Anthozoan Cnidarian Development: the starlet sea anemone Nematostella vectensis

Woods Hole Embryology 2009 Instructor: Mark Q. Martindale

T.A.: Heather Q. Marlow

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Cnidarians and the Evolution of Development Cnidarians are diploblastic animals (possessing only endodermal and ectodermal tissues) with a single gut opening (the mouth). Within the metazoans (multicellular animals), cnidarians are one of the earliest branching taxa. They are among the first taxa with a nervous system, but are overall quite simple morphologically. This morphological simplicity as well as their phylogenetic position make them interesting subjects for evolutionary developmental studies. Cnidarians have considerable cell type diversity as well as a variety of gastrulation modes. As a phyla, cnidarians display extreme diversity in form and life history.

Embryonic Development in Nematostella vectensis

Nematostella vectensis is a anthozoan cnidarian. Anthozoans have only a polyp (adult) stage and a swimming planula (larval) stage, and lack the derived medusa (jellyfish) stage found in other cnidarians. Animals are gonochoric (each animal is one sex) and spawn unfertilized eggs or sperm into the water column. In the laboratory, animals can be cultured in 1/3x artificial sweater, fed newly hatched artermia and can be induced to spawn year round using light and temperature cues. Embryonic development takes five days from the time eggs are spawned through the development of the planula and finally settled polyp stage. The genome of N. vectensis has been sequenced, assembled and annotated by the Joint Genome Institute (Putnam et. al, 2007).

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Nematostella development (Lee et. al, 2007)

Embryonic development in Nematostella vectensis. Various stages of the embryonic development of Nematostella are shown from egg to polyp. Early gastrula to polyp stages are 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 h.p.f.). The first cleavage is unipolar and initiates at one pole of the egg. (C) Four-cell stage. In most cases the first two cleavages are simultaneous and give rise immediately to a 4-cell embryo. (D) Eight-cell stage. (E) Mid-cleavage stage. (F) Late cleavage stage. (G) Blastula stage (4–6 h.p.f.). Cleavage gives rise to a ciliated coeloblastula. (H) Early gastrula stage (12–15 h.p.f.). Gastrulation initiates at one pole of the coeloblastula and proceeds via invagination. (I) Early planula (not, vert, similar 24 h.p.f.). Ectodermal and endodermal layers are well-organized and the planula exhibits directional swimming with the aboral end, distinguished by the apical tuft (arrowhead), forward. (J) Late planula. The planula elongates along the OA axis and the primary mesenteries (arrows) begin to form. The apical tuft persists (arrowhead). (K) Early tentacle-bud stage of polyp. The first four tentacle primordia develop around the mouth at the oral pole. (L) Juvenile polyp. (h.p.f., hours post-fertilization at 25 °C; low

asterisk, site of gastrulation/oral pole; A–H, bar not, vert, similar 60 μm; I–L, bar not, vert, similar 90 μm).

Nematostella Anatomy and Cell Type Diversity (Marlow et. al, 2009)

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Spawning and de-jellying Nematostella Animals are kept at 16C in 1/3xFSW in the dark and are fed freshly hatched artemia 3x per week. A day or two before animals are to be spawned, they are fed with chopped oyster and the water is changed. Animals are placed on a light box for eight hours. Animals are then removed from the light box and water is changed with room temperature 1/3xFSW. Animals are then left out on the benchtop in ambient light. They spawn approximately 2 hours later.

1. Collect spawned egg masses 2. Incubate egg masses in 2-4% cysteine (pH 7.4) on a rocking table in a falcon tube

for ten minutes (must be done before first cleavage or after gastrulation) 3. Rinse eggs 5x in 1/3xFSW 4. Keep eggs in glassware until use for injections or fixation (they will stick to plastic)

Injecting Nematostella embryos 1. Keep embryos in glass dishes until you’re ready to inject them. 2. Scratch several deep grooves on the injection surface of an uncoated 35mM petri dish. 3. Embryos will stick to plastic for the first 20 minutes after they are added to the injection dish. Add approximately 20 to 30 embryos to a dish. Inject as many as possible within the first 20 minutes. When the embryos no longer stick on the plastic, move them into the grooves where they can be injected. 4. From the time they are fertilized to the first cleavage (embryos go directly to four cells), you will have two hours to inject. Beta Catenin Beta catenin is a cell adhesion molecule which is found at the cell surface, but it also has an important role in establishing early embryonic polarity. Beta catenin in Nematostella (as in many other bilaterians) is first found evenly distributed throughout the cytoplasm of the embryo. Due to the localization of the molecule disheveled at the animal pole of the embryo, Beta-catenin becomes localized to the nucleus in cells in the animal half of the embryo. These cells will give rise to the endoderm of the embryo and will also form the site of gastrulation. Disruption of beta-catenin or disheveled localization results in abnormal development of the endoderm. (Wikramanayake et. al, 2004)

Beta-catenin Injection Synthetic Beta-catenin RNA fused to a GFP marker when injected into Nematostella embryos will be translated and fluoresce green. The protein will be localized in the same manner as wildtype beta-catenin protein.

Cleavage stage

Blastula stage

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What to Look For: Embryos will begin fluorescing at late cleavage stages. Initially, the beta-catenin protein will be uniformly expressed throughout the embryo. During the blastula stage, the protein will be localized to the nuclei of cells in one half of the embryo. This marks the animal pole of the embryo. Morpholino Injections We will be injecting MO oligonucleotides at 0.5mM (a good starting concentration ranges from 0.25 to 1.0mM). As a tracer (to determine which cells or embryos you’ve injected), the MO are mixed with fluorescent dextra (red). Pax family transcription factors are homeodomain transcription factors that contain both a homeodomain and a paired domain, both of which bind DNA and influence transcription as well as an octapeptide domain. As in bilaterian animals, there are four classes of Pax genes in cnidarians. Pax genes are often involved in the development of the central nervous system and sensory organs. While two Nematostella Pax genes contain all three domains and are widely expressed in ectoderm, two additional genes (PaxA and PaxC) lack some of these domains and are restricted to specific cell populations in the ectoderm. What to Look For: Examine animals to look for absent or abnormal neural cell populations such as a reduced apical tuft or missing cnidocyte stining cells. PaxC: PaxC is a Nematostella Pax homolog which contains a homeodomain domain and a paired domain but lacks the octapeptide domain. It is expressed in a cell-type specific manner in both embryos, lavae (planulae) and polyps. 3. name: PaxCsplice PaxA: PaxA is a Nematostella Pax homolog which contains only the paired domain and lacks the homeodomain and octapeptide.

4. Morpholino 4 splice blocking name: For PaxAsplice2 (Matus et. al, 2007)

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Six family transcription factors are homeodomain-containing proteins. Three classes of six family genes, Six1/2, Six3/6 and Six4/5 are found in bilaterian animals. When functionally knocked-down, these genes cause striking anterior neural defects. Six1/2 and Six3/6 are involved in neural development in bilaterians while Six4/5 is associated with endomesoderm formation. What to Look For: Six3/6 has a broad aboral expression domain and is involved in neural ectoderm patterning in other animals. Watch for changes in apical tuft morphology or swimming behavior. Six1/2: Also known as sine oculis, this gene has important roles in anterior nervous system development and eye formation in bilaterian animals. 1. Morpholino 1 translation blocking name: six1/2trans Six3/6: Also known as optix, this gene is involved in a number of processes in neural development and eye formation in bilaterians. 2. Morpholino 2 translation blocking name: six3/6trans Sox/Fox genes: The Sox and Forkhead (Fox) gene families are comprised of transcription factors that play important roles in a variety of developmental processes, including germ layer specification, gastrulation, cell fate determination, and morphogenesis. Both the Sox and Fox gene families are divided into subgroups based on the amino acid sequence of their respective DNA-binding domains, the high-mobility group (HMG) box (Sox genes) or Forkhead domain (Fox genes). (Magie, et.al, 2005) What to Look For: FoxA is expressed throughout gastrulation and may play a role in establishing an ectodermal/endodermal boundary. Gastrulation or pharyngeal ectoderm defects are possible. FoxA: FoxA family transcription factors mark the blastopore during gastrulation in early Nematostella embryos and pharyngeal ectoderm in later stage embryos. 5. Name: FoxAtrans

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Forkhead expression in developing Nematostella embryos (Fritzenwanker et.al, 2004)

TGFbeta Signaling: The TGFbeta signaling family plays a diversity of roles during development in bilaterian animals including gastrulation, neural development, establishment of embryonic axes and left-right asymmetry. BMP molecules act as ligands for the TGF-beta receptors and can activate the pathway. BMP2/4: BMP2/4 (Decapentaplegic) is a TGF-beta ligand that acts as a morphogen in development and may act in combination with its antagonist chordin to play an important role during germ layer formation in Nematostella. 6. Name: Bmp2/4splice TGF-beta signaling pathwy member during early Nematostella development

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RNA-binding proteins: RNA binding proteins such as ELAV and Musashi (Msi) bind to 5’ or 3’ UTR regions of mRNA transcripts and influence stability and translation of the transcript. These proteins can have roles in cell differentiation or stem cell maintenace. Musashi: Msi acts to maintain cells in a proliferative stem state. 7. Name: msi93splice

(Marlow et. al, 2009) Pumilio: Pumilio is expressed in a wide variety of animals in the lineage giving rise to germ cells. It has an important functional role in maintaining the germline stem lineage. As in other RNA binding proteins such as Msi, Pumilio acts on the transcript of its target genes to affect translation or splicing. (Yale Passamanek, unpublished data)

Visualizing your Morpholino Injected Embryos To visualize general cell morphologies and ultrastructure you can utilize DIC imaging in live animals or fix your embryos for subsequent staining. Relaxation of Larval and Polyp Stages (72+ hpf): If embryos are older than 72 hours, it will be useful to relax them in magnesium chloride prior to fixing or imagin them.

1. Collect embryos in a small plastic or glass dish 2. Wait for animals to fully relax. Polyps should have extended tentacles. 3. Gently pipet in 7% MgCl2 (dissolved in dH2O) until the amount of magnesium chloride is

approximately 1/3x the total volume. Fixation (general purpose): 1. 4% formaldehyde in 1/3x filtered seawater (FSW) 2. Incubate at 4C for 1 hour

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3. Rinse 3-5 times in phosphate buffered saline solution Phalloidin and DAPI staining: Phalloidin is a mushroom toxin which binds to the actin in cell membranes. It can be ordered conjugated to many different fluorescent markers. DAPI binds to nuclei and is visible under UV. 1. Fix animals as above and then incubate in pre-mixed Phalloidin/DAPI solution for 20min-1hour at room temperature 2. Wash quickly two times in PBS 3. Mount in 70% glycerol and mount on a slide with clay feet Cnidocyte Staining:

1. Fix animals as above except substitute PBS for 1/3xFSW 2. Wash 3x for 5 minutes in Cnidocyte Buffer 3. Stain using pre-made DAPI cnidocyte stain 4. Wash 3x 10 minutes in PBS 5. Clear in 70% glyercol and visualize

References Byrum, C. A., and M. Q. Martindale. 2004. Gastrulation in the Cnidaria and Ctenophora. In C. Stern, Gastrulation. From cells to embryos pp. 33–50. Cold Spring Harbor Press, Cold Spring Harbor. Darling JA, Reitzel AR, Burton PM, Mazza ME, Ryan JF, Sullivan JC, Finnerty JR. Rising starlet: the starlet sea anemone, Nematostella vectensis. Bioessays. 2005 Feb;27(2):211-21. Nicholas H. Putnam, Mansi Srivastava, Uffe Hellsten, Bill Dirks, Jarrod Chapman, Asaf Salamov, Astrid Terry, Harris Shapiro, Erika Lindquist, Vladimir V. Kapitonov, Jerzy Jurka, Grigory Genikhovich, Igor V. Grigoriev, Susan M. Lucas, Robert E. Steele, John R. Finnerty, Ulrich Technau, Mark Q. Martindale, Daniel S. Rokhsar. Sea Anemone Genome Reveals Ancestral Eumetazoan Gene Repertoire and Genomic Organization. Science 6 July 2007: Vol. 317. no. 5834, pp. 86 – 94. Rentzsch F, Fritzenwanker J, Scholz C., Technau U. FGF signaling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. Development 135, 1761-1769 (2008). Joseph F Ryan, Maureen E Mazza, Kevin Pang, David Q Matus, Andreas D Baxevanis, Mark Q Martindale, John R Finnerty. Pre-bilaterian origins of the Hox cluster and the Hox code: evidence from the sea anemone, Nematostella vectensis. PLoS ONE. 01/02/200702/2007; 2(1):e153.

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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 asingle 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 ahydrorhiza 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 begathered 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 seawater.

B. Methods of Observation: If a number of snail-shells bearing ''male'' and "female" colonies are placed in alarge uncovered dish of sea water and left overnight, eggs will usually be shed and fertilized between 7 and9 A.M. on the following morning. Colonies kept in running sea water have been known to shed daily for aweek before becoming exhausted. The shedding can be controlled by light, however, if eggs are desired atsome other time of day. Colonies should be kept in running sea water, under a glowing 100-watt bulb, fromthe time of collection until gametes are needed. They should then be placed in the dark for one or morehours and subsequently re-exposed to light. By the use of a hand lens or dissecting microscope, the sexescan 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 waterand inseminated with one or two drops of water taken from a dish of shedding males. Ballard (1942) givesfurther details of this method for controlling shedding.

A. Asexual Reproduction: The gonosomes, or reproductive individuals, are usually without tentacles andhave 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 oneanother 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 byGoette (1907, 1916).

B. Sexual Reproduction: The maturation of the eggs within the gonophores occurs as a direct response to

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light and can be seen in eggs dissected from colonies placed in the light after several hours of darkness. Insuch eggs, the large germinal vesicle begins to break down soon after the exposure to light. The first polarbody is given off 45 minutes after exposure to light, the second polar body ten minutes later. Occasionallythe 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 bya highly transparent, radially striated jelly, which swells on exposure to sea water. The swelling of this layercauses 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 rightangles to the preceding one. The separating pairs of blastomeres tend to retain broad protoplasmicconnections with one another on the side opposite the cleavage furrow, until just before the succeedingcleavages 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 thesea water, or other unfavorable factors.

Mitotic synchronism quickly disappears. Gastrulation is said to start as early as the 16-cell stage, by mixeddelamination and multipolar proliferation. The gastrula loses its spherical form and remains for a few hoursan irregular mass; then it returns to the spherical form and gradually lengthens into the planula form. Forillustrations 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 thecourse of a few days, it lengthens, one end becoming progressively slimmer, while it rolls and crawls alongthe bottom like a planarian. The large end (which goes first in this movement) is the end which laterproduces the adhesive disc by which the larva attaches for metamorphosis; it becomes the aboral end of thepolyp.

Following attachment of the attenuated planula, there is a delay of a few hours to several days, and then thetapering free end shrinks down almost to the substrate, where it produces a mouth and a succession oftentacles. The new polyp elongates, its attached end meanwhile actively sending out a number ofanastomosing and encrusting hydrorhiza processes from which branch new polyps. For further details ofplanula 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 theHydractiniidae. J. Morph., 92: 241-272.

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

GOETTE, A., 1907. Vergleichende Entwicklungsgeschichte der Geschlechtsindividuen der Hydropolypen.

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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.