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GDF9 Heterodimer GDF15 ZEBRAFISH DORSOVENTRAL PATTERNING Bmper (CV-2) PROVEINS SnapShot: Development BMP in Signaling James A. Dutko and Mary C. Mullins 19104, USA University of Pennsylvania School Medicine, Philadelphia, PA Department of Cell and Developmental Biology,

636 Cell 145, May 13, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.05.001 See online version for legend and references. SnapShot: BMP Signaling in Development James A. Dutko and Mary C. Mullins Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Bone morphogenetic proteins (BMPs), a major subgroup of the transforming growth factor β superfamily, are morphogens that participate in tissue patterning. Here, we highlight the versatility by which BMP signaling patterns tissues of the dorsoventral axis in early embryos, the fly wing disc, the mouse vertebral field, and neuroblasts in the developing neural tube. To activate the BMP signaling pathway, covalently linked BMP dimers (yellow and blue) bind and recruit two type I and type II serine-threonine kinase receptors. BMP molecules assemble into homo- and heterodimers and are then processed in the Golgi by furin-type proteinases, releasing a functional C-terminal ligand domain. In certain contexts, BMP heterodimers are more effective than homodimers in activating the pathway. Each type I receptor (teal and green) contacts both monomers of the ligand, and each type II receptor (maroon) contacts only one monomer. The constitutively active type II receptor phosphorylates a glycine-serine (GS)-rich domain on the type I receptor, activating it to phosphorylate the Smad intracellular effector proteins. The Smad family consists of BMP receptor-activated Smads (R-Smads, including Smads1, 5, and 8 [blue]), the common Smad (Smad4 [green]), and the inhibitory Smads (I-Smads, including Smads6 and 7 [dark blue]). R-Smads contain two MAD homology domains, MH1 (bright blue) and MH2 (light blue), separated by a linker region. Smad4 heterotrimerizes with two phosphorylated R-Smads via their MH2 domains. The heterotrimer subsequently translocates to the nucleus, binds DNA via MH1 domains, and recruits coactivators and corepressors to regulate gene expression. I-Smad6 and 7, which lack N-terminal DNA-binding domains, attenuate BMP signaling by blocking R-Smad phosphorylation and compete with R-Smads for the assembly of the Smad heterotrimer. BMP signaling is also negatively regulated by Smad ubiquitination regulatory factors (Smurf [red]) and phosphatases (dark blue). A throng of secreted molecules regulate the availability of BMP ligands for signaling. These factors function within robust and sophisticated systems to stimulate, attenuate, and refine BMP signaling levels. BMP antagonists, a collection of unrelated secreted proteins that contain cysteine-rich domains (numbered in Chordin), modulate BMP signaling by competing with receptors for ligand binding. Crossveinless-2 (CV-2, BMPER [light green]) can switch between BMP activation and inhibition, binding Chordin-BMP and Twisted gastrulation (TWSG1)-BMP, type I receptors, or heparin sulfate proteoglycans (HSPG). CV-2′s five cysteine-rich Von Willebrand factor type C (VWC1-5) domains and one D domain (VWD) bind BMP pathway proteins and HSPGs, respectively. BMP signaling is also regulated by the extracellular matrix, cell membrane coreceptors, and inhibitory pseudoreceptors.

Zebrafish Dorsoventral Patterning Zebrafish,Xenopus , and Drosophila exhibit related mechanics in the extracellular regulation of BMP signaling during dorsoventral (DV) axial patterning. In zebrafish, DV patterning is a paradigm for the obligate function of BMP heterodimers. A BMP morphogen gradient (blue to red), revealed by phosphorylated Smad1/5, persists throughout gastrulation with high signaling ventrally (blue) and complete signal attenuation dorsally (red). In this context, Bmp2b and Bmp7a function exclusively as a heterodimer with their correspond- ing type I receptors, Alk3/6 and Alk8 (Alk2 paralog). A BMP signaling gradient is formed by a combination of dorsally secreted BMP antagonists (Chordin, Noggin1 [Nog1], and Follistatin-like 1b [Fstl1b]) and ventrally secreted modulators Bmp1a, Tolloids, Twsg1a, and CV-2, which promote BMP signaling, and Sizzled, which opposes BMP signaling. Extracellularly, the signaling gradient is modulated by: (1) the ventral movement of BMP antagonists, (2) Tolloid-mediated cleavage of Chordin, (3) formation of a ternary complex of Twsg1a, Chordin, and BMP, either activating BMP signaling by converting Chordin to a better substrate for Tolloid or inhibiting BMP signaling in the absence of Tolloid, (4) CV-2 antagonism of Chordin, (5) Sizzled inhibition of Tolloid, and (6) maintenance of BMP ligand expression via positive feedback from BMP signaling. Patterning by BMP signaling during gastrulation is progressive temporally along the anteroposterior (AP) axis, with anterior cells patterned early in gastrulation and posterior cells later, suggesting coincident AP and DV fate acquisition. In Drosophila, a BMP transport mechanism generates the peak BMP ligand concentration. First, Sog (Drosophila Chordin) and Twsg bind to BMPs, which facilitates extracellular ligand movement. When the complex encounters Tolloid, it cleaves Sog, releasing the ligand and generating a peak of BMP signal.

Fly Imaginal Wing Disc Patterning The wing disc provides an example of BMP gradient formation through facilitated diffusion that emanates from a linear source of BMP in the absence of secreted BMP antagonists. Wing disc patterning requires two BMP ligands, decapentaplegic (dpp, blue) and glass bottom boat (gbb, orange), and two type I receptors, thickveins (tkv, green) and saxophone (sax, pink). Dpp preferentially binds Tkv, whereas Gbb preferentially binds Sax. dpp is expressed from a narrow stripe of cells at the anterior-posterior (AP) bound- ary, and an extracellular Dpp concentration gradient forms along the AP axis to specify four longitudinal veins at distinct AP positions within the disc. Although gbb is broadly expressed across the disc, only gbb expression overlapping with dpp expression acts in patterning the wing disc, suggesting the function of a Dpp-Gbb heterodimer in vein patterning. The BMP activity profile (red) as revealed by phosphorylated Mad (Smad1/5/8 ortholog) is a bimodal gradient with a low point within thedpp expression domain. The nonuniform and limited expression of Tkv across the wing disc plays an important role in shaping the BMP activity profile. tkv expression is repressed within the dpp expression domain, accounting for the low point of BMP activity between the two peaks of activity. The steeper slope of the gradient in the posterior disc is due to higher Tkv levels in the posterior compared to anterior compartment. The shape of the BMP activity gradient is further controlled by the GPI-anchored HSPGs, Dally and Dally-like, and the secreted Dally-interacting protein, Pentagone. The Dally proteins are required for the extracellular movement of ligand across the disc, whereas Pentagone, expressed in the disc periphery, facilitates distal transport of ligand, shaping the lateral edges of the gradient. Endocytosis is also required for BMP signaling in the wing disc but does not shape the gradient. Although the extracellular mechanisms modulating ligand availability and distribution in the wing disc versus DV patterning are distinct, both rely on multifaceted control combined with feedback regulatory mechanisms to provide robustness to perturbations, which ensures the proper functional output of the BMP signaling gradient in tissue patterning.

Mouse Vertebral Field A model for BMP signaling in the developing vertebral field presents a departure from traditional morphogen gradient models. BMP signaling levels manifest as alternating domains of high and low phospho-Smad (P-Smad1/5/8, yellow) in the vertebral body and the intervertebral disc, respectively. The intervertebral disc cells express BMPs (BMP2, 4, and 7), Chordin (CHRD), Chordin-like 1 and 2 (CHRDL1 and 2), TWSG1, and TLL1 (all in blue). Chordin protein is relocalized in a CV-2-dependent manner from the intervertebral disc to the vertebral body, possibly in a ternary complex with Twsg1 and BMPs. Cells of the vertebral body express CV-2 (green), where it is retained presumably via interaction of its VWD domain with HSPGs. The anti-Chordin activity of CV-2 in the vertebral body is thought to promote the release of BMPs from Chordin and also retain BMP, generating a domain of high BMP signaling.

Neural Tube Patterning The neural tube is another example of a localized source of BMPs forming a gradient that patterns a field of cells. The mechanism by which the BMP activity gradient forms in the dorsal neural tube is not clear; nevertheless, loss and gain of function for BMP ligands and receptors in mouse, chick, and zebrafish cause distinct neural tube cell types to be reduced or expanded, reflecting the BMP signaling levels present. Multiple BMP ligands are expressed from a dorsal organizing center, composed of roof plate cells and overlying ectoderm. BMP antagonists and Sonic hedgehog (Shh) are expressed from the ventral organizing center, the floor plate, and notochord. Together, these reciprocal gradients pattern the neural tube with the two organizing centers, antagonizing one another such that BMPs override induction of ventral markers, and the ventralizing activity of Shh is enhanced in the presence of BMP antagonists.

Protein Bank Identifiers Protein Data Bank Identifiers used in the figure are available at http://www.rcsb.org/pdb/home/home.do: 3H9R, 3EDG, 3G2F, 3BK3, 3HH2, 2H64, 1M4U, 1U5M, 1DD1, 1KHU, 3KMP, 1ZVD. Asterisk indicates an available structure.

636.e1 Cell 145, May 13, 2011 ©2011 Elsevier Inc. DOI 10.1016/j.cell.2011.05.001 SnapShot: BMP Signaling in Development James A. Dutko and Mary C. Mullins Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Acknowledgments

We thank Mullins lab members for helpful suggestions. J.A.D. is supported by NIH Training Program in Developmental Biology, grant 5T32HD007516. M.C.M. is supported by NIH grant R01-GM56326.

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