annelid module oligochaetes - …mblembryology.stowers.org/images/exp...

Embryology 2009 Annelid module – Oligochaetes Dr. Alexa Bely

Annelid module Oligochaetes

MBL Embryology Course – July 2009

Alexa Bely ([email protected])

www.life.umd.edu/biology/faculty/bely/BelyLab.htm

TA: Ed Zattara ([email protected])

Table of Contents page I. Overview of naidine annelids 2

II. Culturing naidines 3

III. Anesthetization and immobilization 4

IV. Amputation 5

V. Specimen fixation 5

VI. Staining muscle and nervous system 5

VII. Labeling of proliferating cells 6

VIII. Labeling of cells undergoing programmed cell death 6

IX. PROTOCOL - Acetyl-tubulin/serotonin/DNA triple staining 7

X. PROTOCOL - Alkaline phosphatase staining 8

XI. PROTOCOL - Phalloidin Staining 8

XII. PROTOCOL - BrdU Incorporation 9

XIII. PROTOCOL - TUNEL staining of apoptotic cells 11

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I. Overview of naidine annelids

Naidine worms (Annelida: Clitellata: Naididae) are a group of small, delicate annelids (segmented worms) that are common in freshwater. Naidines exhibit multiple forms of post-embryonic development including regeneration, asexual reproduction by fission, and indeterminate growth via posterior segment addition. All three of these processes can be studied in the naidines Pristina leidyi and Allonais paraguayensis which will be provided for this lab.

Schematic drawing of a Pristina worm. A body made of serially repeated segments is capped terminally by asegmental portions. The gut and ventral nerve cord run along the whole length of the body.

Allonais paraguayensis reproduces by architomic fission (“simple fission”), by which a

worm breaks into two (or sometimes more) fragments, and each fragment then regenerates the missing structures. This species can regenerate anteriorly and/or posteriorly in 1-2 days, and can do so starting from as few as a couple of mid-body segments.

Pristina leidyi reproduces by paratomic fission, by which new structures are formed prior to physical separation. A new head and tail are intercalated in the middle of the worm’s body, producing a transiently linked chain of individuals. This species can regenerate anteriorly and/or posteriorly in 3-5 days.

Types of fission in naidines. Allonais reproduces asexually through architomy (left), in which the worm splits in two or more pieces, and each re-grows the missing end. In Pristina’s paratomy (right), development of these ends precedes the physical separation of the pieces.

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II. Culturing naidines Pristina leidyi and Allonais paraguayensis are maintained in spring water which can be either purchased directly (e.g., Poland Spring) or made artificially (e.g., 0.35 g/L sea salt in distilled water). Animals are provided pieces of brown paper towel as a substrate to crawl on and are fed regularly with Spirulina powder (Pristina) or wheat grains (Allonais). Worms maintained this way grow continuously, adding segments from the posterior growth zone and developing one or more fission zones in mid-body segments. Growth rate can be modulated by temperature and the amount of food.

1. Start with 4-8” diameter culture dish 2. Half-fill dish with fresh spring water 3. Add 10-100 individuals from a stock culture 4. Add 1-3 pieces of paper towel (~2” x 2”) 5. Add a small amount of Spirulina diluted in spring water to the culture (for Pristina) or

2-3 pieces of cracked wheat grains (Allonais) 6. About once a week, replace half of the water. If worm density is high, discard half of

the worms. 7. About once a week, add a small amount of additional food

• For fast growth cultures of Pristina, add enough food to make water light green and

change water daily. Maximum growth will occur a few days after culture is started.

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III. Anesthetization and immobilization Anesthetizing and immobilizing worms can be useful for observing living specimens or for amputation. Below are three drugs that can be used on naidines for such purposes.

Nicotine: Worms are knocked-out within a minute or two of being transferred to nicotine and remain anesthetized for several minutes. Worms habituate to nicotine, so this drug is only useful for short-term immobilization. The effects of nicotine are fully reversible; moving worms into clean spring water reverses effects within about a minute.

Stock solution: 1 mM in dH2O; store at RT protected from light.

Working solution: 50 μM in spring water (1:20 dilution from stock); prepare and use fresh or within a few days (protect from light if storing several days); gentle swirling can accelerate the process and help avoiding coiling.

Mode of action: Nicotine is an agonist of nicotinic receptors in the peripheral and central nervous system. More information is available at http://www.inchem.org/

Ivermectin: Worms become immobilized after about 10-30 minutes in ivermectin and remain anesthetized for several hours. Check for peristaltic movements in the gut and blood pumping to ensure that the animal is still alive, as worms sometimes die after exposure to ivermectin. The effect of ivermectin is usually reversible; move worms into clean spring water to reverse effects within a few hours.

Stock solution: 50 mM in ethanol; store at 4C.

Working solution: 0.8-2.0 μM in spring water; prepare and use fresh.

Mode of action: Ivermectin potentiates GABA-ergic neural and neuromuscular transmission. More information is available at http://www.inchem.org/

Tetrodotoxin (TTX): Worms become immobilized after 30-120 minutes. Check for peristaltic movements in the gut and blood pumping to ensure that the animal is still alive, as worms sometimes die after exposure to TTX. TTX is suitable for long-term studies; we have had worms survive in TTX for up to 10 days in working solution. The effect of TTX is irreversible except for short exposures.

Stock solution: 3.1 mM in dH2O; store at 4C.

Working solution: 2.0-6.2 μM in spring water; prepare and use fresh.

Mode of action: TTX blocks voltage-sensitive sodium channels and thus the conduction of nerve impulses along nerve fibers and axons.

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IV. Amputation 1. (optional) anesthetize worms in nicotine if desired (see above, section II), processing just a

few worms at a time to minimize nicotine habituation 2. place worm on a glass surface (glass dish with low sides or glass plate) in a small drop of

water 3. either add enough water that the image of the worm isn’t too distorted by water droplet

surface or remove almost all of the water 4. using a stereomicroscope to view the animal, cut at desired location(s) with a scalpel 5. culture amputated worms (singly, if desired) in spring water, without food or paper towel, in a

6-24 well culture plate until the desired timepoint V. Specimen fixation Naidine worms have no hard skeleton, relying instead on a hydrostatic support mechanism based on tandemly arrayed coelomic compartments. Lack of hard support structures imply that the morphology of the animal may change depending on the degree of muscular contraction. Given that strong contraction and even breaking may happen upon fixation, it is recommended that they are relaxed prior to it. Pre-fixation relaxation Prior to fixing animals for any of the following protocols, relax the worms (for optimal morphological preservation) by placing worms in ~500µL of cold relaxant solution for about 10 minutes at 4C (either in fridge or on ice). On adding the cold solution, and before fixation, tap the container gently to separate and loosen up the worms.

Relaxant Solution: 10 mM MgCl2 5 mM NaCl

1 mM KCl 8% EtOH

Fixation In most cases, worms can be fixed using 4% formaldehyde in PBS for 20’-60’. Fixation time may influence results of posterior staining procedures, especially for the TUNEL procedure. VI. Staining muscle and nervous system

Muscle and nervous system stains allow us to follow developmental events taking place during regeneration and fission in naidines. Fluorochrome-conjugated phalloidin binds to F-actin and highlights muscle fibers. Different sets of nervous system elements can be revealed by looking for endogenous alkaline phosphatase activity (using NBT/BCIP), or using immunohistochemical detection of tubulin and serotonin. DNA in cell nuclei can be counterstained with any nuclear dye, like DAPI, Hoechst, YO-YO or TO-PRO.

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VII. Labeling of proliferating cells Patterns of cell proliferation during growth, regeneration and fission can be investigated using bromodeoxyuridine (BrdU), a brominated analogue of thymidine. When BrdU is added to the medium where the worms are cultured, it is metabolized via the pyrimidine salvage pathway and incorporated in S-phase cells during DNA replication. Incorporation is cumulative, with more cells being labeled the longer the incubation in BrdU; labeled cells can be detected with a pulse as short as 15’ in fast growing worms, although longer pulses are recommended. Once incubation is over, worms may be fixed immediately, or changed to a BrdU-free environment for pulse-chase experiments. After fixation, BrdU incorporated in the cells can be detected using monoclonal antibodies against it. VIII. Labeling of cells undergoing programmed cell death Apoptosis, a type of programmed cell death, can be detected in naidines using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. During apoptosis, cleavege of DNA results in small double stranded fragments and single strand breaks (“nicks”) in larger fragments. The TUNEL assay is based on the labeling of these nicks via the addition of FITC-labeled nucleotides to their 3’ ends mediated by the enzyme terminal deoxynucleotidyl transferase (TdT). The labeled specimen can be imaged right away in an epifluorescence or confocal microscope, or treated with anti-FITC antibodies for observation under a brightfield microscope.

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IX: PROTOCOL - Acetyl-tubulin/serotonin/DNA triple staining Purpose: Samples: start/end date:_________ RELAX IN RELAXANT SOLUTION 10 min (optional) FIX IN 4% FORMALDEHYDE 30-60 min. in 1X PBS Wash 5x in PBS, 5-10 min. [can store O/N in PBS if desired] Wash in PBS +0.1% Triton-X (PBTx)

PBTx: 50 ml PBS + 500 µl 10% Triton-X BLOCK in PBTx + 10% normal goat serum for 1 hour. (100 µl NGS in 900 µl PBTx)

NGS should be preincubated at 65C 1 hour, then aliquoted and stored at -20C. Thawed aliquots are stable several months at 4C

INCUBATE IN MOUSE ANTI ACETYLATED-TUBULIN ANTIBODY (1:100 dilution in blocking solution) and ANTI-SEROTONIN RABBIT POLYCLONAL ANTIBODY (1:100 dilution in blocking solution) overnight at 4 C degrees, or 4-8 hs at room temperature with shaking.

(mouse monoclonal anti-tubulin, acetylated, antibody) _________ µl ________ dilution (rabbit polyclonal anti-serotonin antibody) _________ µl ________ dilution __________ hours __________ temp

Wash PBTx at least 3 changes over at least 1-2 hours INCUBATE IN MIXED STAINING SOLUTION containing blocking solution, 1:200 secondary Abs and a nuclear marker

(prepare ∼50 µL per tube) for 2-5 hr at room temperature; protect from light: 45 µL PBTx 5 µL Normal Goat Serum 0.5 µL GOAT ANTI MOUSE- ________ <= conjugate fluorochrome 1 Ex. Max: 0.25 µL GOAT ANTI RABBIT-_________ <= conjugate fluorochrome 2 Ex. Max:

1 µL DAPI 10 ng/mL or TOPRO3 or YOYO Ex. Max: Wash in PBTx at least 3 changes over 10 minutes. Wash in PBS 1x at least 3 changes over 10 minutes. CLEAR IN GLYCEROL ( >1 hour 50% glycerol/1XPBS; then to >75%glycerol/1X PBS glycerol). Store at 4C in the dark in >75%glycerol/1X PBS. MOUNT IN ANTI-FADE MEDIUM; small clay feet or glass beads can be placed between the slide and the coverslip to avoid squashing the stained specimen. ________________________________________________________________________

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X. PROTOCOL - Alkaline phosphatase staining Purpose: Samples: start/end date:_________ RELAX IN RELAXANT SOLUTION 10 min (optional) FIX IN 4% FORMALDEHYDE 30-60 min. in 1X PBS Or FIX IN 1% GLYCERALDEHYDE 30-60 min. 1X PBS Wash 5x in PBS, 5-10 min. [can store O/N in PBS if desired] Wash 3x in PBS +0.1% Triton-X (PBTx)

PBTx: 50 ml PBS + 500 µl 10% Triton-X DEVELOP IN NBT/BCIP STAINING SOLUTION –

• 1 NBT/BCIP tablet (contains NBT, BCIP, Tris ph9.5, MgCl2) • 9 ml dH20 • 1 ml NaCl [1 M] (200 ul 5M NaCl / 800 ul dH2O)

Around 10 mins works fine for Pristina leidyi Wash 3x in PBT 5-10 min Wash 2x in PBS, 5-10 min CLEAR IN GLYCEROL ( >1 hour 50% glycerol/1XPBS; then to >75%glycerol/1X PBS glycerol). Mount in glycerol (75% glycerol/1X PBS). XI. PROTOCOL - Phalloidin Staining Samples: date:_____________ FIX 30-60 min. in 4% formaldehyde in 1X PBS Wash 5x in PBS, 15 min. [can store O/N in PBS if desired] Wash in PBS +0.1% Triton-X (PBTx)

PBTx: 50 ml PBS + 500 µl 10% Triton-X (10% Triton X in MQ H20) BLOCK in PBTx + 10% normal goat serum for 10 min. (100 µl NGS in 900 µl PBTx)


INCUBATE IN PHALLOIDIN (1:100 in PBTx) for 30-120 min at room temp (RT), or overnight at 4C. Alexa Fluor 488 phalloidin: Molecular probes cat #41C1-4 (dissolve in DMSO to 1U/ul and store frozen) _________ µl ________ dilution __________

45 µL PBTx 5 µL Normal Goat Serum 0.5 µL PHALLOIDIN-ALEXA FLUOR ______ <= conjugate fluorochrome 1 Ex. Max:

1 µL DAPI 10 ng/mL or TOPRO/YOYO Ex. Max: Wash in PBTx at least 3 changes over 10 minutes. Wash in PBS at least 3 changes over 10 minutes. CLEAR IN GLYCEROL ( >1 hour 50% glycerol/1XPBS; then to >75%glycerol/1X PBS glycerol). MOUNT IN ANTI-FADE MEDIUM; small clay feet or glass beads can be placed between the slide and the coverslip to avoid squashing the stained specimen.

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XII. PROTOCOL - BrdU Incorporation Purpose: Samples:

Start/end date:__________ - - - day 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - EXPOSE WORMS to 0.1 mg/ml BrdU for _________ hours.

[10 mg/ml BrdU stock: 10 mg/ml BrdU in dH2O]—aliquoted (40ul) and stored at -20C. 0.1 mg/ml BrdU (=326 µM) working solution: 40µL BrdU (10 mg/ml) + 4 ml spring water

RELAX IN RELAXANT SOLUTION 10 min (optional) FIX IN 4% FORMALDEHYDE 30-60 min. in 1X PBS Wash 2x-5x in PBS, 5-10 min. [can store O/N in PBS if desired] DENATURE DNA in HCl denaturation solution for 30 min at 37 C.

HCl denaturation solution: 3 parts concentrated HCl to 1 part dH2O. Make fresh before using (need ~0.5 ml/tube).[Concentrated HCl is typically 37.2% HCl ( 12.1 N). Old stocks may be 30% (9.76 N)]. Use glass Pasteur pipettes to dispense HCl – Do NOT use the regular Gilson pipetters! (HCl corrodes them.) ]

Wash in PBS 4 times until neutral pH (7.0). Use pH paper to determine if neutral pH has been reached. Wash in PBS +0.1% Triton-X (PBTx)

PBTx: 50 ml PBS + 500 µl 10% Triton-X BLOCK in PBTx + 10% normal goat serum for 1 hour. (100 µl NGS in 900 µl PBTx)


INCUBATE IN MOUSE ANTI-BRDU (1:20-1:100 dilution in blocking solution) overnight at 4 degrees, or 4-8 hs at room temperature with shaking.

_________ µL ________ dilution __________ hours temp Wash PBTx at least 3 changes over at least 1-2 hours INCUBATE IN SECONDARY ANTIBODY 1:200 for 2-5 hr at room temperature (dilute antibody in NGS block solution)

(O/N at 4C is OK too) - choose either a fluorochrome conjugate or HRP conjugate _________ µL ________ dilution __________ hours Goat anti-mouse, _____ conjugated

Wash in PBTx at least 3 changes over 30’. [If using HRP conjugate, skip to that section from here] Incubate with 25% glycerol/75% PBS for 10-30min in the dark Incubate with 50% glycerol/50% PBS for 30-60min in the dark Keep in 75% glycerol/25% PBS and store at 4C, protected from light MOUNT IN ANTI-FADE MEDIUM; small clay feet or glass beads can be placed between the slide and the coverslip to avoid squashing the stained specimen.

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- - - - - - - - - - - - - - - - - - -USING HRP CONJUGATE SECONDARY AB- - - - - - - - - - - - - - - - - DEVELOP

• Preincubate 10 min in ~300µL 0.5 mg/ml DAB + 0.064% NiCl in PBTx. (NiCl is optional, makes reaction more sensitive) Preincubation solution (Make FRESH): 1000 µL DAB 0.5 mg/ml in PBTx (aliquots at -20C) 10 µL NiCl 8% (made in dH2O, stored at RT)

• Add 1 µL H2O2 0.3% per 250-300µL and allow color to develop to desired intensity (about 10 mins work fine for Pristina). _________ minutes developing

• Stop reaction by washing multiple times with PBTx, then PBS. (Post-fixing optional)

MOUNT IN GLYCEROL ( >1 hour 50% glycerol/50%PBS; then to >75% glycerol). ________________________________________________________________________

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XIII. PROTOCOL - TUNEL staining of apoptotic cells Purpose: Samples: start/end date:_________ REAGENTS TO BE PREPARED FRESH Permeabilization Solution (0.1% sodium citrate in 1%Triton X-100 in PBS):

• 28.5 mg Na3C6H5O7. 2H2O • 22.5 mL of 1x PBS • 2.5 mL of 10% Triton X-100

Alkaline Phosphatase Buffer (100 mM NaCl, 100 mM Tris, 50 mM MgCl2 in 0.1 % Triton-X):

• 200 µL 5M NaCl • 500 µL 1M Tris • 1 µL MgCl2 • 100 µL 10% Triton-X • 9.35 mL dH2O

Alkaline Phosphatase Color Reaction Solution:

• 1 NBT/BCIP tablet (Roche, cat #1-697-471) (contains NBT, BCIP, Tris ph9.5, MgCl2) • 9 ml dH20 • 1 ml NaCl [1 M] (200 ul 5M NaCl / 800 ul dH2O)

dissolve tablet and keep in dark until ready to use (solution can be stored several days at 4oC) [to make solutions less than 10 ml, use 1/2 tablet dissolved in 1/2 solution] - - - - - - - - - - - - - - - - FIXATION/DEHYDRATION - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

� RELAX IN RELAXANT SOLUTION 10 min (optional) Time in RS:

� FIX IN 4% FORMALDEHYDE 20 min. in 1X PBS Time in FA:

� Wash with 25% methanol/75% PBS



� Wash 2x with 100% methanol

� Store at -20C overnight (optional) Date:

- - - - - - - - - - - - - - - - - -PERMEABILIZATION/TUNEL REACTION- - - - - - - - - - - - - - - - -

� Wash with 75% methanol/25% PBS Date:



� Wash 3x in PBS over 30 minutes W/Time

� INCUBATE IN PERMEABILIZING SOLUTION (0.1% Triton X-100 in 0.1% sodium citrate) for

2 min, 45 sec on ice

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� Wash 2x with PBS Washes

� DIGEST IN PRONASE 0.5mg/mL for 25 min Time

� Stop digestion by washing 2x with 2 mg/mL glycine in PBS Washes


� INCUBATE IN TUNEL REACTION MIX for 60 min at 37C in the dark

x Per tube (50 µL per sample)

45 µL Label Solution Temp

5 µL Enzyme Solution Time


[If using converting signal to ALP, skip to that section from here]

� Incubate with 25% glycerol/75% PBS for 10-30min in the dark Time

� Incubate with 50% glycerol/50% PBS for 30-60min in the dark Time

� Keep in 75% glycerol/25% PBS and store at 4C, protected from light

� MOUNT IN ANTI-FADE MEDIUM; small clay feet or glass beads can be placed between the slide and the

coverslip to avoid squashing the stained specimen.

- - - - - - - - - - - - - - - - - - -ALKALINE PHOSPHATASE SIGNAL CONVERSION- - - - - - - - - - - - - - - - -

[using Roche’s AP conversion kit]

� Wash with PBTx

� INCUBATE in 50 µL of 1:100 dilution in PBTx of mouse anti-FITC, ALP conjugated antibody for 30-60

min at 37C Time

� Wash 3x with PBTx over 5 min W/Time

� Wash 3x with ALP Buffer over 5 min W/Time

� DEVELOP by adding 1ml of ALP Color Reaction Solution in the dark (~5min) Time

� Wash 3x with PBTx over 5 min W/Time


� CLEAR IN GLYCEROL ( >1 hour 50% glycerol/1XPBS; then to >75%glycerol/1X PBS glycerol).

� MOUNT IN GLYCEROL; small clay feet or glass beads can be placed between the slide and the coverslip to

avoid squashing the stained specimen.

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