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Identification of Genes required for Cytoplasmic Localization

in Early C. elegans Embryos

Kenneth J. KemphuesJames R. PriessDiane G. Morton

Niansheng Cheng

皓宇。宇瑄。銘崧

C. elegans

• Hermaphrodite & Male

• Research was begun in 1974 by Sydney Brenner.• It has since been used extensively as a model organism.

http://herkules.oulu.fi/isbn9514267567/html/i183412.html

http://www.sfu.ca/biology/faculty/hutter/hutterlab/research/Celegans.html

Development• Fertilization

• First Cleavage

• Second Cleavage

• Axe Determination Anterior & Posterior Dorsal & Ventral Left & Right

Fertilization• Fertilization determines AP axis.• After fertilization, the two pronucleis join together.

http://www.mbg.cornell.edu/cals/mbg/research/kemphues-lab/movies.cfm/kjk1_wt

First Cleavage• Then the mitotic spindle forms, and it migrates posteriorly. • Owing to the migration, the two daughter blastomeres are produced

in different sizes after the first cleavage.


Second Cleavage• At the second cleavage, AB divides transversely and P1 divides

longitudinally. AB always divides before P1.


Second Cleavage

• Cell-Cell Interaction determines the DV axis.

Principles of Development (Lewis Wholper) Chapter 5

Third Cleavage• The LR axis.


P Granule

• Large ribonucleoprotein complexes destined to the germ line.

• One of the cell fate determinants.

• Mitotic spindle migrates posteriorly.

• Daughter cells are produced in different sizes after the first cleavage.

• AB＞ P1

• P granules are localized to P1.

• AB divides transversely and P1 divides longitudinally at second cleavage.

• AB always divides before P1.

• P granules are localized to P2.

Background

• There must be some genes controlling the developmental progresses described before.

Aim

To identify the genes required for cytoplasmic localization in early C. elegans embryos

Aim-1

Identifying the genes

Aim-1• Genes required for cytoplasmic localization in the early

cleavages are expected to be expressed during oogenesis.

→ Maternal Genes

• Mutations in such genes are likely to be maternal effect lethal mutations.

• Screening method

+: egl-23 or lin-2m: recessive maternal effect lethal mutation

egl-23(lin-2): fertilize but don’t lay egg

Screening

egl: egl-23 egl/egl EMS

egl/egl Self-fertilization

egl/egl egl/egl egl/egl

examine defects

Defects

• Equal First Cleavage

• Altered Second Cleavages

• Abnormal Localization of P Granule

• Abnormal Differentiation

• Grandchildless Phenotype

Aim 2

Observing the defects

Equal First Cleavage

Equal First Cleavage

• Blastomere Size Measurement – Zeiss Photomicroscope III (PM III)– Planimeter

• Spindle Movement Measurement http://www.microscopy-uk.org.uk/mag/artnov07/dw-pm3.html

DIC=NIC

• Differential interference contrast microscopy (DIC)• Nomarski Interference Contrast (NIC) • Nomarski microscopy

• Unstained, transparent samples• Appearing black to white on a grey background • Similar to phase contrast microscopy

(without the bright diffraction halo)• Emphasizing lines and edges

though not providing a topographically accurate image

http://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy

Table-2 Relative Sizes of AB Blastomeres

3757±3par-4(it33)

2152±1par-3(e2074)

3551±2par-2(it5)

3953±2par-1(b274)

5757±2N2(wild type)

No. of embryosAB/TotalGenotype

Percent egg length

Altered Second Cleavages

Conclusion

• Abnormal positioning of the early mitotic spindlessize

• Altered timing of early cleavage

• The par embryos contribute significantly to later pattern abnormalities.

Abnormal Localization of

P Granule

P granule localization

• Normal: the posterior pole

• Par mutants: immunofluorescence of 4-cell embryos stained with anti-P granule antibody

http://www.mun.ca/biology/scarr/4241_Devo_Germ_Celegans.html

Wild type par-1 mutant par-2 mutant

par-3 mutantpar-3 mutant par-4 mutant

Result

• par-1: P granules are distributed

everywhere• par-2: no or incomplete localization• par-3: in either 2 middle or 2 polar

blastomere• par-4: resemble par-1, but more P

granules are in posterior-most cells

WT

Conclusion

• par mutants are fail to localize P granules properly.

Abnormal Differentiated Cells

Production

• Intestinal differentiation is most severely affected.

• The failure to produce intestinal cells correlates with the strength of the mutation.

•Method:

Normarski micrograph

•Result:

par mutation may affect the location of the original cells

• Method: Immunostaining with

different antibodies.• Figure C,D

Sensory neuron• Figure E,F Pharyngeal muscles• Figure G,H Birefringent granules

Conclusion

• Detailed cell lineage analysis of par embryos has not yet been undertaken.

• par mutants may affect differentiated cell types.

Grandchildless Phenotype

• Observation: Normarski microscopy

• Result: Many par mutant larvae develop into

morphologically normal adults but lack mature gametes.

Wild type hermaphrodite

par-3 mutant hermaphrodite

O： oocyte S： spermatheca E： embryos V： vulva I： intestine

GS： somatically derived gonad sheath

http://www.wormatlas.org/handbook/fig.s/ReprodFIG1.jpg

Conclusion

• Mutations at the four par loci lead to abnormalities, such as cleavage pattern, timing of cleavages, and partitioning of P granules.

• At terminal stage, par embryos exhibit different phenotypes of the differentiated cells, such as neuron and muscle.

• The germ line seems also be specially sensitive to mutations in the par genes. All incompletely expressed mutations result in a grandchildless phenotype.

• The par genes function in a common process requires for proper timing and pattering of cleavages, intestinal differentiation, and P granule localization.

Continued Research

• Actin microfilament have been shown to be required for the pattern of P granule location and the proper positioning of the mitotic spindle…….

• So par genes may connect to actin…….

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