STATEMENT OF RESEARCH 1

Organ fields

Statement of Research E. M. De Robertis

From the renewal of an HHMI Investigatorship (October 2009)

Molecular Mechanisms of Embryonic Self-regulation

During the period under review, we discovered a biochemical pathway of

interacting extracellular proteins that explains, for the first time, the molecular

mechanism of self-regulation of a morphogenetic field. Three new principles

emerged. First, we found that long-range cell-cell communication during dorsal-

ventral (D-V) patterning occurs through the action of extracellular regulators of

growth factors that control the flow of BMPs, which are then released by Tolloid

proteinases at a distance from where they were produced1-4. Second, we discovered

that the D-V BMP and the anterior-posterior (A-P) Wnt signaling gradients are

integrated via phosphorylations of the transcription factor Smad1/5/8 by BMP

receptor (BMPR) and GSK3, a protein kinase regulated by Wnt5,6. Third, we

uncovered unexpected connections between cell division and protein degradation7

that open new cell biological directions of great promise for the next funding

period.

Within the organism, cells proliferate, differentiate and

die as part of groups of hundreds or thousands of cells, called

morphogenetic fields, that have the remarkable property of self-regenerating after

perturbations. Fields were first identified by Ross Harrison in amphibian embryos8. He

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found that half of the mesoderm of a future forelimb would induce a whole limb - not

half a limb - after transplantation8,9. The Xenopus blastula also forms a morphogenetic

field, which can be bisected and still give rise to well-patterned

identical twins10,11. Understanding self-regulation has been the

long-term goal of my research12.

Chordin as the key to Spemann’s Organizer

Embryonic cell differentiation is regulated by a D-V activity gradient of

BMPs (Bone Morphogenetic Proteins, part of the TGF-β superfamily of growth

factors), transduced through phosphorylation of the transcription factors Smad1/5/8.

This morphogen gradient determines embryonic tissues13-15.

For example, in the ectoderm, BMP inhibition induces

Central Nervous System (CNS) while high BMP signals

induce epidermis. The precise allocation of tissue types requires a reproducible and

resilient molecular mechanism.

In 1924, Hans Spemann and Hilde Mangold performed a transplantation

experiment that started a new era in embryology. They discovered that a small

group of dorsal cells, called the organizer, was able to respecify the host’s

embryonic field, inducing a Siamese twin including a complete CNS16. Spemann

received the 1935 Nobel Prize in Physiology for this discovery of embryonic

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induction by organizer. Using a Xenopus Spemann

organizer cDNA library, we isolated many secreted

molecules expressed in this tissue, such as Chordin, Frzb,

Crescent, and Cerberus11. Surprisingly, most of these proteins were novel and

functioned as inhibitors of growth factors, rather than as growth factors as we had

expected.

The key molecule for understanding embryonic induction by organizer tissue is

Chordin17, a BMP antagonist secreted in prodigious amounts by the organizer

(estimated at 33 nM in the gastrula extracellular space if uniformly distributed, but

reaching much higher levels on the dorsal side2). As shown in the previous period,

transplanted organizers depleted of Chordin (using antisense morpholino oligos18)

lose all embryonic inductive power19. Chordin and a co-factor called Twisted

gastrulation (Tsg) form a ternary complex with BMPs, via the Cysteine-rich (CR)

modules in Chordin20,21. This inhibits binding to BMPR and the complex can

diffuse in the intercellular space. Inhibition is

reversible, for Zinc metalloproteinases of the

Tolloid (Tld) family can cleave Chordin at two

specific sites, releasing BMPs for signaling22.

Parallel work by other researchers in

Drosophila23, zebrafish13, and various

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organisms24 has established that the Chd/Tsg/BMP/Tld biochemical pathway

constitutes the ancestral embryonic D-V patterning mechanism.

Self-regulation is mediated by dorsal and ventral signaling centers

During the period being considered, we showed that embryonic self-

regulation requires a ventral signaling center. A duel between the dorsal organizer

and the ventral center - which secrete distinct sets of antagonists and BMPs under

opposite transcriptional control – explains self-regulation. A molecular see-saw, in

which BMP signals activate ventral

genes and repress dorsal ones, generates

a self-adjusting gradient of BMP

activity1,2. We elucidated the

extracellular biochemical pathway

illustrated nearby, in which blue arrows

indicate transcriptional regulation by

Smad1/5/8, black arrows show the direct

protein-protein interactions demonstrated

biochemically in our laboratory, and red

arrows denote the flux of Chordin/Bmp

complexes towards more ventral regions, as discussed below.

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Self-regulation through opposite transcriptional control of BMPs

The ventral center expresses BMP4 and BMP7 and the dorsal center BMP2

and ADMP25-27. Expression of BMPs in dorsal tissue is paradoxical, because this is

the region of the embryo where BMP signaling is lowest. Bruno Reversade and I

found that when all four BMPs are depleted simultaneously, the embryonic field

collapses and the entire embryo becomes covered in CNS1. By grafting wild-type

tissues into BMP-depleted embryos, we demonstrated that both the dorsal and

ventral centers serve as sources of

BMPs that diffuse over long distances

in the embryo, rescuing CNS (marked

by Sox2) and epidermal (cytokeratin)

patterning. These two sources of BMP

signals originating from opposite poles of the embryo generate a self-regulating

gradient1.

Extracellular D-V communication via Sizzled, Crossveinless-2 and Tolloid

During this period we also found that the BMP gradient is established and

self-regulated through three novel extracellular protein-protein interactions.

Sizzled is a Xenopus sFRP (secreted Frizzled-related protein) expressed

ventrally28. With Hojoon Lee we discovered that Sizzled binds to tolloids via its

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frizzled domain2. In enzyme kinetic analyses, Sizzled was a competitive inhibitor of

Chordin cleavage, with a Ki in the same range (20 nM) as the Km of the Tolloid

enzyme for its substrate2. This reaction provides self-regulation, for when BMP

levels increase, Sizzled expression is upregulated, causing Chordin levels to

increase, inhibiting BMP and restoring the gradient.

Crossveinless-2 (CV2) is a ventral center secreted protein that contains five

Chordin-like CR modules, first cloned in Drosophila by Seth Blair29 and

subsequently by us in the mouse30. CV2 has pro-BMP effects in Drosophila wing

cross veins23,29. However, overexpressed CV2 is a strong BMP inhibitor that binds

BMPs preventing BMPR binding31-33. CV2 remains tethered via glypicans/Dally to

the surface of the cells that secrete it31,32, mediating endocytosis of BMPs34. With

Andrea Ambrosio we found the molecular explanation for the pro-BMP effects of

CV2: it binds with high affinity (1-2 nM) to Chordin protein3. CV2 binds with even

higher affinity to Chordin/BMP complexes or Chordin

fragments cleaved by Tolloid protease. Thus, CV2

functions as a molecular sink, concentrating

Chordin/BMP complexes diffusing from more dorsal

regions3. Once in the ventral side, BMPs are released

from Chordin by Tolloid, allowing maximal BMP signaling.

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Tolloid chordinase activity is critical to D-V patterning, so we developed a

new fluorogenic substrate, mimicking a Chordin cleavage sequence, for enzyme

kinetic studies4. We discovered that BMP4 is a non-competitive

inhibitor of Tolloid or BMP1 (an alternatively spliced form of this

enzyme). BMPs were initially purified as bone-inducing proteins

from bone matrix. BMP2-7 were TGF-β-related, but why the BMP1

proteinase co-purified as well had remained a mystery35. We found

that BMP4 binds specifically to the “CUB” domains of BMP1/Tolloid with high

affinity (KD 15-20 nM), establishing an enzymatic

negative feedback loop in the ventral side4.

These three novel protein-protein interactions – Sizzled-Tolloid, CV2-

Chordin, and Tolloid-BMP4 – help explain how the dorsal and ventral centers

communicate over long distances to maintain a self-adjusting signaling gradient.

MAPK activation inhibits Smad1, explaining “heterologous” neural inducers

We became interested in Smad1 after finding that neural induction by IGF

and FGF is caused by MAPK phosphorylations in the central “linker” region of

Smad136 that inhibit its nuclear activity37,38. During the 1930s, amphibian

experimental embryology ground to a halt when it was found that dead organizer

tissue, or non-specific substances such as nucleoproteins, sterols, and even sand

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particles, could cause CNS differentiation when placed in contact with salamander

ectodermal explants10,11. We found that the neural induction caused by dissociation

of Xenopus ectodermal cells is due to sustained activation of MAPK and Smad1/5/8

inhibition39. Using Ambystoma maculatum embryos over three breeding seasons

(Jan.-Feb.), Cecilia Hurtado and I showed that “heterologous” neural inductions,

such as culturing ectoderm attached to a glass surface or sandwiched around sand

particles, were caused by MAPK/Erk activation40. The heterologous inducers that

had brought down Spemann’s edifice have finally found a molecular explanation:

the activation of MAPK linker phosphorylations that inhibit Smad1/5/8.

Integrating A-P and D-V positional information via Smad1 phosphorylations

When twins are generated, the D-V and A-P body axes are seamlessly

integrated. Christof Niehrs (Heidelberg) has proposed that the A-P axis is

established by a gradient of Wnt, maximal in the posterior41. During our

investigations on neural induction, we noticed GSK3 sites in Smad1/5/8 that could

be primed by the MAPK linker sites5 (GSK3 phosphorylates substrates containing

Serine or Threonine four amino acids upstream of a pre-phosphorylated residue42).

This was very exciting, because the canonical Wnt pathway signals by regulating

the activity of GSK343.

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With Luis Fuentealba and Edward Eivers, we generated high-titer phospho-

specific antibodies for human pSmad1GSK3 and pSmad1MAPK, and Drosophila

pMadGSK3 and pMadMAPK. This allowed us to identify a novel cell biological

pathway that terminates Smad1 signaling5,6. After

activation of BMPR, Smad1 is sequentially

phosphorylated at C-terminal, MAPK, and GSK3 sites.

Phosphorylation by GSK3 is required for

polyubiquitinylation by Smurf1 E3 ligase, transport along

microtubules to the centrosomes, and proteasomal

degradation5. Epistatic experiments in Xenopus5 and

Drosophila6 showed that Wnt/Wg signaling inhibits Smad1/Mad phosphorylation

by GSK3, and that Smad1/Mad are required for many aspects of Wnt signaling.

Thus, we have discovered a new branch of the “canonical” Wnt pathway

(previously believed to signal exclusively by increasing β-Catenin levels), in which

Wnt increases the duration of the BMP/Smad1 signal.

Embryonic positional information may be encoded by a system of Cartesian

coordinates in which the D-V BMP and the A-P Wnt

gradients are integrated at the level of Smad1/5/8

phosphorylations5. In this view, the BMP gradient

determines the intensity (or amplitude) and the Wnt

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gradient the duration of Smad1/5/8 signals24. Smad1/5/8 are transcription factors

that coordinate the expression of hundreds of downstream target genes; this hard-

wired system of signaling integration may provide resilience to the embryonic body

plan.

Conclusions

During the period under review we discovered a biochemical pathway for the

self-regulation of the Xenopus embryonic field. Self-regulation is mediated by

extracellular signaling between dorsal and ventral signaling centers that secrete

cocktails of BMPs and their regulators. These centers self-adjust to signaling

changes in one another because they are under opposite transcriptional control by

BMPs. The D-V BMP gradient is integrated with the A-P Wnt gradient

intracellularly through phosphorylations of Smad1/5/8 by GSK3, such that Wnt

increases the duration of the BMP signals.

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