Cleavage, Blastula & Gastrulation in Invertebrates

Cleavage, Blastula & Gastrulation in Invertebrates

Chapters 8, 9

I. Overview of Early Development

•Events of Cleavage

•Yolk

•Cleavage Patterns

•Control of cell division

•Cytoskeleton in mitosis

•Gastrulation

•Axis formation

I A. Events of Cleavage

•Rapid rate of cell division (mitosis)

–Frog egg from zygote to 43,000 cells in 40 hours

•No overall growth of embryo

•Lack of cell growth between division

•Cells get progressively smaller

•Cytoplasmic volume to nuclear volume ratio decreases

–Sea urchin decreases 91 fold (550:1 to 6:1)

–Amphibian decreases 1,000 fold

•Reduction in cytoplasmic : nuclear volume ratio affects timing of nuclear events (transcription)

–Xenopus, transcription occurs after 12 divisions (midblastula transition)

–If double chromatin, transcription occurs after 11 divisions

•Biochemical events

–Increased DNA synthesis

–Increased protein synthesis

–Not much initial RNA synthesis

•Biochemical events

–Actinomysin D (transcription block) does not block cleavage

•Conclusion?

–Puromycin (translation block) does block cleavage

•Conclusion?

I B. Yolk

Areas of egg

•Animal pole/hemisphere

–Low yolk concentration

–Where polar bodies given off

•Vegetal pole/hemisphere

–Higher yolk concentration

–Orients to bottom (down), as yolk dense

Amounts of yolk

•Microlecithal

–Very small amounts of yolk

–Echinoderms, mammals

•Mesolecithal

–Moderate amount of yolk

–amphibians

•Macrolecithal

–Very large amounts of yolk

–Birds, reptiles, some fish

Distribution of yolk

•Isolecithal

–Evenly distributed throughout cytoplasm

–Echinoderms, mammals

•Telolecithal

–Concentrated at one end

–Amphibians, birds, reptiles, fish

•Centrolecithal

–Concentrated in middle

–Insects

I C. Cleavage Patterns

•Patterns affected by

–Cytoplasmic factors altering angle of mitotic spindle

–Amount and distribution of yolk

Cleavage of two main types

–Holoblastic

•Furrows completely through egg/embryo

•Microlecithal and mesolecithal

•Four main variants

–Radial

–Spiral

–Bilateral

–Rotational

Figure 8.4(1) Summary of the Main Patterns of Cleavage

Radial vs. Spiral Cleavage

Cleavage of two main types

–Meroblastic

•Furrows NOT completely through egg/embryo

•Macrolecithal

•Discoidal: one limited area of surface divides (telolecithal)

•Superficial: entire surface area divides (centrolecithal)

Figure 8.4(3) Summary of the Main Patterns of Cleavage

Superficial Cleavage

I D. Control of Cell Division

Cell Cycle

•Rapid division

•S and M, no G1 or G2

Mitosis-Promoting Factor (MPF)

•High in M

•Low in S

MPF has two subunits

•Cyclin B

•Cyclin-dependent kinase

•During M, cyclin B activates Kinase

•Kinase activates mitotic activity via phosphorylating target proteins:

–Histones

–Nuclear envelope lamin proteins

–Cytoplasmic myosin

•Leads to chromatin condensation

•These changes lead to

–Chromatin condensation

–Depolymerization of nuclear envelope

–Organization of spindle

•Cyclin degraded at end of M, cell shifts to S period as kinase inactive

•Proteins that regulate cyclin already present in egg cytoplasm

•At mid-blastula transition

–Stored cyclin regulators used up, new transcription and translation occurs (nuclear-dependent)

–G1 and G2 added to cell cycle

–New mRNA’s transcribed (necessary for gastrulation)

I E. Cytoskeleton in Mitosis

Two major events

•Karyokinesis

•Cytokinesis

Karyokinesis

•Microtubule-based (tubulin subunits)

•Inhibited by colchicine, nocodazole

Cytokinesis

•Microfilament-based (actin), at plasma membrane

•Inhibited by cytochalasin B

I F. Gastrulation

•Rearrangement of cells into layers

•Endoderm and mesoderm brought inside

•Ectoderm spreads over surface

•Five major mechanisms for cell movement

•Invagination: infolding

•Involution

•Inward movement of expanding outer layer

•Cells “roll over” edge and spread inward

•Ingression

•Migration of individual cells

•Delamination

•Splitting of sheet into 2 or more layers

•Epiboly

•Movement of sheet as unit over surface

•Encloses deeper layers

I G. Axis Formation

3 axes

•Anterior/posterior

•Dosal/ventral

•Left/right

Figure 8.6 Axes of a Bilaterally Symmetrical Animal

II. Early Sea Urchin Development

•Cleavage

•Gastrulation

II A. Cleavage

Early cleavage is radial holoblastic

•1st meridional

•2nd meridional, perpendicular to 1st cleavage

•3rd equatorial

•4th

–Animal tier meridional  mesomeres

–Vegetal equatorial but unequal  upper macromeres & lower micromeres

Figure 8.7 Cleavage in the Sea Urchin

•First two cleavages

Further in cleavage

•Blastula at 128 cell stage (7 cleavages), with large central blastocoel

•Cells pretty much same size

•Tight junctions hold cells together

After 9-10 cleavages, get mid-blastula transition

•New mRNA synthesis

•Synchrony of cell division lost

•Cilia form

Later, cells at vegetal pole thicken as vegetal plate

Blastomere determination

•60 cell embryo, cell fate can still be altered

•If cells remain in place, fate predictable

•Fate Map

–Animal pole  ectoderm

–Veg1  ectoderm or endoderm

–Veg2  endoderm, coelom, secondary mesenchyme

–Large micromeres  primary mesenchyme (skeleton)

–Small micromeres  mesoderm (coelom)

Figure 8.9(1) Fate Map And Cell Lineage of the Sea Urchin

Figure 8.9(2) Fate Map And Cell Lineage of the Sea Urchin

Micromere fate already determined, unlike rest of cells of blastula

•Isolate micromeres  skeletal spicules form

•Transplant micromeres to animal pole of different embryo

–Get second site of gastrulation

–Animal pole cells converted to endoderm

Figure 8.10 Ability of the Micromeres to Induce a Secondary Axis in Sea Urchin Embryos

•Animal hemisphere alone Dauerblastula (animalized embryo), no mesoderm or endoderm

•Animal hemisphere plus micromeres  fairly normal embryo, animal hemisphere cells become endoderm

•Signal from micromeres is β-catenin, a transcription factor activated by Wnt pathway

•Presence of β-catenin specifies vegetal half structures

•Treat embryo with lithium chloride

–β-catenin accumulates in all cells

–Get only endoderm and mesoderm

–“exogastrula” develops

•Prevent β-catenin from entering nuclei

–Get only ectoderm

–Dauerblastula develops

Axis specification

•Animal/vegetal set up prior to fertilization

•Anterior/posterior set up prior to fertilization

•Dorsal/ventral and left/right specified after fertilization

•Vegetal plate thickens

•Primary mesenchyme cells (PMC) separate from vegetal plate

•PMC ingress into blastocoel

•Ingression due to changes in cell-cell adhesion

–Lose adhesion to hyaline layer

–Gain adhesion to proteins lining blastocoel (basal lamina)

II B. Gastrulation

Invagination

•Vegetal plate bends inward and extends ¼ to ½ way into blastocoel

•Opening is blastopore

•Cavity of invagination is archenteron

Figure 8.20(1) Invagination of the Vegetal Plate

Figure 8.20(2) Invagination of the Vegetal Plate

Invagination

•Gut tube thins out & extends towards opposite wall via convergent extension

–Cells mitose and thin out

Figure 8.22 Cell Rearrangement During the Extension of theArchenteron in Sea Urchin Embryos

Invagination

•Final extension of gut tube

–Secondary mesenchyme cells at archenteron tip send out filopodia

–Filopodia extend and touch/connect to specific region of outer wall

–Filopodia then shorten and pull archenteron

Figure 8.24 Mid-Gastrula Stage of Lytechinus pictus

III. Early Snail Development

•Cleavage

•Gastrulation

III A. Cleavage

•Spiral pattern

•Cleavage planes

–Rotate 90o

–NOT parallel or perpendicular to animal-vegetal axis

–Oblique

•Solid blastula (stereoblastula)

•Cleavages 1 and 2 nearly meridional, 4 large blastomeres (A, B, C, D)

•Cleavage 3 equatorial and unequal

–Small micromeres above

–Large macromeres below

–Micromeres sit in furrow between macromeres

Figure 8.25(1) Spiral Cleavage of the Mollusc Trochus

Figure 8.25(2) Spiral Cleavage of the Mollusc Trochus

Figure 8.26(1) Spiral Cleavage of the Snail Ilyanassa

Left-Right Control of cleavage planes

•Two alternatives

–Left (sinistral)

–Right (dextral)

•Controlled by cytoplasmic factors

Figure 8.27(1) Looking Down on the Animal Pole of (A) Left-Coiling and (B) Right-Coiling Snails

Figure 8.27(2) Looking Down on the Animal Pole of (A) Left-Coiling and (B) Right-Coiling Snails

Left-Right Control of cleavage planes

•Phenotype controlled by mother genotype

•Her genotype controls egg cytoplasmic factors that affect offspring cleavage orientation

•Dextral (D) dominant to sinistral (d)

•Individual’s phenotype NOT reflective of own genotype

•Individual’s phenotype reflective of MOTHER’s genotype

•Thus, dextral snails had DD or Dd mother

•Sinistral snails had dd mother

•Inject cytoplasm of DD female into eggs of dd females, offspring dextral, not sinistral

•Reciprocal cross did NOT produce same outcome

•Offspring phenotype reflection of maternal genotype (but not maternal phenotype)

•Example of maternal inheritance (non-mendelian)

Polar Lobe

•Ventral extension from cells of early embryo

•From zygote prior to 1st cleavage, ends up attached to CD cell after 1st cleavage, then resorbed

•Again from CD cell after 1st cleavage, ends up attached to D cell after 2nd cleavage, then resorbed

Figure 8.32(1) Polar Lobe Formation in Certain Mollusc Embryos

•E.B. Wilson removed polar lobe at 2 cell stage

•Embryo developed but lacked mesoderm

•Conclusion?

•Autonomous/Conditional?

III B. Gastrulation

•Solid blastula

•Micromeres at animal pole move via epiboly over surface

•Enclose lower and inner cells

Figure 8.35 Gastrulation in Crepidula

IV. Early Tunicate Development

•Cleavage

•Gastrulation

•Fate Maps & Cytoplasmic Rearrangement

IV A. Cleavage

•Bilateral, holoblastic

•Mirror-image sides (L and R)

•1st cleavage meridional, L and R halves

•2nd cleavage meridional, offset towards back

–Large anterior and small posterior cells

Figure 8.36 Bilateral Symmetry in the Egg of the Tunicate Styela partita

IV B. Gastrulation

•Invagination of endoderm

•Involution of mesoderm

•Epiboly of ectoderm

Figure 8.42 Gastrulation in the Tunicate

IV C. Fate Maps & Cytoplasmic Rearrangement

Following sperm penetration, cytoplasm rearranges

•Clear  ectoderm

•Slate gray  endoderm

•Yolk  chordamesoderm

•Yellow crescent  mesoderm of tail muscles

•Light gray  neural tube and notochord

Figure 8.37(1) Cytoplasmic Rearrangement in the Fertilized Egg of Styela partita

Figure 8.37(2) Cytoplasmic Rearrangement in the Fertilized Egg of Styela partita

•Macho-1 mRNA localized in vegetal pole

•Ends up in B4.1 blastomere (tail muscles)

•Add antisense RNA to unfertilized egg

•Embryos lack tail musculature, tails severely shortened

•Type of specification of cell fate?

Figure 8.39 Autonomous Specification by a Morphogenetic Factor

•β-catenin found in vegetal A4.1 and B4.1 blastomeres

•Transcription factor, necessary for endoderm specification

•Where else have we seen β-catenin specify endoderm?

V. Early Caenorhabditis elegans Development

•Cleavage

•Anterior-Posterior Axis Formation

V A. Cleavage in Caenorhabditis elegans

•Rotational holoblastic cleavage

•1st cleavage meridional, offset towards posterior pole

–Anterior AB (somatic)

–Posterior P1 (stem)

•1st cleavage, posterior to left

•2nd cleavage

–AB divides equatorial

–P1 divides meridional, gives off somatic (EMS) and stem cell

•Eventually 558 cells at hatching

•Can trace lineage of each cell

•Makes for good model system

V B. Anterior-Posterior axis Formation in Caenorhabditis elegans

Anterior-Posterior

•Egg has elongated axis (Ant/Post)

•However, determination of which is anterior and which is posterior is delayed

•Sperm entry determines posterior end

–Sperm centriole moves sperm nucleus to one end  posterior

–Spindle fibers position the PAR maternal proteins (PAR = partitioning)

Anterior-Posterior

•PAR proteins also localize P-granules

•P-granules evenly distributed prior to fertilization

–Ribonucleoprotein complexes

–Translation regulators

–Specify germ cells

Anterior-Posterior

•Migrate to posterior end to future P1 cell, then to P2 cell, P3, then P4 (future germ cell)

•Movement dependent upon microfilaments (blocked with Cytochalasin D)

•Movement NOT dependent upon microfilaments (not blocked with demecolchicine)

Figure 8.44(1) Segregation of the P-granules into the Germ LineLineage of the C. elegans Embryo

Figure 8.44(2) Segregation of the P-granules into the Germ LineLineage of the C. elegans Embryo

Figure 8.44(2) Segregation of the P-granules into the Germ LineLineage of the C. elegans Embryo

V. Early Drosophila Development

•Cleavage

•Gastrulation

•Body Axis Formation

V A. Drosophila Cleavage

•Superficial cleavage (no initial cellularization)

•Zygote nucleus in central portion of egg

•Divides 8 times  256 nuclei

•Nuclei migrate towards periphery, continue to divide

Early Cleavage Animation

Early Cleavage Time Lapse Photography

•During 9th division, 5 nuclei reach posterior end, form pole cells germ cells

•Remaining nuclei continue to divide at periphery, forming syncytial blastoderm

•After 13th division, cell membranes form and partition cytoplasm  cellular blastoderm

•Single layer of cells around central yolk

Figure 9.3(1) Formation of the Cellular Blastoderm in Drosophila

Figure 9.1 Laser Confocal Micrographs of Stained Chromatin ShowingSuperficial Cleavage in a Drosophila Embryo

V B. Drosophila Gastrulation

•Begins during mid-blastula transition

•Invagination on ventral surface (ventral furrow)

–Pinches off

–Forms tube  mesoderm

•Two pockets invaginate

–Anterior, fore and midgut

–Posterior, hindgut endoderm and pole cells

•Germ band (ectoderm and mesoderm) forms posterior to prosencephalon

•Posterior section divides into segments

•Larval segments correspond to adult segments

Figure 9.7 Comparison of Larval and Adult Segmentation in Drosophila

V C. Drosophila Body Axis Formation

•Anterior/Posterior and Dorsal/Ventral determined by position of egg within follicle cells (nurse cells)

•Nurse cells lay down morphogenetic gradients of mRNA

V C 1. Anterior/Posterior Axis

•Specification of segment fate determined by

–General anterior/posterior (bicoid, hunchback, nanos, caudal)

–Gradients activate Gap genes for major body regions

–Major body region activate pair rule genes (parasegments)

–Parasegments activate segment polarity (segments)

–Specific structures for each segment via homeotic genes

Figure 9.8(1) Model of Drosophila Anterior-Posterior Pattern Formation

•Maternal effect sets up gradients

•Gradients activate

•Gap genes, activate

•Pair rule genes (parasegments)

V C 1a. General Anterior-Posterior

•Due to maternal mRNA (and translated proteins)

•Anterior mRNA’s

–Bicoid concentrated anterior

–Hunchback throughout egg

•Posterior mRNA’s

–Nanos concentrated posterior

–Caudal throughout egg

•All 4 mRNA’s translated at fertilization

Bicoid

•Translated, get bicoid protein gradient

•Bicoid protein gradient has two effects

–Increased translation of hunchback mRNA at anterior end, and hunchback binds to DNA

–Inhibited translation of caudal mRNA at anterior end

•Result is gradient of hunchback protein with high end at anterior

•So bicoid mRNA from ovary results in bicoid protein gradient

•If bicoid not expressed

–No hunchback gradient

–Anterior end develops as posterior end (telson)

•Bicoid deficient embryos lack heads

•Adding bicoid mRNAto bicoid-deficient embryo allows normal development

Nanos

•Translated, get nanos protein gradient highest at posterior end

•Nanos protein inhibits hunchback mRNA translation in posterior end

–Nanos binds to 3’ UTR, deadenylates

•Absence of nanos yields embryo without abdomen

Terminal Gene Group

•Acron (anterior); telson (posterior)

•Torso gene mRNA deposited evenly in egg by follicle cells

•mRNA translated throughout, inactive protein in plasma membrane

•Follicle cells produce torso-like protein at anterior and posterior ends, activate specific genes

–Absence results in lack of acron and telson

Figure 9.12(1) Anterior-Posterior Pattern Generation by the DrosophilaMaternal Effect Genes

Figure 9.12(2) Anterior-Posterior Pattern Generation by the DrosophilaMaternal Effect Genes

V C 1b. Gap Genes (Major Body Regions)

•Activated by proteins of anterior/posterior axis (hunchback, caudal)

•Divides embryo into broad regions (several segments)

•Examples include Krüppel

V C 1c. Pair Rule Genes (Parasegments)

•Activated by products of Gap genes

•Subdivide broad regions into parasegments

–Posterior region of 1 segment + anterior region of next segment

•Transcription of these genes results in “zebra-like” pattern (7 stripes, 14 parasegments) where expressed and NOT expressed

•Eight known genes, each with unique pattern of expression (e.g., fushi tarazu)

V C 1d. Segment Polarity Genes (Segments)

•Gene expression for Anterior-Posterior, Gap genes, and Pair-rule genes occurs in syncytial blastoderm

•Segment polarity genes expressed once blastoderm cellularizes

•Segment polarity gene expression controlled by presence/absence of pair-rule gene products

V C 1e. Homeotic Genes (Segment-specific Structures)

•Specify specific structures for each segment

•Two gene complexes (Hom-C)

–Antennapedia: head and 1st two thoracic segments

–Bithorax complex: 3rd thoracic segment and posterior

•Initial expression influenced by gap genes and pair-rule genes

Figure 9.28 Homeotic Gene Expression in Drosophila

•Posterior homeotic genes inhibit effects of anterior genes in posterior segments

•If lose posterior gene, phenotypic effects of anterior gene extended posterior

•Ultrabithorax (Ubx) deletion mutant

–3rd thoracic segment converted into 2nd thoracic segment

–Get two second segments, two sets wings

Figure 9.29 A Four-winged Fruit Fly Constructed by Putting Together ThreeMutations in cis Regulators of the Ultrabithorax Gene

•Homeotic mutants known back to 1800’s!

•Named by William Bateson (1894)

•Convert one body segment into having structures of another segment (e.g., legs on head instead of antenna)

•Homeodomain proteins are transcription factors

•Contain 60 aa sequence (domain) that binds to DNA

•Can form homeobox region of DNA (180 bp for the 60 codons) where homeodomain binds

•Found in genes across the animal kingdom in determining anterior-posterior axis

V C 2. Dorsal-Ventral Axis

•Major effect due to protein from dorsal mRNA deposited in egg by follicle cells

•Translated about 90 minutes after fertilization

•Evenly spread throughout cytoplasm

•However, only in ventral cells is dorsal protein moved to nucleus

•In nucleus dorsal protein

–Stimulates production of ventral cell type genes

–Supresses dorsal cell type genes

•If dorsal protein NOT moved into nucleus, instead get

–Supression of genes for ventral type cells

–Activation of genes for dorsal type cells

Mutants

•Dorsal protein does not enter cells

– all cells dorsalize

•Dorsal protein enters all cells

– all cells ventralize

Last updated 17 March 2004

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