Developmental biology

animal
plant

Growth and development

Growth (成長)

Development

embryogenesis (胚形成)
juvenile
adult vegetative
adult reproductive

Ham, Johan: brought in gonorrhea ejaculate to Leeuwenhoek

Homeobox (ホメオボックス)

cell determination in the embryo

Testis and sperm (精巣・精子)

Sperm or spermatozoon (pl. –a) (精子)

Egg (卵)

Organization of egg cytoplams (卵細胞質)

Fig. 63. Redistribution of cytoplasmic inclusions in the egg of Cynthia partita following centrifugation. a, Egg immediately after centrifugation; b, centrifuged egg 22 hours later (the yellow granules, shown black, have taken up a subequational position on one side of the egg). 1st m, Spindle of first meiotic division. (Conklin 1931)

Species                             
Arbacia lixula
Echinus esculentus
Paracentrotus lividus
Strongylocentrotus
    droebachiensis
S. purpuratus
Sphaerechinus grarrularis
Heliocidaris crassispina
Echinarachnius parma
Hemicentrotus pulcherrimus
Pseudocentrotus depressus
Echinocardium cordatum
Brissopsis lyrifera               
Rana temporaria
R. esculenta
R. japonica
R. clamitans
Discoglossus pictus
Bufo bufo
B. vulgaris formosus
Axolotl
Triturus cristatus                  

Fig. 3.2. Part of a chromosome from a growing egg cell (oocyte) in the ovary of a salamander showing some of the variety of different kinds of “lampbrush loops” that are formed along the chromosome.

Theories on fertilization

acrosin → dissolution of envelope
lindin → attachment → membrane fusion

└* fertilization potential (fast block against polyspermy)
└* intracellular Ca⁺⁺ level → (a) (b)
└** alkalization of cytoplasm

└ actin polymerization → displacement of cytoplasmic
____________________ constitutions
└ activation of various enzymes
__(ca pH 7.2 = optimum pH for various enzymes)
mRNA is unmasked by this mechanisms (unmasking)

Ex. DNA polymerase → chromosome formation

└ veteline delaminase → fertilization membran
_____________________(block against polyspermy)
└ sperm receptor hydrolase
_______(attacks binder and slows block against polyspermy)

→ O₂ uptake

(O₂ uptake of fertilized egg)/(O₂ uptake of unfertilized egg)

Phallusia mamillata = 2.0
Rana platyrrhina = 1.0
Bufo bufo (metaphase of
    2nd mat.) = 1.0
Fundulus heteroclitus (division) = 1.0
Oryzias latipes = 1.0
Fucus vesiculosus = 1.9
Strongylocentrotua purpuratue = 3.7
Psammechinus miliaris = 3.6
Paracentrotus lividus = 4.7
Arbacia punculata = 4.5
Dendraster excentricus = 3.0

              HCl                            NaOH
Concentration  N/   N/   N/   N/  N/ Normal   N/   N/    N/   N/  N/
in sea water  500 1000 2000 4000 104 sea     104  4000  2000 1000 500
                                     water
Cleaved egg %  0   2.5  8   22.5 45    53     84  92.5  88.5  89   0

Fig. 60. Displacement of organ rudiments in an embryo of Cynthia partita as a result of centrifugation of the egg. n, neural system; p, eyp pigment; ch, autochord; ect, ectoderm; ms, muscle cells. (Conklin 1931)

Preparation________Eggs reaching blastula stage (%)
Whole tissue extract__________94
Large granules______________35.2
Medium and small granules____19.5
Supernatant_________________0
Phosphate buffer_____________0.3
Pricked "dry"________________ 0

    Stage                Amount of DNA  Condition of nucleus

    1st polar body         127 ±  3            2n
    Sperinatozoid           61 ±  1            1n
    Cleavage, interphase   210 ±  9            2n-4n
    Cleavage, prophase     263 ± 10            4n
    Cleavage, telophase    124 ±  3            2n

Asymmetric property (非対称性)

Fig. 13. "Retarded nucleation" in Triton. Zygote nucleus pushed to one side by the constriction of the egg. (a) first cleavage. In the nucleated half; (b) further cleavages In this part, the other half Is still imcleaved; (c) passage of one nucleus from the nucleated into the non-nucleated half, beginning of development In the latter; (d) the two embryo. that developed from the constricted (収縮された, 窄まった) egg; the one produced by the half In whIch nucleation was retarded is considerably younger, but normally built. (Spemann and Tannhauser 1938)

Development of gonad (生殖腺発達)

1. Origin of primordial germ cell (gamete) (始原生殖細胞の起源)

2. sex differentiation (性分化)

Pre-embryo (初期胚)

substance           water  dry    protein     total   glycogen   fat    ash
                                      subs  nitrogen  carbs                                  
Unfertilized egg    77.3   22.7    10.7      5.43       5.13     4.82  0.34
Blastula (12 hrs)   77.3   22.7    10.2      5.46        4.8      4.43  2.07
Pluteus                 78.8   21.2      9.7       3.4       traces   3.69  3.56

Substance                                                       Egg   Hatched tadpole
Total nitrogen                                                   162            159
Extractable nitrogen (N of active protoplasm)   40            42.8
Nonprotein nitrogen (in nucleic acid?)                4              5.0
Total carbohydrates                                          104             58
Glycogen                                                          73.2            31

mammal (哺乳類)

yolk sac (卵黄嚢), amnion (amniotic sac, 羊膜), chorion (漿膜), allantois (尿膜)

Neural induction (神経組織誘導)

discovered organizer, by using hair loop, glass needle, pipette, etc.

Organizer (形成体)

neural induction
embryonic induction
primary induction
⇒ not only dorsal lip but also the others can be organizer

Analysis for nature of induction (誘導特性解析)

Technical

Renal system (腎臓系)

gonad (♂/♀) (mesodermal) ┅┅┅ different origin
┃_________↑ incursion_______┇
┃_________primodial germ cells (始原生殖細胞) → sperm or egg
┣ ovary ━━━━━┓
┗ testis ━━━ sperm or egg determined by the surrounding tissues
__⇑ hormones
[testis, mesonephros – urinary duct, Wolffian duct]
[ovary, pronephros – Müllarian duct, oviduct]

Metaphros (kidney, 腎臓)

vreteric bud - mesencyme - branching - collecting duct
(尿管芽)______┗━━━━━┛
_____________condensation (forming duct)
______________________→ uriniferous tubule

a) epithelium: singular separation (分離単独) → no differentiation / mesenchyme: singular separation → no differentiation
b) ureter: dependent on metanephic mesenchyme
c) uriniferous tuble [replacement is possible to salivary gland epithelium, spinal cord]: not dedepnent on ureter (ureteric bud)
⇒ the formation of urinary duct is "permissive"
mesenchyme: cell adhesion is weak prior to the formation of ureteric bud

Pattern formation (パターン形成)

a) destroyed 1/2 limb bud → normal limb
b) slit limb bud vertically → duplicated limb
c) combined 2 lim buds in harmonous orientation → normal limb
d) rotated limb field at 180o and transplanted →

AP-axis: neural tube closure stage / DB-axis: tail but stage

Proximal-distal: forming limb (need re-consideration)

A-P / D-V, somatic layer mesoderm, epidermis epithelium

+/O (normal)    ×         +/O (normal)
      ┏━━━━╋━━━━┓
     +/+             +/O           O/O
  normal        normal   poor regeneration
                                   (males = sterile / females = maternal effect)
        ♀ O/O         ×         ♂ (+/O)
            ┏━━━┻━━━┓
          +/O                        O/O
arrested gastrula    arrested gastrula

Cell-mediated immunity (cellular immunity)

Humoral immune response: plasma cell → Ig circulation
Cellular immune resposne: lymphocytes → Ab

Genetic basis of cellular immune response

                                              Accept   Reject

    1) isograft (= autograft), isogenic          O
    2) allograft                                         O
    3) AA × BB (P)         Transplant P → F1   O
       → AB (F1)           F1 → P                      O*
    4) → AA, AB, BB        F2 → F1             O

*: reject the substances that have not present in itself

Morphogenesis (形態発生)

Polarity (極性)

young: shoot apical meristem (root as well as stem)
young: root apical meristem

Plant movement (植物の運動)

Embryogenesis (胚形成)

P (parallel variation) - correlated to physiological changes
E (excision) - terminated physiological processes after the removal
S (substitution) - physiological processes are resumed by exogenous addition of substances
I (isolation) - the reaction is activated by exogenous substances
G (generality) - the substance and reaction widely observed
S (specificity) - not be replaced by other natural substances
→ When these conditions are satisfied, the hormone (or related substance) works physiological

Hormonal interactions

H₁ + H₂ → E₁ + E₂ (additive) vs E₁ × E₂ (synergistic)

Ex. high concentration auxin → ethylene production↑

→ the specific enzyme(s)↑ → inactive bound auxin↑
→ maintaining low-leveled auxin in vivo

1. Auxin (オーキシン)

IAA crystallized - confirmed that IAA is a hormone

Synthesis

Function

Changes in RNA metabolism induced by auxin

[³H]-UMP incorporated (pmol/30 min/mg protein)           
RNA polymearase = 88    + PM-IAA = 146
                                         + PM-2,4-D supernatant = 138
                                         + PM-3,5-D supernatant = 96  

PM, plasma membrane fraction

RNA polymerase____________________________________ 72
+ PM suspended in TGMED__________________________ 162
+ Repelleted PM resuspended in TGMED________________116
+ TGMED supernatant after PM repelleted________________74
+ PM suspended in TGMED + 0.1 μM 2, 4-D_____________ 236
+ Repelleted PM resuspended in TGMED + 0.1 μM 2, 4-D___162
+ TGMED suspended after PM repelleted with 0.1 μM 2, 4-D_106
+ TGMED + 0.1 μM 2, 4-D_____________________________84

PM, plasma membrane fraction

+ α-amanitin*                                         133
+ PM-2,4-D supernatant                        227
+ PM-2,4-D supernatant + α-amanitin* 140
+ α-amanitin*                                         146
+ PM-IAA supernatant                           267
+ PM-IAA supernatant + α-amanitin*    140

RNA polymeraase 160


RNA polymerase   186

* α-amanitin acts as the inhibitor of mRNA synthesis. Each assay contained 2 μg of α-amanitin

2. Gibberellin (ジベレリン)

α-amylase formation induced by gibberellin

3. Cytokinin (CK, サイトカイニン)

4. Abscisic acid (アブシジン酸)

Synthesis

Function

5. Ethylene (エチレン)

indcued by a volatile element in matured apple

1959 research on ethylene biosynthesis
1967 auxin is the trigger of ethylene production

trade name = ethrel → applying research on gardening

Etylene production (nl/g/hr) on climacteric plants
Ex.                      apple  apricot  kiwi fruit  pear  tomato
pre-climacteric      0.1     0.03      < 0.1    0.5-0.6    0.2
climacteric           100       0.4         50      50-100   120
temperature (°C)   12        20         20         20         20

slightly different among the varieties and/or cultivars

Etylene production (nl/g/hr) on non-climacteric plants
Ex. blueberry = 0.04-0.05. cucumber = 0.02-0.16. orange = 0.02-0.06

Conditions

break point on Arrhenius plot → phase transition (sometimes 11.4°C)

O₂ is required for ethylene production

Ex. wheat treading (麦踏), classically known

grew short when the shoots were swept by hand or brush
induced by ethylene productionk - inhibiting shoot elongation

most plants supressed growth ⇒ thigmomorphogenesis

→ epinasty (上偏成長), induced by ethylene

Inhibition responses
    ethylene synthesis in vegetative
        tissue and non-climacteric fruit
    flower development in most plants
    auxin transport
    shoot and root elongation
    normal orientation of cell wall
        microfibrils

Biosynthesis

mannose, mannitol, citric acid → ethylene

                                                            |← hemicellulose
methionine + ATP → SAM¹ → ACC² → etylene → PG³
______________________________|→ galactose

¹: S-adenosyl methionine
²: amino cyclo propane
³: polygalacturonase

C1 ⇒ carbon dioxide (CO₂)
C2 ⇒ formic acid
C3 and C4 ⇒ ethylene

  methionine       NH₃
                            |
CH₃-S-CH₂-CH₂-CH-COO-
 ^{5          4      3      2     1}

these two experiments cofirmed: ACC = precursor of ethylene

Metabolism

ethylene (¹⁴C₂H₄) abosorved by plants → confirmed the uptake
emitted CO₂ = non-radioactive
⇒ CO₂ as a resultant of assimilation is not derived from ethylene

uptake by benzene and toluene

radioactive etylene → not moved for long distance

                           _/O_\
H₂C=CH₂ → H₂C=CH₂ → HO-CH₂-CH₂-OH

Ethylene in soil (土壌中エチレン)

                        [plant exudes  sugars] ⬅ [nutrients rebind to clay
[micro-organism ⬇                     ⬆                       and organic matter]
   activity increases]               [micro-organism         ⬆
   ⬇                                         activity resumes]  [iron re-oxidises]
[microsite becomes                                   ⬆            ⬆
 anaerobic]                [micro-organism     [O₂ diffuses back
   ⬇                             activity ceases] ➡ into site]
[iron reduces to                ⬆                             ⬆
 soluble ferrous] ➡ [ethylene gas released]  [plant takes up nutrients]
   ⬇                                                                   ⬆
[releases nutrients bound to clay and organic matter into soil H₂O]

adjust energy turnover and nutrient use efficiency
inhibit pathogen growth in soil
response of aboveground organs to immersion ≈ ethylene

6. Florigen (フロリゲン)

Hormonal control at hormone level

RA + RB = RAB ← additive response (R: response, AB: hormones)
RA + RB < RAB ← synergistic response
IAA, ABA, Cytokinin havce complementary functions

Somatic division or somatic cell division (体細胞分裂)

G₁ (gap₁) → S (synthesis) → G₂ (gap₂) → M (mitosis)
common for most eukaryotes

prophase → metaphase → anaphase → telophase

Meiotic division

germination rate

a few percent under dark
nearly 100% under light after imbibition duirng 24-48 hrs

exposed to radiation of which wavelengths ≥ 700 nm (far-red, FR)

→ inhibited seed germination

(Institute in Beltsville, Maryland)

P_r ↔ P_fr
P_r → P₆₈₉ → P_fr → P₆₅₀ → P_r = circulated
= trigger reaction

examined the effects of R/FR on seed germination

(this cultivar does not germinate the seeds under the darkness)

R: exposed to red at 1 W/m² for 1 min
FR: exposed to far-red at 1 W/m² for 4 min

light treatment       R   +FR   +R  +FR  +R   +FR  +R  
germination (%)   81.0  4.5  54.5   3.5  65.0  3.5 72.5

R: induced seed germination ⇔ FR: inhibited seed germination

Dark(control) NUV : B +NUV +B +NUV +B +NUV
       100           95    11    93    38     86   34    91

⇒ presence of mycochrome

Ⓧ + ◎ + UV irradiation →

                                Amoclae   F hoclies
Dry weight                    5-8        2.5-4
Protein                        2-5         1.5-2 ↓
RNA (mg)                      1           0.4
Total carbohydrate       0.5         0.4 =
Cellulose                       0           0.1
Mucopolysaccharide     0          0.08
DNA                             36           30        
de novo Ex. temporal changes in the synthesis patterns of actin (structural protein)

Fig. Decrease in dry weight and oxygen consumption

the most of energy during the emergence process is from protei. carbonhyborates are used least
→ expression of genes that are for storage-specific proteins and the RNAs

³H-DNA → thermal denaturation for single strand → sharing force, ca 400 nucleotides → remnant, return to double strand (= repeated sequence) → hydroxyappite column
⇒ ³H-labeled single strand = DNA (unique sequence)

→ probe DNA-RNA hybrid

                                          gr    aggr pseudo culm     all  gr + culm
% of genome present
  as RNA transcripts (%)  30.2  27.4    34.2   35.6    56.0  49.0      
Estimated number of
  genes expressed           8600 7800 10000 11000 11000              

gr = growth. aggr = aggregation. culm = culmination. pseudo = pseudo-plasmodium. all = all stage

Growth + (Growth + Culmination) = 65.8: common genes present
2.86 × 10⁷ nucleotide pair → one gene = 10³ bp (M.W. = 4300) ⇒ unique gene = 286 × 10⁴

Key enzyme (キーエンザイム)

enzymes related to carbohydrate synthesis (cf. PSV: prespore vacuole) - absent in the stalk

nuclear-cytoplasm interaction after the nuclear transfer

Higher plants (高等植物)

de novo: gene = active → m-RNA → protein (α-amylase)

Double fertilization and division pattern

α-amylase (α-アミラーゼ)

Ribophorin in pea epicotyl

             Free              Membrane
             ---------------   ---------------
             M  Sp  Lp  Total  M  Sp  Lp  Total  Total

    Control               480               90     570
    BA                    441              103     544
    GA                    454              126     580
    IAA                   258              323     581
    NAA                   294              272     566

Total amount is constant - (possibility) free-bound induced by hormone
Inhibition factor: Rabit reticulocycle – cell-free system

Heme = globrin, Hemine
in-vitro system does not proceed when hormone is not supplied from the outside

Elongation factor (伸長因子)

                   2 days     4       6  
No addition   816     382    168
2 days         8080      ↔   6580
4                      ↓                 ↓
6                 1953       ↔   1800
S-100: sup of 10⁵ g → translation
No differences of ribosomes + aged S-100 is not effective → presene of inhibitor

Shoot apex (茎頂)

characterized by the presence of an apical meristem at the topmost part of the root and the shoot

Relationships between stem and root (茎と根の関係)

IAA 0.002 ppm → root
Adenine 40 ppm → stem (leaf)
IAA 0.005 ppm + Adenine 40 ppm → stem, leaf, root

_____________Callus___Roots___Leaves
IAA (mg/l)______3______3________0.03
Kinetin (mg/l)___0.2_____0.02_____ 1

1) embryoid (胚様体)
2) origin = unicell (plant): (need to obtain the evidence)

tobacco pith cells → culture single cell → can form both bud and root

medium: auxin, kinetin + agar → to liquid: differentiate root, stem and leaf
in general, the differentiation occurs under auxin-free condition ← substances for growth regulation

→ mature plant/embryoid → callus mass

Defoliation (落葉)

Lamina senescence____Abscission layer(zone)
↓___________________↓
IAA production →→→→ Stimulate senescence
____________________↓
_________C₂H₄(exo) ⇔ C₂H₄ production
____________________↓
____________________Increase cellulase
____________________↓
____________________Cell separation

cell differentiation ┌ plant hormones
⇓_____________ ↓_______________specific gene activation
functional specification ← specific enzyme
↓____________________↑
specific structure__specific protein ← specific mRNAs _______________ or polypeptide___ ↑
______________________________specific transcription
Fig. Conceptual framework for predicted cell differentiation processes

Flower shedding (落花)

Fruit shedding (落果)

ABC model