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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.

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

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

longitudinally. AB always divides before P1.

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

Second Cleavage

• Cell-Cell Interaction determines the DV axis.

Principles of Development (Lewis Wholper) Chapter 5

Third Cleavage• The LR axis.

Principles of Development (Lewis Wholper) Chapter 5

Principles of Development (Lewis Wholper) Chapter 5

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

Genes

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

Altered Second Cleavages

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…….