Protocols

Contents

1. Methods to study larval development arrest or delay in C. elegans (Dr Nagagireesh Bojanala)

i. Gonad development in C. elegans hermaphrodite

ii. Vulva development in C. elegans hermaphodite

2. Hoechst staining to study cuticle permeability in C. elegans (Anjali Sandhu)

3. Lipid droplet staining in C. elegans (Madhu Dasgupta)

4. Protocol to study swarming in Pseudomonas aeruginosa (Shubham Joge)

5. MATLAB17 to extract features of P. aeruginosa swarm (Divakar Badal)- COMING UP SOON

DIC based study of gonad development in C. elegans larvae

At hatching, the L1 larvae have a gonad primordium made up of four cells, (Z1 to Z4). The outer somatic gonad precursor cells (Z1 and Z4) surround the central primordial germ cells (Z2 and Z3) (Hubbard and Greenwald, 2005). All these cells remain mitotically quiescent till mid-L1 stage. By the end of L1 stage, Z1 and Z4 proliferate to produce 2 two distal tip cells (DTCs) and 10 proximal cells that comprise gonad primordium. These cells undergo rearrangements in late L2. At early L3, rapid extension of gonad arms occurs along with rapid germline proliferation. The DTCs send signals for germline proliferation. By late L3 stage, both gonad arms extend their processes dorsally up and make a turn towards the midline. At mid L4 stage, both arms are in close proximity to the midline without touching each other. During late L4 stage, both arms cross over the midline and overlap with each other.

larval stage - gonadal cell

early to mid L1 4 cells (Z1 Z2 Z3 Z4)

mid to late L1 - 6 to 12 cells ( only somatic gonad precursors Z1 n Z4 divide, germ cell pre Z2, Z3 not)

early to late L2 - 12 cells ( cell rearrangements occurs, 2 DTCs - distal tip cells, and 10 proximal cells)

early to late L3 - DTCs give signal to germ cell pre Z2 Z3 starts to proliferate to produce syncytial germ cells.

References:

Hubbard EJA, Greenstein D (2005) Introduction to the germ line. WormBook2005:14.http://dev.wormbook.org/chapters/www_introgermline/introgermline.ht

Methods to study larval development arrest or delay in C. elegans

Rationale: In wild type N2 strain of C. elegans, progression through larval development can be traced very easily by measuring increase in body length (L1,250µm; L2(360-380) µm; L3 (490-510) µm ; L4 (620-650) µm). However, this progression is delayed or halted under starvation, or altered signaling (insulin IGF-1/DAF-2 signaling) etc. The scaling between time and body size is also altered in mutations affecting TGF beta pathway or collagen (dumpy) genes where mutant animals are smaller or wider or both than the N2 animals at the same development stage. This necessitates use of parameters other than body length to study larval developmental progression. C. elegans cell lineage tracing can be used to study coordinated and stage specific organ formation during development. Thus, cell division in specific lineages can be used to call out transitions through larval development. Here we show the development of C. elegans hermaphrodite goand and vulva to trace early and late larval stages.

Microscopy and egl-17::cfp based lineage tracing of vulva development (Dr Nagagireesh Bojanala)

C. elegans vulva formation is a paradigm for studying cell fate specification and organogenesis (Sternberg, 2005). As shown in the accompanying figure, six Pn.p cells, P3.p to P6.p, are equipotent to adopt vulva fates. But, during mid L3 stage, the gonadal cell called Anchor Cell (AC) secretes a gradient of LIN-3/EGF (Epidermal Growth Factor) which induces vulval fates in P5.p, P6.p and P7.p. The P6.p cell that sits below the AC receives the highest concentration of EGF and adopts 1⁰ cell fate, and P5.p/P7.p get intermediate EGF signal and adopt 2⁰ fates. The 1⁰ cell fate allows P6.p to undergo three rounds of mitotic divisions, starting from early L3 to early L4, resulting in 8 daughter cells. The 2⁰ cell fate allows P5.p/P7.p to divide three times and produce 7 cells each. The final round of cell division occurs in an invariant fashion, all four P6.pxx cells divide transversely in a ‘TTTT’ fashion producing EFFE lineages. But, the P5/7.p cells divide in LLTN fashion with mirror image symmetry along the antero-posterior axis producing ABCD lineages (L, longitudinal division; T, transverse division; N-no division). Thus, after final cell divisions, P(5-7).p cells produce 22 daughter cells, which undergo morphogenesis later to produce the functional vulva in adult animals (Sharma Kishore et.al., 1999).

DIC imaging to study vulva morphogenesis

Morphogenesis of the vulva occurs from late L3 to midL4 stage, which takes ~10 hrs. The initiation of vulva lumen formation starts around late L3 with advent of P6.pxx cells/EFFE moving dorsally upwards and movement of ABCD/DCBA cells towards the vulva midline. Later further cell migration and morphogenesis events results in bell shaped/early L4 and Christmas tree shaped/mid L4 vulvae. Finally, at late L4/A transition the 22 cells undergo eversion to form functional vulva that help in egg-laying and copulation. Vulva morphology of the bell shaped opening and Christmas tree stage can be visualized under DIC (indicated by arrow and arrowhead)

Pegl-17 reporter based study of vulva morphogenesis

LIN-3/EGF produced from the AC will elicit conserved EGFR/RAS signaling in the VPCs. LET-23 is the EGF receptor and LET-60 is the Ras molecule in worms. Within the VPCs, Ras upregulates EGL-17/FGF in the nucleus, suggesting egl-17 as a direct transcriptional target for vulva cell fates (Burdine RD et.al., 1998). First expression of egl-17 is briefly seen in P6.p before AC induction (late L2), but in midL3, P6.p (Pn.p/1cell) expression appears highly distinct and correlates with levels of LIN-3. Similar levels were seen in P6.px (2 cell/midL3), P6.pxx (4 cell/mid to late L3), and P6.pxxx (8 cell/early L4). Surprisingly, at midL4 stage, egl-17 expression switches to 2⁰ cell fated cell, VulC and D, which is influenced by LIN-12/Notch signaling. Thus, based on the temporal and spatial activation of egl-17 promoter, larval stage can be assigned to individual animals. DIC image of the same animals also reports the shape of vulva opening.

CUTICLE PERMEABILITY ASSAY

Rationale: Hoechst 33258 ^(352/461) is a cell permeable nucleic acid counterstain which emits blue fluorescence. In C. elegans, a collagen and peptidoglycan rich layer of cuticle lines the whole body of the worm as well as the lumen of the pharynx. In wild type N2 animals, the cuticle is impermeable to this stain and cannot access pharyngeal or hypodermis nuclei (panel A). When the cuticle permeability is compromised, e.g. in bus-2 mutant, Hoechst 33258 can cross the cuticle within 30 minutes and Hoechst bound pharyngeal and hypodermis nuclei can be easily seen. Thus, this stain can be used to study other genes or cuticle component which may be important to maintain the permeability barrier.

Protocol:

1. At the young adult stage, wash worms off of the plate using 1ml of M9 buffer and transfer to a 1.5ml microfuge tube. Allow the worms to settle by gravity or spin at 3K rpm for 1 min.

2. Wash the worm with M9 buffer 2-3 times to get rid of E. coli.

3. To a 100ul suspension of worm in M9, add 1ul of Hoechst 33258 stain (10ug/ml final concentration) and rock gently at room temperature for 30 mins covered in aluminum foil (From here on, perform all the steps under dim light condition).

4. Remove the stain solution.

5. Wash the worms with M9 buffer 3-4 times to get rid of the unbound stain.

6. Mount worms in 50% glycerol on an agar pad and place a glass cover slip. Pharyngeal and hypodermis nuclei in the stained worms can be viewed at 40x magnification (or higher). bus-2 mutant should be used as a positive control (shown in the panels, B and C).

Reference:

Hiroki Moribe, John Yochem, Hiromi Yamada, Yo Tabuse, Toyoshi Fujimoto, Eisuke Mekada (2004). Tetraspanin protein (TSP-15) is required for epidermal integrity in Caenorhabditis elegans. Journal of Cell Science 2004 117: 5209-5220; doi: 10.1242/jcs.01403

LIPID DROPLET STAINING IN C. elegans (Madhumanti Dasgupta)

Lipid droplets are fat storage organelles conserved across eukaryotes. These organelles have a single phospholipid monolayer and they store neutral lipids such as TAGs and cholesterol esters. In C. elegans, they are predominantly found in the intestine and in the hypodermis. Fatty acids are released from TAGs in lipid droplets by the action of lipases during growth, calorie restriction or starvation. The lipid droplet homeostasis is susceptible to caloric restriction, starvation and possibly to other stresses.

Lipids droplets can be studied biochemically and by microscopy. Microscopy involved staining of lipid droplets in live or fixed worms with a number of lipophilic dyes such as Oil Red O, Sudan Black, Nile Red and BODIPY. Oil Red O has been shown to stain neutral lipids (Rourke et al). Nile Red and BODIPY stain also lysosomal related organelles while staining fixed worms (Rourke et al.). In our laboratory, we use Oil Red O as the preferred choice of dye for studying lipid mobilization under different stresses. Oil Red O is a fat soluble diazo dye, which differentially is more soluble in fat as compared to water in which the stain is dissolved. We utilize BODIPY staining method as a complementary approach. BODIPY is a lipophilic fluorochrome which binds to the lipid core of the droplets.

Reagents/recipes :

2X MRWB Buffer (1ml) with 2% Paraformaldehyde (PFA)

· 2M KCl – 80µl

· 2M NaCl – 20µl

· 0.1M Na2EGTA – 140µl

· 0.05M spermidine trihydrochloride – 20µl

· 4mM spermine -100µl

· 0.1M Na-PIPES – 300µl

· ß-mercaptoethanol – 2µl

· Water – 330µl

· Add 20mg of PFA and add 6-7 drops of 5N NaOH so that PFA dissolves

60% Oil Red O stain

0.5g/100ml stock of ORO in isopropanol is equilibrated over 4-5 days by rotating the tubes at RT. Prior to use, ORO is diluted to 60% with water, equilibriated for at least 1 hour and filtered using 0.45µm syringe filter.

Method:

1. At the L4 larva or the young adult stage, worms are washed and collected in a microfuge tube using 1ml of 1X PBS. Washing step is repeated two more times. During this step, prepare 60% ORO diluted in water and keep it for rotation for 1 hour.

2. To permeabilize the cuticle, worms are resuspended in 140µl of PBS and 140µl of 2X MRWB containing 2% PFA. The worms, immersed in the fixative, are rotated (covered with foil) at RT for 1 hour.

3. After 1 hour, worms are allowed to settle, MRWB buffer is removed and worms are washed with 1ml PBS three times.

4. Worms are resuspended in 60% Isopropanol and incubated at RT (with rotation) for 15 minutes.

5. Isopropanol is removed and 1ml of freshly diluted, equilibrated and filtered 60% ORO is added to worms. The worms are stained under rotation at RT overnight.

6. Next day, worms are allowed to settle and the stain is removed. Worms are washed with PBS + 0.01% Triton three times first, followed by two more washes using 1X PBS.

7. The samples are mounted on a glass slide in 50% glycerol and covered with a cover slip. Slides are viewed under 10X magnification, bright field microscope. Images can be captured using a true color camera.

8. Image J can be used to quantify oil red O stained lipid droplets in C. elegans. Convert your images to 8 bit format, threshold and then quantify the mean pixel density.

9. Controls to be used for ORO staining: daf-2 (e1370) strain has more lipid droplets than wild type N2 animals, and can be visualized and quantified using Oil Red O Stain. Starvation for 8-12 hours leads to depletion of more than 90% of lipid droplets in wild type N2 animals and can also be used to ensure that your ORO stain and protocol works.

Reference : The protocol was adapted and modified from E. J. O’Rourke et al., 2009, Cell Metabolism

BODIPY STAINING OF LIPIDs in C. elegans :

1. 1mg/ml stock solution of BODIPY ^493/503 is prepared in DMSO.

2. At the young adult stage, worms are washed in M9 buffer and fixed in 4% paraformaldehyde (PFA) solution for 15 mins.

3. Worm in the fixative are exposed to three cycles of freeze thaw.

4. PFA solution is removed by washing three times with M9. Worms are incubated in 500µl of 1µg/ml BODIPY ^{493/ 503} in M9 for 1 hour at room temperature.

5. The stain is removed and the worms are washed three times with M9 buffer.

6. The worms are mounted on a glass slide in 50% glycerol, covered with a cover slip and observed under fluorescence microscope with FITC filter (excitation 490, emission 525)

Reference : Method is adapted from M. Klapper et al., 2011, Journal of lipid research