Bio 127 Section III Late Development Germ Line

Bio 127 - Section III Late Development Germ Line Development Gilbert 9 e – Chapter 16

Section 4 Encompasses : • • Development of the Tetrapod Limb Sex Determination The Saga of the Germ Line Post-Embryonic Development

Student Learning Objectives 1. You should understand that sexual reproduction requiring the fusion of gametes from male and female gonads occurs in specific organisms. 2. You should understand that the primordial germ cells that give rise to gametes arise outside of the gonads and must migrate to them. 3. You should understand that in most organisms the primordial germ cells are specified conditionally, while in some they are specified autonomously by cytoplasmic determinants in the egg. 4. You should understand that migration of the germ cells from their site of origin to the gonads is an essential part of reproductive success.

• In all plants and some animals, somatic cells can readily form new organisms – Cnidarians, flatworms, tunicates • In many animals, there is an early division between somatic and germ cells – Insects, roundworms, vertebrates

• Two step process; – Primordial germ cells (PGCs) are determined in a specific location in the embryo – PGCs migrate to the gonad and become the progenitor population for eggs and sperm

Two Methods of Germ Cell Determination • Autonomous Specification – Egg cytoplasmic determinants – Called ‘Germ Plasm’ – Nematodes, flies, frogs • Conditional Specification – Signals from surrounding cells – Majority of sexually reproducing organisms – Including mammals

The nematode Caenorhabditis elegans Remember cleavage and gastrulation: Asymmetrical divisions produce a stem cell (P-lineage), “founder” cell. Stem cell divisions are meridional Founder cell divisions are equatorial

Gastrulation in C. elegans

P-granules hold cytoplasmic determinants in C. elegans PIE-1: Blocks all transcription, thus all differentiation Germ plasm also has blocks to translation, stem cell factors, controls for asymmetric divisions and meiosis inducing agents. P 4 Blue is DNA marker, Green is P-granule marker

Blue stain marks transcriptional activity P 4

Synctitial cleavage in Drosophila is followed by cellularization

Pole plasm forms during cellularization - mitochondria - fibrils - polar granules no transcription no translation germline stabilization anterior posterior

Localization of germ cell-less (gcl) gene products Human males with mutant homolog are often sterile

Germ plasm at the vegetal pole of frog embryos Marker for frog homolog of fly/worm translation blocker, Nanos

• The frog cells that take up these granules will become PGCs and migrate to the gonads as the kidney forms – Again, no transcription or translation – Therefore, no differentiation

Conditional Specification of mammalian PGCs • Posterior of epiblast at the junction of the primitive streak and extraembryonic ectoderm – Cells are no different from other epiblast – Specified in gastrulation before 3 layers form – Wnts from endoderm make them competent – BMPs from extraembryo ectoderm finish it

Picture the blastocyst full of yolk. . .

Poor old Henson discovered this node as well but didn’t get the naming rights

Conditional Specification of mammalian PGCs • Same deal as the others: – Repress differentiation by repressing gene expression • Specified outside embryo forming cells – Once expression is shut down they can go back into embryo and not respond to signals

Germ Cell Migration • • • Drosophila Zebrafish Frogs Mice Birds and Reptiles

Germ Cell Migration: Drosophila As the endoderm invaginates, the ectoderm and mesoderm extend and converge to wrap around the dorsal side to form the “germ band”

Germ Cell Migration: Drosophila - mitochondria - fibrils - polar granules no transcription no translation germline stabilization anterior posterior

Germ Cell Migration: Drosophila Germ cells passively ride endoderm

Germ Cell Migration: Drosophila Endoderm expresses repellent molecules Germ band is retracting PGCs and gonad progenitors in 2 migration streams

Germ Cell Migration: Drosophila Combination of chemoattraction and repulsion drive them to gonad E-cadherin MET forms epithelium around PGCs

Germ Cell Migration: Drosophila • Both mesoderm and PGCs divide through the larval stage, differentiate at metamorphosis • At larval-pupal transition anterior PGCs in gonad become germ line stem cells • In ovaries, the cells attach top stromal cap testes, the cells attach to hub cells In

Remember: Zebrafish development occurs very rapidly 24 -hours from 1 cell to vertebrate embryo!

Germ Cell Migration: Zebrafish specification: germ plasm determination: PGCs by 32 -cells four clusters join into two migration of bilateral clusters into developing gonad follows signal Sdf-1 using receptor CXCR 4

Remember: Germ plasm at vegetal pole in frogs Marker for frog homolog of fly/worm translation blocker, Nanos

Germ Cell Migration: Frogs During cleavage the germ plasm rises up until it ends up in the endoderm at top and back near lip

Germ Cell Migration: Frogs The endoderm below mesdoderm are PGCs

Germ Cell Migration: Frogs Migration anterior to gonads at endoderm-mesoderm boundary ~30 PGCs reach gonads by fibronectin and Sdf-1

• Remember Sdf-1 • Soluble signal whose receptor is CXCR 4 • Common signal for vertebrate germ cells • Also used by humans to call HSC to bone marrow, guide lymphocytes, MSC?

Germ Cell Migration: Mice PGCs formed in extraembryonic epiblast 10 -100 cells @ Day 6. 5 in mice

Germ Cell Migration: Mice Once formed, they migrate directly into the hindgut endoderm and migrate anteriorly through Day 9 dividing the entire time They leave the gut by the dorsal mesentary and enter the genital ridges by Day 12 as 2500 -5000 PGCs.

Germ Cell Migration: Mice • The travelling stem cell niche – Support cells travel with PGCs to maintain the undifferentiated stem cell phenotype – They secrete stem cell factor (SCF) – The cells follow fibronectin trail – Sdf-1 also required

Germ Cell Migration: Birds and Reptiles Germ line cells determined in the area pellucida, migrate to hypoblast Migrate to gonads via blood stream when extraembryonic vessels form

Germ Cell Migration: Birds and Reptiles Sdf-1 from intermediate mesoderm draws them out of vessels and through the mesodermal tissues to the gonad

Bio 127 - Section III Late Development Post-Embryonic Development Gilbert 9 e – Chapter 15

Section 4 Encompasses : • • Development of the Tetrapod Limb Sex Determination The Saga of the Germ Line Post-Embryonic Development

Student Learning Objectives 1. You should understand that development never stops during the life of the organism and that three major processes occur in the postembryonic animal: metamorphosis, regeneration and aging. 2. You should understand the Direct Development involves young organisms with the same body plan as the adult; whereas Indirect Development involves major changes to form the adult body plan. 3. Indirect Development, or metamorphosis, is hormonal reactivation of 4. You should understand that regeneration is the reactivation of developmental process to restore missing tissues. 5. You should understand that aging and physiological senescence are an interplay of genetic and environmental influences.

Metamorphosis • Development of a larval stage and an adult stage specialized for different functions – Larvae often specialized for growth, dispersal, etc. – Adults usually specialized for reproduction – Example Cecropia moths: • Larvae are wingless eating machines • Adults have one day to mate – don’t even have mouth parts! • Two major types of larvae – Primary: little to no similarity to adult (sea urchins) – Secondary: add and subtract parts from similar form • (insects, amphibians)

Metamorphosis: Sea Urchins Pluteus Larvae

Metamorphosis: Sea Urchins Primary Larvae: No trace of adult morphology

Metamorphosis: Amphibians • Hormone(s): T 3 and T 4 • Four Major Morphological Processes – Growth of new structures – Cell death in existing structures – Remodeling of existing structures – Biochemical respecification • Shift in the genes expressed and the physiological functions they control

Metamorphosis: Amphibians Tadpole eyes are on the sides of the head, frog eyes are on the front and top Binocular Vision New neurons differentiate and form new ipsilateral tracts

Metamorphosis: Amphibians • Cell death in existing structures – Apoptosis • T 3 induces apoptosis in tail and gills • Apoptosis occurs in gut epithelium due to ECM loss – Phagocytosis • Macrophages finish off the cells of the tail • Also destroy larval RBCs to make fresh ones with the adult hemoglobin protein

Metamorphosis: Amphibians Remodeling: - Eyes - Skull - Skeleton - Gut - Sensory

Metamorphosis: Amphibians Biochemical Respecification NH 3 = ammonia, amino group NH 4+ = ammonium ion T 3 causes a shift in transcription factor expression that upregulates these genes.

Metamorphosis: Amphibians 2 NH 3 + CO 2 + H 2 O (urea)

Actions of thyroxine (T 4) and tri-iodothyronine (T 3) Thyroid receptors are found in the nucleus where they are bound to inactive promoters. When thyroid hormone enters such a cell, it will bind to receptor and the combination is an active transcription factor for that specific gene. Cells that make high levels of deiodinase II are more responsive to thyroid stimulation. Cells that make high levels of deiodinase III are less responsive

Actions of thyroxine (T 4) and tri-iodothyronine (T 3) • What genes you have your thyroid receptors on is very important to your function – Limb muscle cells grow in response to thyroid hormones – Tail muscle apoptoses in response to thyroid hormones • How much deiodinase II you make is very important – Limb buds make a lot and respond to early low levels of T 4 – Tails make very little and wait for later very high levels of T 4 – This is good! • How a tissue is organized before T 4 is very important – Thyroid hormones make skin apoptose – Head and body have basal stem cells, tail does not

• Your tail degenerates during week 4 of gestation in much the same fashion as the frogs!

Metamorphosis: Amphibians • Some amphibian species have evolved alternatives to metamorphosis: Heterochrony – Neoteny: Normal gonadal maturity, retention of juvenile form – Progenesis: Accelerated gonadal maturity, retention of juvenile form – Direct Development: No larval form

Metamorphosis: Amphibians Neoteny in the Mexican axolotl (salamander) Normal adult with juvenile form. Adult form not seen in nature, resulting from T 4 in the pond.

Metamorphosis: Amphibians Direct development in a common Puerto Rican frog Two views of the developing limb buds within the egg Frogs, not tadpoles, hatch from the eggs. Very large, nutrient-rich eggs allow skipping larval food gathering stage.

Metamorphosis: Insects Adult = imago

Metamorphosis: Insects • Larva are eating machines to provide energy for non-feeding pupal development • They have both doomed larval cells and rudiments of imaginal cells for adult – The larval cells will apoptose in the pupa – Imaginal disc cells will form wings, legs, antennae, eye, head, thorax and genitalia – Histioblasts will form adult abdomen – Imaginal cell clusters in each organ will form adult organ as the larval organ degenerates

Metamorphosis: Insects Locations and developmental fates of imaginal discs and imaginal tissues in the third instar larva of Drosophila

Metamorphosis: Insects Leg Imaginal Disc before pupa during pupa Epidermis cells form a hollow tube that coils Telescopes out in pupa

Metamorphosis: Insects

Metamorphosis: Insects A view into the minds of fly-guys. . The cells at center of the disc secrete Wingless (Wnt) and Decapentaplegic (TGF-B) This causes different expression levels of transcription factors Dachsund (green) and Distal-less (red).

Metamorphosis: Insects • Like amphibians, control is hormonal in insects – Presence of juvenile-hormone makes a larval molt – Shift to steroid 20 -hydroxyecdysone gives pupal molt – Differential timing in the development of pupal structures is due to 20 E receptor expression timing

Regeneration • Restoration of missing tissues • Post-embryonic reactivation of development • Occurs in some form in all species – Stem cell-mediated regeneration – Epimorphosis: adult cell de- and re-differentiation – Morphallaxis: adult cell repatterning – Compensatory regeneration: adult cell division

Regeneration: Epimorphosis flatworms, salamanders The surviving cells lose their specification and form an undifferentiated tissue bulge

Regeneration : Epimorphosis Vertebrate limb development from apical ectodermal cap (ridge)

Regeneration: Morphallaxis The hydra is constantly regenerating cells that are sloughed off. Only the head and foot aren’t moving

Regeneration: Morphallaxis • Any cells along the length can become head, body or foot • If you cut a hydra up all pieces will form all structures • Each piece has a dual gradient already established and both ends are specified

Regeneration: Morphallaxis The bud always forms roughly the same distance anterior-posterior due to the combined gradient

Regeneration: Compensatory Regeneration • No dedifferentiation occurs in liver regeneration • All five cell types produce more of themselves • • • hepatocytes duct cells fat-storing (Ito) cells endothelial cells Kupffer macrophages • Progenitor cell back-up plan: Oval cells

Aging • Time-related deterioration of physiological functions necessary for survival and fertilization – Life span vs. senescence • Combination of: – Mutations – Environmental factors – Random epigenetic changes