Keynote: The comparative analysis reveals independence of developmental processes during early development in frogs (ISMB 2009)

Eugenia del Pino

Studied “marsupial” frogs, found in the gardens of her university.

Introduction: Ecuador, Evolution & Embryonic Development. Ecuador is a small country in the northern part of S. America between Columbia and Peru, and the Andes run from north to south. The capital city is well over 2,000 m high. The Galapagos islands belong to Ecuador, and were never connected to the mainland as the islands are volcanic in origin. Darwin noted differences between the animals from these islands and those from the continent. And, there are no native frogs in the Galapagos, as the ocean was a barrier they could not naturally overcome. Together with Brazil and Columbia, Ecuador has the world’s largest diversity of frogs. Ecuador has 10-20% of all the terrestrial vertebrates.

There are foam-nesting frogs (Engystomops), Dendrobatid frogs (Colostethus), and Marsupial frogs (Gastrotheca) are just some of the types of frogs. She’ll compare these to X. laevis, which comes from S. Africa as the early development of this frog is better known than that of humans.

Part 1: The biology of different frogs. Of the 4 frog types mentioned, 2 develop rapidly – these are the aquatic X.laevis and the foam-nest E.randi. Both frogs are exposed to predation, and in the case of E.randi the dessication of the foam nest, and thus it is a benefit to have rapid development. The eggs are embedded in the foam nest, and can be collected and studied in the lab. After 2 days the eggs hatch. Interestingly, the embryos are white for camoflage.

The other two types mentioned have a slower development. Dendrobatid frogs include the poison arrow frogs, though they don’t study the poisonous ones. The males find an appropriate nest spot, then call the females. Males care for the embryos for 20 days. Tadpoles hatch from the nest and attach to the back of the father, and he takes them to the water. Female marsupial frogs have the pouch on the back which opens at the base of her back. The male pushes the eggs into her pouch with his feet. The mother transports the embroys for 4 months, and then puts them in the water by opening the pouch with her hind legs. The reproductive physiology of the frog resembles that of mammals. The physiology of the eggs does too – the eggs are much bigger (can get to 1 cm in diameter, when Xenopus are 1.2 mm in diameter). Eggs and embryos are white, and it is difficult to see the features of development because of that.

The dorsal blastopore lip (DBL) is a very important structure in the embryo, and the embryonic disk develops (the latter of which is not a characteristic of other frogs – it is a characteristic of chicken embryos).

Part 2: Comparison of frog development. Start by showing changes from gastrula to neurula in X.laevis. Then we were shown a video of this from Williams and Smith in Cambridge. Gastrulation begins with development of the DBL, then cells come to the DBL and move inside via involution. The lip grows from the ventral sides until it is a circle, and then involution occurs all around the embryo. Then the shape changes to that of a tadpole.

Why talk about gastrulation? It is a common process among vertebrates – it is during this time that the body plan is established with ectoderm, mesoderm, and endoderm. At the end of gastrulation, dorsal/ventral sides have been determined. A major problem of early development is to perform the change from sphere to elongated tadpole. The important movements are called the dorsal convergence and extension (CE). The planar cell polarity pathway controls CE. An important gene in this is Brachyury, which is expressed in the notochord and the entire future mesoderm.

The DBL is also called the “organizer” due to the work of Spemann-Mangold in 1924, who transplanted the DBL to the ventral side of a urodele gastrula, and the ventral side was then organized into a second embryo. Spemann got the Nobel Prize in 1935 (Mangold had died by that time). If the early DBL is transplanted, only the head is duplicated. If the late DBL is transplanted, only the trunk and tail are duplicated. Therefore the organizer is said to contain two separate parts. By 1991, a in-situ hybridization was developed for the study of frog embryos. During that time, Goosecoid was shown to be expressed in the DBL, and if it was expressed on the side, you get duplication: definitely an organizer gene. Then, in 1992, Lim1 was found to work in the same way. Since then, many more genes have found to be important both in DBL and in the ventral side. Organizer genes are conserved across vertebrates.

Comparison among the different frogs comes next. Time from fertilization to end of gastrulation: Xenopus (14 hrs),  foam-nesting (24 hrs), dendrobatid (4 days), marsupial frog (14 days). But, is the morphology of the gastrula similar? The cavity formed during gastrulation is the Archenteron (“the primitive cut”). This cavity is elongated during gastrulation. In the slowly-developing frogs there is delayed elongation of the archenteron. In other words, slow development is accompanied by a delay in archenteron elongation. Immunostaining is useful as a comparative tool for this work. Polyclonial antibodies were made for both Lim1 and Brachyury (from Tokyo and Tubingen, respectively). These are TFs and accumulated in nuclei. Notochord elongation and therefore dorsal CE begins in the midgastrula in rapidy-developing frogs. Conversely, notochord elongation and CE occur after blastopore closure in slowly-developing frogs. The pressure of elongating the body and finishing development isn’t as strong in slowly-developing frogs.

Implications of this work: frog gastrulation is modular, and CE is not essential for gastrulation. The important aspect of gastrulation is involution. Lim1 expression in the DBL is conserved. Lim1 expression in the mid-gastrula of rapidly-developing frogs. The head and trunk organizers are separable from each other. Therefore, there are different ways to make a frog 🙂 Embryonic development are difficult to study and model. Comparative approaches help a lot. However, there is a lack of molecular data for exotic frogs.

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Please note that this post is merely my notes on the presentation. They are not guaranteed to be correct, and unless explicitly stated are not my opinions. They do not reflect the opinions of my employers. Any errors you can happily assume to be mine and no-one else’s. I’m happy to correct any errors you may spot – just let me know!

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