Cnidarian and Ctenophore Lab Manual MBL Embryology Course 2010

Faculty: Mark Q. Martindale ([email protected]) Jonathan Q. Henry ([email protected])

TA: Kevin Pang ([email protected]) Jason Wever ([email protected])

Introduction

Cnidarians and ctenophores are two early-diverging metazoan phyla. Studying their respective development, using both traditional and new molecular tools, is improving our understanding of early metazoan evolution. While these two groups were initially combined as the ʻCoelenterataʼ, they are currently believed to be separate phyla. They are both important for understanding the evolution of axial properties and germ layers.

Cnidarians are a diverse phyla that include Anthozoans (corals, sea anemones), Staurozoans (stalked jellyfish), Scyphozoans (jellyfish), Hydrozoans (hydroids, Hydra), and Cubozoans (box jellyfish). Currently believed to be the sister-group of the Bilateria, they are relatively simple morphologically, while genomically quite complex. There is a huge diversity of life history strategeies and developmental modes within the Cnidaria.

Ctenophores, on the other hand, are believed to have branched off prior to the Cnidaria. While most cnidarians undergo ʻregulativeʼ development, ctenophores have a phylum-specific stereotyped cleavage program and mostly ʻmosaicʼ development. They represent one of the earliest groups to posses a nervous system and true muscles (non- epithelial).

I. Nematostella vectensis (Cnidaria: Anthozoa) II. Hydractinia echinata (Cnidaria: Hydrozoa) III. Mnemiopsis leidyi (Ctenophora)

Embryonic development of the cnidarian, Nematostella vectensis

Nematostella vectensis is an anthozoan cnidarian. Anthozoans have a polyp stage and a swimming planula stage. They lack the medusa (jellyfish) stage found in other cnidarians. It is currently believed that anthozoans, with just a polyp stage, reflect the ancestral cnidarian condition. The genome of Nematostella vectensis has been sequenced by the Joint Genome Institute (Putnam et al, 2007). Animals have separate sexes and will release eggs or sperm directly into the water column. They can be cultured in the lab using 1/3x filtered seawater (FSW) and fed with newly hatched artemia. Spawning can be induced year round using light/temperature cues. Development takes approximately five days from fertilized egg to settled polyp. (see Hand and Uhlinger 1992, 1994).

Embryonic development in Nematostella vectenis (Lee et al, 2007)

Development of Nematostella shown from egg to polyp, oriented with the oral pole to the left. (A) Unfertilized egg. The female pronucleus is visible adjacent to the cell membrane at one pole of the egg (arrowhead). (B) First cleavage (1-2 hpf). The first cleavage is unipolar and originates at one pole of the egg. (C) 4-cell stage. In most cases the first two cleavages are simultaneous and will give rise to a 4-cell embryo. (D) 8-cell stage. (E-F) Mid and late cleavage stages. (G) Blastula stage (4-6 hpf). Cleavage gives rise to a ciliated coeloblastula. (H) Early gastrula stage (12-15 hpf). Gastrulation initiates at one pole (animal pole, future oral pole) and proceeds via invagination. (I) Early planula stage (~24 hpf). Ectodermal and endodermal layers are well-organized and the planula exhibits directional swimming, with the aboral end forward, distinguished by the apical tuft (arrowhead). (J) Late planula. The planula elongates along the oral-aboral axis, and the primary mesenteries (arrows) begin to form. (K) Early tentacle bud stage. The first four tentacle primordia develop at the oral pole surrounding the mouth. (L) Juvenile polyp, with the primary mesenteries visible (arrows). (hpf, hours post-fertilization when raised at 25°C; *, site of gastrulation/oral pole; A-H bar ~60 µm; I-L bar ~90 µm)

Body plan and germ layers (Martindale 2005; Martindale et al, 2004)

Lateral View (oral pole up) Cross section through pharynx

Neural anatomy and cell type diversity (Marlow et al, 2009)

Techniques: Induction of spawning Animals are kept in the dark at 16°C in 1/3x FSW. They are fed freshly hatched artemia 3 times per week. The day before animals are to be spawned, they are fed chopped oyster and have their water changed. Bowls of animals are placed on a light box for 8 hours. They are removed from the light box, have their water changed, and kept out on the bench. They will spawn approximately 2 hours later (~10 hours after initial light exposure). Eggs and sperm are both released from the gastric cavity. Eggs are surrounded by a mass of jelly.

De-jellying Nematostella -Collect egg masses -Make up solution of 2-4% cysteine in 1/3 FSW (pH 7.4) -Pipet egg masses into a 15 ml conical tube with cysteine solution and place on a rocker for 10 minutes (must be done before first cleavage or after gastrulation or blastomeres will separate) -Rinse eggs 3-5 times with 1/3x FSW

Injecting Nematostella -Keep embryos in glass dishes until ready to inject them -Scratch several deep grooves on the injection surface of an UNCOATED 35 mm petri dish -Add approximately 20-30 embryos to the dish -Embryos will stick to the plastic for the first 20 min or so after they are added to the dish -When they are no longer sticky, move the embryos into the grooves -Inject as many as possible -From fertilization to first cleavage(s), there is approximately 2 hours

Beta-catenin:GFP mRNA Beta-catenin is a dual-function molecule, involved in both cell adhesion as well as axial patterning. In Nematostella, beta-catenin protein is initially expressed uniformly in the cytoplasm of the early embryo. Due to the early localization of Dishevelled at the animal pole, beta-catenin is stabilized in animal hemisphere, where it eventually gets translocated to the nucleus, thereby specifying the site of gastrulation and endomesoderm formation. Disruption of beta-catenin and/or Dishevelled localization results in abnormalities in endomesoderm formation (Wikramanayake et al 2003; Lee et al 2007). Injection of the Beta-catenin:GFP mRNA will show the protein distribution of Beta-catenin protein during development. Embryos will begin to express the construct at late cleavage stages, initially uniformly in the cytoplasm. During the late blastula stage, the protein will be localized to the nuclei in half the embryo (animal hemisphere) and eventually degraded in the other half.

Morpholino antisense oligonucleotides -MOs will be injected at 0.5 mM along with red fluorescent dextran

1. BMP2/4/Dpp (Splice-blocking) This TGF-β ligand is expressed asymmetrically from gastrulation and through development and may be playing a role in germ layer and axial specification. This morpholino should result in a block or delay in gastrulation.

2. AshA (Translation-blocking) Achaete-scute proneural homolog (bHLH family), expressed around 24 hpf weakly in the ectoderm (ubiquitously), then enriched in the oral and tentacular region. Likely involved in promoting neural markers/cnidocyte differentiation/possibly tentacle formation.

3. AshD (Translation-blocking) Another Achaete-scute homolog, expressed at 24 hpf weakly in the oral endodermal ring. During development, in planula stages it is expressed in the forming pharynx and the endodermal portion of the directive mesenteries. In polyps it is also expressed in the directive mesenteries. Likely promotes nematostome/cnidocyte formation, may also be involved in sensory and ganglion neuron development.

4. SoxB2 (Translation-blocking) This HMG-box transcription factor is expressed at gastrulation and through polyp stages in individual ectodermal and endodermal cells (see Magie et al, 2005). It is likely involved in neural development, however over-expression experiments have showed a delay in gastrulation. This would suggest knockdown would lead to expanded endoderm formation.

5. Six4/5 (Translation-blocking) Sine oculis-related homeobox 4/5 is expressed strictly zygotically in the presumptive endomesoderm starting at the blastula stage (10 hpf) and later in the entire endomesoderm after gastrulation. This gene is downregulated following U0125 treatment (MAPK inhibitor, which blocks gastrulation), suggesting it plays a role in gastrulation movements or endomesodermal downstream targets.

6. Ets1 (Splice-blocking) Ets and pointed domain transcription factor, expressed in two separate domains – in the presumptive endomesoderm and future aboral pole starting at the blastula stage (similar in expression to sprouty). Ets1 is a putative downstream target of the MAPK pathway and therefore may be required for gastrulation or endomesoderm specification.

7. Myc (Splice-blocking) C-myc is a bHLH class transcription factor, known for its role as an oncogene and in stem cell regulation. It is expressed around the edge of the tentacle field beginning in the late planula stage. Knockdown is predicted to affect tentacle formation.

8. Grainyhead1 (Translation-blocking) This transcription factor is primarily known for its role in Drosophila and mouse wound healing as well as ectoderm specification. It is expressed maternally, around the blastopore at gastrulation, and then becomes localized to the endoderm later in development. It may play a role in cell shape change or cell movement, and could possibly block gastrulation.

Fixation For larval and polyp stages (72+ hpf), relax embryos with magnesium chloride prior to fixation or imaging. In a small dish, gently pipet 7% magnesium chloride to the animals (about a third of the volume) and wait for animals to relax. They will either stop swimming (planula) or their tentacles will fully extend (polyps).

-Fix with 4% formaldehyde in 1/3x FSW for 1 hour at 4°C -Wash 3-5 times with PBS

Phalloidin/DAPI (or Hoechst) -After fixation, incubate with Phalloidin/DAPI for 20 min to 1 hr at room temperature -Wash twice quickly with PBS -Mount in 70% glycerol before visualization

Cnidocyte staining -Fix in 4% formaldehyde in 1/3x FSW, plus 10 mM EDTA (pH 7.6) for 1 hr -Wash 3-5 times in Tris buffer (10 mM NaCl, 10 mM Tris pH 7.6) -Stain for 10-20 min with 140 mM DAPI in Tris buffer -Wash 3 times in PBS -Mount in 70% glycerol before visualization

References:

Hand C, Uhlinger K (1992) The culture, sexual and asexual reproduction, and growth of the sea anemone Nematostella vectensis. Biol Bull 182, 169-176.

Hand C, Uhlinger K (1994) The unique, widely distributed sea anemone, Nematostella vectensis Stephenson: a review, new facts, and questions. Estuaries 17, 501-508

Lee PN, Kumburegama S, Marlow HQ, Martindale MQ, Wikramanayake AH (2007) Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Dev Biol 310, 169-186.

Magie CR, Pang K, Martindale MQ (2005) Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis. Dev Genes Evol 215, 618-630.

Marlow HQ, Srivastava M, Matus DQ, Rokhsar D, Martindale MQ (2009) Anatomy and development of the nervous system of Nematostella vectensis, an anthozoan cnidarian. Dev Neurobiol 69, 235-254.

Martindale MQ (2005) The evolution of metazoan axial properties. Nat Rev Genet 6, 917-927.

Martindale MQ, Pang K, Finnerty JR (2004) Investigating the origins of triploblasty: ʻmesodermalʼ gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum Cnidaria; class, Anthozoa). Development 131, 2463-2674.

Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, Salamov A, Terry A, Shapiro H, Lindquist E, Kapitonov VV, Jurka J, Genikhovich G, Grigoriev IV, Lucas SM, Steel RE, Finnerty JR, Technau U, Martindale MQ, Rokhsar DS (2007) Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86-94.

Wikramanayake AH, Hong M, Lee PN, Pang K, Byrum CA, Bince JM, Xu R, Martindale MQ (2003) An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation. Nature 426, 446-450.

Table of Contents

COELENTERATA

(HYDROZOA)

Hydractinia echinata

(Degenerate medusae)

Colonies of this form are fairly common on the Littorina shells inhabited by the small hermit crab, Pagurus. There are three types of individuals in the fully developed colony: (1) ordinary polyps (feeders), with a single whorl of tentacles; (2) thread-like coiling forms with no mouth and an apical knob of nematocysts (stingers, commonest around the lip of the shell); and (3) gonosomes. All three types arise singly from a hydrorhiza network covered by a rust-red spine-studded crust (Nutting, 1901).

The snails on which the colonies grow are common in the littoral near Woods Hole, Mass., and can be gathered in considerable numbers at Sheep Pen Harbor and Tarpaulin Cove.

A. Care of adults Colonies may be kept in large beakers or other deep vessels, supplied with running sea water.

B. Methods of Observation: If a number of snail-shells bearing ''male'' and "female" colonies are placed in a large uncovered dish of sea water and left overnight, eggs will usually be shed and fertilized between 7 and 9 A.M. on the following morning. Colonies kept in running sea water have been known to shed daily for a week before becoming exhausted. The shedding can be controlled by light, however, if eggs are desired at some other time of day. Colonies should be kept in running sea water, under a glowing 100-watt bulb, from the time of collection until gametes are needed. They should then be placed in the dark for one or more hours and subsequently re-exposed to light. By the use of a hand lens or dissecting microscope, the sexes can be segregated to separate fingerbowls of fresh sea water. The males will shed 50 minutes after re-illumination, the females five minutes later. The eggs should be transferred to fingerbowls of fresh sea water and inseminated with one or two drops of water taken from a dish of shedding males. Ballard (1942) gives further details of this method for controlling shedding.

A. Asexual Reproduction: The gonosomes, or reproductive individuals, are usually without tentacles and have a large knob of nematocysts on the proboscis; each bears a number of gonophores, which are medusa-buds reduced to the status of sporosacs. Ripe "male" and "female" colonies can be distinguished from one another with the unaided eye, since the eggs within the sporosacs are dull green against the red hydrorhiza, and the sperm, when mature, are a white mass. For details of gonophore development, see the papers by Goette (1907, 1916).

B. Sexual Reproduction: The maturation of the eggs within the gonophores occurs as a direct response to light and can be seen in eggs dissected from colonies placed in the light after several hours of darkness. In such eggs, the large germinal vesicle begins to break down soon after the exposure to light. The first polar body is given off 45 minutes after exposure to light, the second polar body ten minutes later. Occasionally the first polar body may divide. The eggs are shed immediately after the second maturation division (Ballard, 1942).

The eggs are yolky and usually green; occasionally grey, orange or pink ova are shed. Teissier and Teissier (1927) give the average egg-diameter as between 160 and 170 microns. When shed, the eggs are covered by a highly transparent, radially striated jelly, which swells on exposure to sea water. The swelling of this layer causes the polar bodies to be lifted from the egg surface and they are soon lost. Cleavage may be irregular, but usually the somewhat amoeboid egg undergoes three equal, total cleavages, each of which is at right angles to the preceding one. The separating pairs of blastomeres tend to retain broad protoplasmic connections with one another on the side opposite the cleavage furrow, until just before the succeeding cleavages begin. There is much variation in the time and degree of shifting of positions of the blastomeres, but the bizarre cleavage patterns often seen in the laboratory are commonly the result of evaporation of the sea water, or other unfavorable factors.

Mitotic synchronism quickly disappears. Gastrulation is said to start as early as the 16-cell stage, by mixed delamination and multipolar proliferation. The gastrula loses its spherical form and remains for a few hours an irregular mass; then it returns to the spherical form and gradually lengthens into the planula form. For illustrations of the cleavage pattern, see the papers by Beckwith (1914) and Bunting (1894).

C. Later Stages of Development and Metamorphosis: At the end of 24 hours the embryo is a "preplanula" (Teissier and Teissier, 1927) with an elongated oval form, recognizable polarity and ciliation. During the course of a few days, it lengthens, one end becoming progressively slimmer, while it rolls and crawls along the bottom like a planarian. The large end (which goes first in this movement) is the end which later produces the adhesive disc by which the larva attaches for metamorphosis; it becomes the aboral end of the polyp.

Following attachment of the attenuated planula, there is a delay of a few hours to several days, and then the tapering free end shrinks down almost to the substrate, where it produces a mouth and a succession of tentacles. The new polyp elongates, its attached end meanwhile actively sending out a number of anastomosing and encrusting hydrorhiza processes from which branch new polyps. For further details of planula development and metamorphosis, see the paper by Teissier and Teissier (1927) .

BALLARD, W. W., 1942. The mechanism for synchronous spawning in Hydractinia and Pennaria. Biol. Bull., 82: 329-339.

BECKWITH, C. J., 1914. The genesis of the plasma-structure in the egg of Hydractinia echinata. J. Morph., 25: 189-251. BERRILL, N. J., 1953. Growth and form in gymnoblastic hydroids. VI.. Polymorphism within the Hydractiniidae. J. Morph., 92: 241-272.

BUNTING, M., 1894. The origin of sex cells in Hydractinia and Podocoryne, and the development of Hydractinia. J. Morph., 9: 203-236.

GOETTE, A., 1907. Vergleichende Entwicklungsgeschichte der Geschlechtsindividuen der Hydropolypen. Zeitschr. f. wiss. Zool., 87: 1-336.

GOETTE, A., 1916. Die Gattungen Podocoryne, Stylactis und Hydractinia. Zool. Jahrb. abt. Syst., Geog., Okol. der Tiere, 39: 443-510.

NUTTING, C. C., 1901. The hydroids of the Woods Hole region. Bull. U. S. Fish Comm., 19: 325-386.

TEISSIER, L., AND G. TEISSIER, 1927. Les principales étapes du développement d Hydractinia echinata (Flem.). Bull. Soc. Zool. France, 52: 537-547. Ctenophores (“comb-bearers”): Mnemiopsis leidyi

This species of ctenophore is found along the Atlantic coast of North and South America, and easily caught off the NOAA jetty or in Eel Pond during the summer months. Adults are self-fertile hermaphrodites and can reach up to 12 cm in length. Located beneath the comb plates are the gonads (male and female) as well as photocytes (light producing cells), as ctenophores display a striking blue-green bioluminescence when disturbed. Development is relatively quick, from egg to cydippid larva in less than 24 hours. Spawning is induced by a light regime. Adults are caught during the day and kept under constant light. After they are placed in the dark, they will spawn in approximately 9 hours. Sperm are released just before the eggs (200 µm), therefore getting unfertilized eggs is impossible. Each adult can spawn thousands of eggs at one time. Eggs are surrounded by a vitelline membrane, which must be removed with forceps prior to any manipulation.

Body plan (Martindale 2005)

Lateral view Aboral view

Ctenophore development

Cleavages are unipolar, with the first cleavage starting at the animal (oral) pole. This first cleavage corresponds to the sagittal (oesophageal) plane, while the second cleavage corresponds to the tentacular plane. The third cleavage is unequal and oblique, giving rise to the E (end) and M (middle) macromeres. The next three divisions are highly unequal and give rise to micromeres at the aboral (vegetal) pole.

First cleavage occurs approximately 1 hour after eggs are released, and subsequent cleavages are every 10-20 minutes.

Gastrulation occurs via epiboly, as the micromeres surround and migrate over the macromeres (e-h). Gastrulation occurs at approximately 3-4 hpf (h). Just prior to gastrulation, the macromeres will give off a set of micromeres at the oral pole, which will give rise to the musculature, the putative ʻmesoderm.ʼ Cells continue dividing, with ectodermal thickenings giving rise to the future tentacle bulbs (k-l). The pharynx also begins to invaginate (j-l). Asterisks mark the animal pole/oral pole/blastopore. Comb plates form and they begin to swim around at 8-10 hpf (l). They will hatch out of their membranes at 14-20 hpf.

Experiments and Tips:

-Ctenophore embryos should be raised in filtered seawater (FSW). May need to rinse them a few times to make sure there is not too much mucus or debris in the dish.

-Eggs are approximately 200 µm in size. They are centrolecithal, with a large clear yolky center surrounded by a thin layer of cytoplasm.

-For any manipulations, the vitelline membrane must be removed. Use forceps to carefully remove the membrane. This is best done on eggs before they start cleaving. Once “shucked”, embryos must be kept on gelatin-coated dishes or else they will stick to the bottom. They are more fragile outside of their membranes, so be careful moving/pipetting/transporting.

Possible experiments include:

-blastomere separations or ablations

-injection of diI and following the lineage

-time-lapse microscopy (donʼt need to remove from membranes)

-inhibitor soaking treatments (cytochalasin B, aphidicolin, actinomycin D, puromycin)

-Phalloidin and/or antibody staining

Fixation: 4% formaldehyde in FSW for 30 min to 1 hr; wash 3-5 times with PBS (for cydippids, need to relax with a few drops 6.5% magnesium chloride 10 min prior to fixation)

-we have a few untested morpholinos (Glis, Six-class homeobox, TGF-β receptor), however injecting aqueous solutions is much more complicated than diI