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Cnidarian and Ctenophore Lab Manual MBL Embryology Course 2010

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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.
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