at this junction form neural crest closed neural tube Neural crest cells detach and migrate as single cells between the mesodermal tissues to become: 1) sensory and autonomic nervous systems 2) skull 3) pigment cells Closure not simultaneous 4) Cartilage → bone

Only vertebrate Cell adhesion molecular expressed dependent Closed tube detaches – change Epidermal and neural plate/tube interactions may generate crest cells in adhesion molecule 23 expression 24

6 Zebrafish ( Danio rerio) -- A Vertebrate Model

•It is 3 cm long

•Short generation time

•Large clutch size

•External fertilization

•Transparent embryos

•Rapid development

http://zfin.org/ and http://www.nih.gov/science/models/zebrafish/ 25 26

Sphere

29h

48h 27 28

7 Fish (Zebrafish) embryo: •Human disease model •Transgenics •Reverse genetics tool

Fig. 2.26

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The development of Zebrafish Characterization of Fish embryo

Telolecithal: most of the egg cell is occupied by yolk Zebrafish Meroblastic: the cell divisions not completely divide the egg development occurs Discoidal: since only the blastodisc becomes the embryo, this type of very rapidly. In 24 hr meroblastic cleavage is call discoidal. hours of embryogenesis, Cleavage can take place only in the blastodisc, a thin region of yolk free shown here, the 1 cytoplasm at the animal pole of the egg. cell zygote becomes into a vertebrate embryo with a tadpole-like form.

Fig. 2.27 Cleavage of the zebrafish embryo 31 32

8 Fish embryo: blastula stage Three cell populations: At about the 10th cell division -- the onset of the About 10 cell division, the onset of mid-blastula transition: gene transcription MBT begins, divisions slow and cell move. And formed three distinct cell mid-blastula transition populations: 1. Yolk syncytial layer (YSL) (1)YSL (yolk syncytial layer): location of vegetal edge of the blastoderm and 2. Deep cells -- forming the embryos proper fusion produces a ring of nuclei within the part of the yolk cell cytoplasm 3. Envel ope l ayers (EVL) -- fithidlforming the epidermal that just beneath the blastoderm. It is important for directing some of the cell movement of gastrulation. ANIMAL BODY Internal YSL: the yolk syncytial nuclei move under the blastoderm External YSL: some cell move vegetally, stay ahead of the blastoderm margin (2)Enveloping layer (EVL): Made up of the most superficial cell from the blastoderm, which form an epithelial sheet a single cell layer thick.

(3) Deep cells Blastoderm Both YSL and EVL are the deep cells, that give rise to the embryo 4 hpf: hours post-fertilization 33 proper. 34

Fish embryo: gastrulation The fate map of the deep cells after mixing has stopped

The blastoderm at 30% completion of Internal epiboly (4.8 hr) YSL

This stage, no mesoderm, ectoderm

The fate of the early blastoderm cells are not determined. After much cell mixing during cleavage

35 36

9 Types of cell movement during gastrulation

Close-up of the marginal region Formation of the hypoblast, either by involution of cells at the Invagination marggpgin of the epibolizing Involution balstoderm or by Ingression delamination and Delamination ingression of cells from Eiboly: ectoderm covers embryo the epiblast (6hr) The formation of germ layers is started. 37 38

About 90% epiboly (9 hr), mesoderm Types of cell movement during gastrulation can be seen surrounding the yolk, between the endoderm and ectoderm

Complete gastrulation (10.3hr)

Invagination IltiInvolution Ingression Delamination Eiboly: ectoderm covers embryo

39 40

10 Fish embryo: gastrulation

Fig 2.28 Epiboly and gastrulation in the zebrafish

Mesodermal cell Convergence and extension in the gastrula. After fertilization → cell cleavage → spreading out of the layer of cell (d(expressed snail gene) (epiboly) → upper half of the yolk become covered by a cup-shaped flank the notochord blastoderm→ gastrulation by involution of cell → fromed a ring around (A) Dorsal view of convergence and externsion movements during gastrulation. Epiboly the edge of the blastoderm → involuting cell converge on the dorsal spreads the blastoderm over the yolk; involution or ingression generates the midline to form the body of the embryo hypoblast; convergence and extension bring the hypoblast and epiblast cells to the dorsal side to form the embryonic shield. (B) Convergent extension of the embryo; it is show by cells expression the gene no tail 41 (a gene is expressed by notochord cells) 42

Types of cell movement during gastrulation

Invagination Involution Ingression Delamination Eiboly: ectoderm covers embryo

43 44

11 Chick (bird) embryo: the blastodisc (blastoderm) Chicken The blastodisc arises through cleavage (20 hrs.). The blastodisc can be divided into two areas: 1) the area pellucida (a light area) surrounded by 2) the area opaca (a dark ring).

犁溝

yolk

45 Fig. 2.10 46

The life cycle of the chicken (Fig.2.11)

47 48

12 Chick (bird) embryo: the blastodisc (blastoderm) Discoidal meroblastic cleavage in a chick egg The posterior marginal zone forms at the junction of the area pellucida and the area opaca and defines the dorsal side and posterior end of the embryo.

The hypoblast (the source of extra- Germinal embryonic tissues) develops as a layer on top of yolk and develops opaca pellucida opaca from cells from the posterior marginal layer and the overlying cells of the blastoderm. It come from two sources: the posterior marginal zone, which lies at the ectoderm junction between the opaca and pellucida at the posterior of the endoderm embryo. It develop to extra- embryonic structure and related with epiblast. Fig. 2.12 49 50

Primitive streak Types of cell movement during gastrulation

Formation of two-layered blastoderm of the chick embryo Germinal (A,B) Primary hypoblast cells delaminate individually to form islands of cell beneath the epiblast (C) Secondary hypoblast cells from posterior margin → migrate beneath the epiblast and incorporated the poly- Invagination invagination islands → move Involution anterior; Ingression As the hypoblast moves Delamination anteriorly → epiblast cell Eiboly: ectoderm covers embryo collect at the region anterior to Koller’s sickle to form the primitive streak 51 52

13 Chick embryo: the primitive streak Chick embryo: the primitive streak

The primitive streak is a slit or line on the disc which lays down the A/P axis. (posterior) When the primitive streak reaches its greatest length (forward), the anterior end begins to regress back to the posterior end. Onset of gastrulation This structure begins to form from the posterior marginal zone and Primitive streak form at posterior → forward formation → enough extends to a point in the central region of the disc. length close and regress → Hensen’ s node → backward Cells move towards the streak, and mesoderm and endoderm regression → formation of head, somites and notochord… (Fig. internalize at this site. 2.14) The anterior end of the regressing streak is known as Hensen's Unlike amplibians, cell Node. not only proliferation but also growth in size, during gastrulation in bird and mammals.

Primitive streak 53 54

Cell movement of the primitive streak of the The major lineages of the mesoderm chick embryo

Head, somite

Circulatory body cavity system

Scler Myo tome Cartilage skeletal dermis

55 56

14 Chick embryo: gastrulation

As Hensen's Node moves toward the posterior, several structures form behind it: 1) The head fold (from ectoderm and endoderm) 2) The notochord and somites (from mesoderm) 3) The neural tube forms above the notochord (from ectoderm) (The anterior structures are formed first while the posterior structures are completed last.) 4) Neural folds fuse at the dorsal midline and neural crest cells migrate away 5) The head fold separate, gut forms and heart pieces fuse to form heart.

57 58

Chick embryo: neurulation Fig.2.18 Development of the chick embryo

notochord

Neural plate → neural fold → meet midline

Intermediate somites mesoderm→ kidney Splanchnic mesoderm → heat Somite star formation

13 somites

20 somites 40 somites Hensen’s node

59 60

15 Mouse embryo Chick embryo: extra-embryonic structure

Amnion and amniotic cavity provide mechanical protection Chorion maintain shell Allantois bridge for oxygen and waste Vitelline vein take nutrient form yolk to embryo Umbilical vein take oxygen to embryo Fig.2.20

Egg is small, 100mm very small Egg surrounded by protective external coat, zona pellucida 61 62

Development of a human embryo form fertilization to implantation Mouse embryo: fertilization

Fertilization occurs in oviduct. (Fig. 11.26) Cleavage occurs in oviduct: 1st at 24 hours and every 12 hours after that to form the morula (a ball of cells). (Fig. 2.21) • Blastomere compaction happens at 8 cell stage. • Smooth inner membranes and outer membranes are covered with microvilli.

(c) Morula. After further cleavage (b) Four-cell stage. Remnants of the divisions, the embryo is a mitotic spindle can be seen multicellular ball that is still between the two cells that have surrounded by the fertilization just completed the second envelope. The blastocoel cavity cleavage division. has begun to form. 63 64

16 Mouse embryo: In 16 cell morula →

• Cleavage partitions the cytoplasm of one large cell At ~16 cell morula, has two group cells. A small group of internal cell – Into many smaller cells called blastomeres mass (ICM) surrounded by a large group of external (trophectoderm) cells. Trophectoderm: becomes extra-embryonic tissues (such as placenta). Inner cell mass (ICM): becomes the embryo plus some extra- embryonic tissues. The morula (~32 cell stage) has 2 cell fates: 1) inner 8 cells (Inner Cell Mass) 2) outer ~20 cells (trophectoderm).

blastocyst

(a) Fertilized egg. Shown here is the (b) Four-cell stage. Remnants of the (c) Morula. After further cleavage (d) Blastula. A single layer of cells zygote shortly before the first mitotic spindle can be seen divisions, the embryo is a surrounds a large blastocoel cleavage division, surrounded between the two cells that have multicellular ball that is still cavity. Although not visible here, by the fertilization envelope. just completed the second surrounded by the fertilization the fertilization envelope is still The nucleus is visible in the cleavage division. envelope. The blastocoel cavity present; the embryo will soon center. has begun to form. hatch from it and begin swimming.

65 66

Mouse embryo: blastocyst Mouse embryo: post-implantation In the blastocyst (~3½ days), the trophectoderm and ICM are established. Fluid is pumped in to expand cavity and increase the size of the blastocyst. Uterine wall blastocyst: preimplantation (3½ - 4½ days) The surface of ICM will become the primitive endoderm while the remaining becomes primitive ectoderm (= epiblast). Implantation occurs. The zona pellucida is discarded and blastocyst hypoblast attaches to uterine wall. Development of a human embryo form fertilization to implantation

Implantation → trophoblast giant cell invade → trophoectoderm grows → ectoplacental cone & extra-embryonic ectoderm → primitive endoderm cover inner surface of trophectoderm → to visceral endoderm • In the first two days post-implantation, the mural trophectoderm (cells that are not in contact with the ECM) gives rise to polyploid trophoblast giant cells. • The rest of trophectoderm becomes the ectoplacental cone and the extra-embryonic ectoderm which give rise to the placenta. • Primitive mesoderm migrates: 1) to cover inner surface of mural trophectoderm to become the parietal (腔壁) endoderm and 2) to cover egg cylinder and epiblast to become the viseral endoderm 67 • Six days after fertilization, the epiblast is cup-shaped. 68

17 Mouse embryo: gastrulation

6½ days after fertilization: The primitive streak forms at the start of gastrulation at the future posterior end. (Inside cup is future dorsal side) Cells move through the streak and spread forward and laterally between the ectoderm and the visceral endoderm to form the mesoderm. Later, the definitive endoderm (from epiblast) will replace the visceral endoderm. The primitive steak first elongates, then at the anterior tip of the primitive streak, the node forms. (The node formed from anterior → posterior) Then notochord and somites form anterior to the node (A/P axis). Cells migrate through mesoderm to form endoderm (gut).

Epiblast move through the primitive streak to give rise to the mesoderm and definitive endoderm.

69 70 Amnion Chorion Allantois Fig. 2.23

Mouse embryo: late embryogenesis (neurulation) Mouse embryo: final stages of gastrulation

• By 8½ days after fertilization, 1) the neural folds form at anterior and dorsal, and 1. Complex folding 2) the embryonic endoderm internalizes to form the gut. 2. Initially on the ventral surface of embryo • 9 days after fertilization embryogenesis is complete. 3. Internalize to form the gut 4. Heat and liver move into their positions 5. Head becomes distinct Fig. 2.24 6. Embryo surrounded by extra-embryonic membrane

A P

D Amnion Chorion Fig. 2.25 Primitive streak extend→ produce Organogenesis in the anterior part extra-embryonic structure Neural folds formation Allantois →chorion, amino, allantois The primitive streak similar to chick (node = Hensen’s node) 71 72

18 Diagram showing the timing of human monozygotic twinning with relation to Formation of the notochord in the mouse extra-embryonic membrane

Amnion Chorion Allantois 73 74

Model organism: invertebrate Drosophila melanogaster: early embryogenesis

The Drosophila egg is the shape of a sausage . Meroblastic (superficial) cleavage and centrolecithal It has a micropyle at the anterior end (site of sperm entry). With fertilization, the fusion of nuclei is followed by rapid mitotic divisions (9 minutes) and no cytoplasmic cleavage. A syncytium is formed (many nuclei/common cytoplasm). After nine divisions, nuclei move to the periphery to form the syncytial blastoderm .

Fig. 2.30 After fertilization, no cell was form, but rapid nuclear Life cycle of Drosophila Fig. 2.29 75 division in a cytoplasm 76

19 Box 2A Drosophila: embryogenesis

By 13 mitoses the membranes sprout to surround the nuclei to form cells (cellular blastoderm). ~15 cells at posterior (= pole cells) are sequestered and become the germline. During first ~3 hrs large molecules such as proteins can move between nuclei until the cellularization occurs. Single layer of cells give rise to all tissues (syncytium ). Gastrulation starts at ~3 hrs. Mesoderm forms from ventral tissue, midgut from endoderm at the anterior and posterior ends, ectoderm remains on outside. During gastrulation, the ventral blastoderm (germ band), comprises extension. The mesodermal tube forms from ventral tissue then cells separate and move to internal locations under the ectoderm.

77 78

Drosophila melanogaster: gastrulation

The mesoderm becomes muscle and connective tissues. In insects, nerve cord lies ventrally (vertebrates: dorsal). Neuroblasts form a layer between mesoderm and outer ectoderm. midgut (anterior & posterior) grow from threads and fuse. = anterior and posterior midgut ectoderm becomes epidermis. No cell division occurs during gastrulation. Afterward, division restarts. Future mesoderm invaginate ventral region → intrnalized tube → cell leave tube and migrate under the ectoderm The surface of ventral blastoderm → cell leave and form a layer between ventral ectoderm and mesoderm

Anterior and posterior invaginate and fuse → gut Midgut →region endoderm Foregut and hindgut → ectodermal origin 79 80

20 Future mesoderm Fig. 2.31 Gastrulation Ventral view invaginate ventral region → Dorsal view internalized tube → cell leave tube and migrate under the ectoderm germline

The surface of ventral blastoderm → cell leave and form a layer between ventral ectoderm and mesoderm →nervous system

Anterior and posterior invaginate and fuse → gut Midgut →region endoderm Foregut and hindgut → ectodermal origin 81 82

Drosophila melanogaster: segmentation

The germ band (ventral blastoderm) is main trunk region. Drosophila melanogaster: larvae Germ band extension pushes posterior end over dorsal side. The first signs of segmentation grooves appear to outline The larvae hatch at 24 hrs post-fertilization. parasegments (early embryo) which give rise to segments (late embryo). Larval structures of note include: Segments are formed from the posterior of one parasegment and the The anterior end is the acron. anterior of the next. (formed form posterior to anterior) The posterior end is the telson. Along with the head, the larvae has 3 thoractic segments and 8 abdominal segments. The ventral side of the larvae has denticle belts, alternating patches of denticle hairs and cuticle on each segment , used for Fig. 2.32 locomotion.

There are 14 parasegments: Fig. 2.33 3 mouth, 3 thorax, 8 abdominal. 83 84

21 Drosophila melanogaster: metamorphosis imaginal discs Three instar stages of larval life are separated by molts. • 1st instar 2nd instar 3rd instar molt molt 3rd instar larvae forms pupae (pupa) to undergo metamorphosis. The adult tissues arise from imaginal discs and histoblasts. imaginal discs: small sheets of epidermis (~40 cells each of cellular blastoderm) which grow throughout larval life. Imaginal discs: 6 leg, 2 wing, 2 haltere, 2 eye-antenna, plus genital, head discs histoblasts and ~10 histoblasts: nest of cells in the abdomen which give rise to the abdominal segments.

Larval epidermis degeneration begins prior to imaginal disc eversion Imaginal disc cells and histoblasts will replace the larval epidermis Antenna Formation of adult abdominal segments - gene expression in haltere histoblasts Fig. 2.34 Imaginal discs vs. adult structure Genitalia Imaginal discs 85 86

Caenorhabditis elegans: the model of nematode

THE WORM

After gastrulation

In case of self-fertilization there are ~ 0.1 - 0.3% male worms in the population. Fig. 2.35 Life cycle of nematode http://www.wormatlas.org/handbook/contents.htm 87 88

22 the model of nematode Press Release: The 2002 Nobel Prize in Physiology or Medicine 7 October 2002 The Nobel Assembly at Karolinska Institutet has today decided to award Small nematodes that are 1 mm long and 70 µm in diameter. The Nobel Prize in Physiology or Medicine for 2002 jointly to 19,000 gene Sydney Brenner, H. Robert Horvitz and John E. Sulston Small number of cell (558, first larval stage) TfbdthidTransparency of embryo, and growth rapid for their discoveries concerning The adult hermaphrodite (maless can develop) undergo rapid "genetic regulation of organ development and programmed cell development. death" The egg has a 50 µm diameter which forms a polar body after fertilization, nuclear fusion occurs followed by a set pattern of cleavage. The normal pattern of cell division has been mapped. Many cells undergo programmed cell death.

Hermaphrodite: 959 cells from 1090 somatic nuclei of which 131 undergo programmed cell death; 300 germ cells undergo apoptosis; 116 of the 131 dying cells are cells of the nervous 1927 1947 1942 system and ectoderm 89 90

Molecular Regulation of Apoptosis C. elegans mutagenize Non- apoptotic apoptotic

wildtype CED mutants (Cell Death abnormality)

91 92

23 Fig. 2.36 Cleavage of the nematode embryo

Fertilization →polar bodies formation → asymmetric cleavage → anterior AB cell, smaller posterior P1 cell

DIC image

Fig. 2.38 elegans larva at the L1 stage.

Fig.2.37 Anus Cell lineage and cell fate Pharynx in the early nematode Primordium embryo

93 94

Invertebrate: Sea Urchin Sea Urchin: blastula formation

Radial holoblastic cleavage (isolecithal) The blastula stage of sea urchin development begins at the 128 cells. The 4th cleavage, very different from the first three. In animal pole, four cell Blastulation: The cells form a hollow sphere surrounding a central cavity divide to 8 blastomeres and with the same volume (the 8 cells also called (blastocoel). Every cell contact with proteinaceous fluid of the bastoceol mesomeres). In vegetal pole, undergoes an unequal cleavage to four large (inside) and with the hyaline layer on the outside. cells (macromeres) and four small cells (micromeres). About 9th or 10th cleavage, cells become specified and they end develop cilia. Ciliated blastula → rotate within fertilization envelop (E→F) → vegetal pole of The animal mesomeres divide Bastula become thicken equatorially to produced (forming vegetal plate) → two tiers: an1 and an2. then animal pole synthesis The vegetal macromeres and secret hatching divide a small cluster enzyme → digest beneath the large tier. (not fertilization envelope → equal) embryo is a free swimming 4th hatched blastula. cleavage 128 cells blastula.

Meridionally

rotate 95 96

24 Fate maps and the determination of sea urchin blastomeres

Fate map of the zygote Late blastula with ciliary tuft and flattened vegetal plate

blastula

Fate map and cell lineage of the sea urchin.

97 98

Formation of syncytial cables by primary mesenchyme cells of sea urchin

SEM of spicules formed by the fusing of primary mesenchyme cells into syyyncytial cables

Gastrulation star C: SEM of primary mesenchyme cells enmeshed in the extracellular matrix of earlyyg gastrula. D: Gastrula-stage mesenchyme cell migration Prism-stage larva Pluteus larva The extracellular matrix fibrils of the bastocoel lie parallel to the animal-vegetal axix 99 100

25 Ingression of primary mesenchyme cells Invagination of the vegetal plate

CSPG release → into inner lamina → osmotic gradient ↑→ absorb water → swell inner lamina SEM of external surface ,but outer lamina attached does not swell → ofthf the earl y gas trul a inward

Fertilization envelope

101 CSPG: chondroitin sulfate proteoglycan 102

Entire sequence of Identification of developmentally important genes gastrulation in sea urchin The developmental genetics of Drosophila and mice are best known. Homologous genes identified in these organisms are found in other species. Dominant (or semi-dominant) mutations: one copy of mutant gene produces mutant state. These are more easily recoginzed, they don’t cause the eayly death of the embryo in the heterozygous. Recessive mutations: two copies of a mutant gene gives the mutant state.

Allele: The gene is contributed by the male and female Homozygous: both alleles of a pair carry the mutation Heterozygous: just one copy of the mutant gene is present

103 104

26 Recessive mutation vs. Semi-dominant mutation

-/-

Most mutations are recessive, but usually die in embryo. 105 106

Developmental gene can be identified by induced mutation and screening

Genetic screening to produced homozygous mutant zerbrafish embryo

Heterozygous

Embryos homozygou s the induced mutation will heterozygous be found in the offspring of 25% of the matings 107 108

27 Mutagenesis and genetic screening strategy for identifying developmental mutants in Dorsophila main patterns of cleavage

phylotypic stage DTS: dominant temperature- sensitive mutation, up 29oC Time vs. developmental events → death b: a non-developmental lethal TflltditltiTypes of cell movement during gastrulation recessive Primitive streak

gastrulation

Neurulation ethyl methane sulfonate human monozygotic twinning

Syncytium

imaginal discs and histoblasts

109 Dominant (or semi-dominant) mutations 110

28