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Developmental biology has contributed greatly to evolutionary biology in the past century. With the discovery that vertebrates share Hox genes with Drosophila in 1984, it became apparent that all animals evolved from variations of an ancestral embryonic patterning genetic tool-kit. In the dorsal-ventral (D-V) axis, a fundamental experiment was the Spemann-Mangold organizer transplant performed in 1924. Almost a century later, D-V genes have been subjected to saturating molecular screens in Xenopus and extensive genetic screens in zebrafish. A network of secreted growth factor antagonists has emerged, and we review here in detail the Chordin/Tolloid/BMP pathway. Chordin establishes a morphogen gradient spanning the entire embryo that was present even in the cnidarian Nematostella. This ancient system was present in Urbilateria, the last common ancestor of the protostome and deuterostome bilateral animals. We suggest that Urbilateria had a complex life cycle with an adult benthic form on the sea bottom, and also a primary larval pelagic or planktonic phase to disperse the species in the marine milieu. Larvae with two rows of cilia beating in opposite directions to entrap food particles, an apical sensory organ, and a rudimentary eye, are present in many protostome and deuterostome phyla. Although the larval phase has been lost multiple times in evolution, and larvae can adopt traits present in their adult forms, the simplest explanation is that Urbilateria had a pelago-benthic life cycle. The use of conserved developmental patterning systems likely placed evolutionary constraints in the animal forms that evolved by natural selection.
Fig. 1. Molecular screens of dorsal and ventral fragments of the early Xenopus laevis gastrula have yielded a plethora of novel genes involved in embryonic patterning. Screenings by multiple laboratories have been saturating and can be compared to the classical saturating genetic screens of zygotic genes that pattern the cuticle in Drosophila. The dorsal/ventral ratios of 40,000 protein-coding transcripts are listed in the Supplemental Tables of Ding et al. (2017). The genes mentioned here are not listed in order of enrichment but instead prioritized because they have been the subject of mechanistic studies. The Spemann organizer proved to be a rich source of Wnt, BMP and Nodal secreted inhibitors, revealing an unexpected importance of growth factor antagonists in embryonic patterning.
Fig. 2. The Chordin/Tolloid/Sizzled/BMP D-V morphogenetic signaling pathway in Xenopus. There are dorsal and ventral BMPs, which are antagonized by copious secretion of Chordin in the dorsal side. Black arrows indicate direct protein-protein interactions determined by biochemical methods. The patterning system is reinforced by transcriptional regulation indicated by blue arrows. In the ventral side BMP signaling is high and drives the transcription of BMP4/7, Crossveinless-2, xTolloid-related (Xlr) metalloproteinase, and Sizzled, a feedback inhibitor of Tolloid. The red arrow indicates the flux or facilitated diffusion of Chordin/BMP towards the ventral side, where Tolloid cleaves Chordin and releases BMPs for peak signaling. The rate limiting step is the Tolloid enzyme and the system is driven by the degradation of Chordin in the ventral side. This highly regulated long-range morphogen pathway ensures that the BMP gradient remains constant even during epiboly when the circular blastopore becomes increasingly smaller in size as it encloses the yolky endoderm, and when the embryo develops at variable temperatures.
Fig. 3. The Chordin extracellular protein gradient and its reciprocal nuclear phospho-Smad1/5/8 gradient. (A) Optical sections at mid-gastrula stained with an anti-Chordin affinity-purified antibody or (B) an antibody that recognizes the C-tail of the Smad1/5/8 transcription factors only after they have been phosphorylated by BMP receptors. Note that the Chordin gradient extends over a long distance, in this case 2 mm of circumference. Chordin is highly concentrated within the extracellular matrix that separates the ectoderm from endomesoderm (arrowheads). Experimental analyses showed that Tolloid is the key regulator of the BMP gradient. Horizontal optical sections of Xenopus mid-gastrula embryos (stage 11). Images reproduced with permission from Plouhinec et al. (2013); copyright 2013 Proceedings of the National Academy of Sciences.
Fig. 4. Transplantation of lineage-traced wild-type Xenopus organizer or ventral signaling center into host embryos depleted of BMP2/4/7/ADMP (BMP MOs), in which the entire epidermis becomes neural tissue (marked by Sox2), are able to restore epidermal differentiation (marked by Cytokeratin) at a distance of the graft. (A) Embryo at the neural plate stage showing epidermal differentiation. (B) The combined depletion of BMP2/4/7/ADMP morpholinos eliminates epidermal differentiation and the entire ectoderm becomes neural tissue. (C) A wild-type graft labelled with nuclear LacZ gives rise to notochord (lineage marked by LacZ), yet the organizer-secreted BMPs do not signal because they are blocked by Chordin and are only released in the ventral side by the action of Tolloid after long-range diffusion. (D) Wild-type embryo showing neural tissue marked by the pan-neural marker Sox2. (E) Upon depletion of four BMPs the entire ectoderm becomes neural, showing that neural differentiation is repressed by BMP signaling. (F) Transplantation of wild-type ventral center tissue restored epidermis in the graft and surrounding epidermis repressing neural differentiation and restoring D-V patterning. These experiments show that ventral BMPs, as well as dorsal BMPs, are able to diffuse over long distances in the Xenopus embryo. Images reproduced with permission from Reversade and De Robertis, Cell 2005; copyright 2005 Elsevier.
