The ventral to dorsal BMP activity gradient in the early ...The ventral to dorsal BMP activity...

Developmental Biology 378 (2013) 170–182

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Developmental Biology

0012-16http://d

n CorrE-m

journal homepage: www.elsevier.com/locate/developmentalbiology

The ventral to dorsal BMP activity gradient in the early zebrafish embryois determined by graded expression of BMP ligands

Marie-Christine Ramel, Caroline S. Hill n

Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3LY, United Kingdom

a r t i c l e i n f o

Article history:Received 18 December 2012Received in revised form26 February 2013Accepted 4 March 2013Available online 13 March 2013

Keywords:BMPGradientZebrafishBMP responsive elementBMP2B

06 & 2013 Elsevier Inc.x.doi.org/10.1016/j.ydbio.2013.03.003

esponding author. Fax: þ44 20 7269 3093.ail address: [email protected] (C.S. H

Open access under CC BY-NC

a b s t r a c t

In the early zebrafish embryo, a ventral to dorsal gradient of bone morphogenetic protein (BMP) activityis established, which is essential for the specification of cell fates along this axis. To visualise andmechanistically determine how this BMP activity gradient forms, we have used a transgenic zebrafishline that expresses monomeric red fluorescent protein (mRFP) under the control of well-characterisedBMP responsive elements. We demonstrate that mRFP expression in this line faithfully reports BMP andGDF signalling at both early and late stages of development. Taking advantage of the unstable nature ofmRFP transcripts, we use in situ hybridisation to reveal the dynamic spatio-temporal pattern of BMPactivity and establish the timing and sequence of events that lead to the formation of the BMP activitygradient. We show that the BMP transcriptional activity gradient is established between 30% and 40%epiboly stages and that it is preceded by graded mRNA expression of the BMP ligands. Both Dharma andFGF signalling contribute to graded bmp transcription during these early stages and it is subsequentlymaintained through autocrine BMP signalling. We show that BMP2B protein is also expressed in agradient as early as blastula stages, but do not find any evidence of diffusion of this BMP to generate theBMP transcriptional activity gradient. Thus, in contrast to diffusion/transport-based models of BMPgradient formation in Drosophila, our results indicate that the establishment of the BMP activity gradientin early zebrafish embryos is determined by graded expression of the BMP ligands.

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Introduction

During the development of multicellular organisms, a recurrentmechanism for tissue patterning is the formation of morphogengradients. The original definition of a morphogen is a moleculeproduced at a localised source which then diffuses into thesurrounding tissue to provide positional information and specifydifferent cell fates in a dose-dependent manner (Wolpert, 2011).Bone morphogenetic proteins (BMPs) have been described asmorphogens and BMP gradients have been documented in variousdeveloping organisms such as the sea urchin, Drosophila, Xenopus,zebrafish, and mouse (Ramel and Hill, 2012). The best understoodmodels of BMP gradient formation are the Drosophila BMP activitygradients. In the Drosophila embryo, the BMP gradient that isrequired for the specification of dorsal and lateral tissues involvesthe redistribution of the BMP ligands within a uniform expressiondomain (O'Connor et al., 2006). In the developing Drosophila wing,a BMP gradient forms across the Anterior/Posterior (A/P) axiswhich extends beyond the source of the BMP ligand Decapenta-plegic (Dpp; BMP4 orthologue). It is still debated whether this

ill).

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involves free diffusion, restricted diffusion, and/or transcytosis ofDpp (Erickson, 2011; Kicheva and Gonzalez-Gaitan, 2008;Schwank et al., 2011; Yan and Lin, 2009). Much less is understoodabout how BMP gradients are formed in vertebrate embryos. Thereis increasing evidence that the establishment of these gradients ishighly complex and finely regulated (Wolpert, 2011), and thatmechanisms operating in Drosophila are not sufficient to explainthem. A prerequisite for studying gradients in early vertebrateembryos is a sensitive molecular tool for directly visualising them.

The BMPs, along with the related growth and differentiationfactors (GDFs), constitute a subfamily of the transforming growthfactor β (TGF-β) superfamily (Schmierer and Hill, 2007). BMPs andGDFs signal through heteromeric serine/threonine kinase receptorcomplexes comprising type II and type I receptors (also calledALKs). Ligand binding promotes phosphorylation and activation ofthe type I receptor by the type II receptor, leading to phosphoryla-tion of a subset of receptor-activated Smads (R-Smads) (Moustakasand Heldin, 2009). Once activated, the R-Smads form heteromericcomplexes with Smad4, which accumulate in the nucleus andregulate target gene transcription. Importantly, the Smads con-stantly shuttle between the cytoplasm and the nucleus in both thepresence and absence of signalling and the levels of activatedcomplexes in the nucleus are determined by the relative activitiesof the receptor kinases in the cytoplasm and a nuclear phosphatase.

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M.-C. Ramel, C.S. Hill / Developmental Biology 378 (2013) 170–182 171

In the presence of signal the Smad nucleocytoplasmic shuttlingprovides a sensing mechanism for receptor activity (Schmierer andHill, 2007). For many years, TGF-β superfamily signalling was dividedinto two distinct branches with respect to the R-Smads: BMPs andGDFs were thought to signal exclusively through Smad1, Smad5 andSmad8, whilst TGF-β, Activin and Nodals signalled through Smad2 andSmad3 (Schmierer and Hill, 2007). However, it is now established thatin most cell types TGF-β additionally robustly activates Smad1 andSmad5 (Bharathy et al., 2008; Daly et al., 2008; Goumans et al., 2002;Liu et al., 2009; Wrighton et al., 2009). Hence, monitoring thephosphorylation status of a particular R-Smad is not a reliable wayto discriminate signalling by different ligands. A more specific methodis to exploit reporter plasmids with binding sites for transcriptionfactors specifically activated by the pathway in question. For BMP/GDFpathways, such a reporter was generated in which BMP responsiveelements (BREs) from the mouse Id1 enhancer, which bind phos-phorylated Smad1/5–Smad4 complexes, drive luciferase expression(Korchynskyi and ten Dijke, 2002). Most importantly, this BRE-luciferase reporter exclusively responds to BMP/GDF signals and notto TGF-β signals, despite the latter's ability to phosphorylate andactivate Smad1/5 (Daly et al., 2008; Gronroos et al., 2012; Liu et al.,2009). We have recently generated a fluorescent reporter transgeniczebrafish line using a modified version of this BRE-luciferase reporter(BRE-mRFP) (Wu et al., 2011).

In zebrafish embryos, a BMP activity gradient that is strongerventrally during the blastula and gastrula stages is required topattern the embryo along its dorsal/ventral (D/V) axis. Theexistence of a BMP activity gradient is evident when monitoringthe expression of BMP target genes during gastrulation, such asthe bmp ligands themselves or the ventral ectoderm markers gata2and ΔNP63 (Bakkers et al., 2002; Nguyen et al., 1998). The firstdirect visualisation of the zebrafish BMP activity gradient wasobtained using anti-phosphorylated Smad1/5 (p-Smad1/5) immu-nostaining (Tucker et al., 2008). This study concluded that aventral to dorsal gradient of nuclear p-Smad1/5 is set up between4.75 h and 5 h post-fertilisation (hpf; 30–40% epiboly stages).However, it is not known if the newly established BMP activitygradient (as detected with p-Smad1/5) is also reflected in tran-scriptional output as early as 30–40% epiboly, nor how it is formed.

Here we exploit our BRE-mRFP transgenic reporter line todetermine the mechanism by which the ventral to dorsal BMPactivity gradient is established. We first definitively prove that mRFPexpression faithfully reports BMP and GDF signalling at both earlyand late stages of development. Then, taking advantage of theunstable nature of mRFP mRNA and the sensitivity of its detection,we readily visualise the ventral to dorsal BMP transcriptional activitygradient that is generated during blastula stages, showing it isestablished by the 40% epiboly stage. We demonstrate using singleand double in situ hybridisation (ISH) that the graded BMP tran-scriptional activity occurs in a pre-established domain of graded bmpligand transcripts. Using anti-BMP2B antibody staining, we also showthat graded bmp ligand transcription results in graded proteinexpression. Moreover, in contrast to the mechanisms of BMP gradientformation in Drosophila, our results provide no evidence for diffusionor re-distribution of the BMP ligands during BMP gradient formation.Finally, we define the sequence of signalling events that lead tograded bmp transcription to generate a robust BMP activity gradient.

Materials and methods

Fish maintenance, screening, strains and crosses

The zebrafish colony was maintained as described (Westerfield,2000). Founders for the BRE-mRFP transgene (20% of F0 screened,n¼49) were identified by crossing injected adult zebrafish to wild-

type and screening their progeny for mRFP fluorescence at 24 hpf.Experiments described here used F2, F3, or F4 progeny of fivedifferent founders. The swrTA72A allele is a strong loss-of-functionallele corresponding to an ENU-induced point mutation in thezebrafish bmp2b termination codon (Kishimoto et al., 1997).

Plasmids, mRNAs, morpholinos and injections

The mRFP ORF was amplified from pcDNA-mRFP-N and clonedinto pGL3-BRE-luciferase (Korchynskyi and ten Dijke, 2002). BRE-mRFP was then amplified from pGL3-BRE-mRFP and cloned intopT2KXIGΔin. To generate the BRE-mRFP transgenic line thepT2KXIGΔin-BRE-mRFP plasmid DNA (20 ng/μl) was co-injectedwith transposase mRNA (25 ng/μl) as described (Kawakami et al.,2004; Urasaki et al., 2006). The noggin1 and chordin ORFs wereamplified from mid-gastrulation stage cDNA and cloned intopGEM-T and pCS2þ , respectively. The following mRNAs wereinjected: CA-alk8 (7.5 ng/μl) (Bauer et al., 2001), bmp2b (50 ng/μl)(Wu et al., 2011), chordin (320 ng/μl), membrane mRFP (50 ng/μl)(Kieserman and Wallingford, 2009) and cherry H2B (80 ng/μl)(Woolner and Papalopulu, 2012). The following MOs (Genetools,LLC) were used: dharma (Shih et al., 2010), chordin (Nasevicius andEkker, 2000), gdf6a (French et al., 2009), bmp2b (Imai and Talbot,2001) and bmp4 (Chocron et al., 2007). All MOs were injected at6 ng/nl except the gdf6A and bmp2b MOs which were at 3 ng/nl. Avolume of approximately 2.5 nl of DNA, mRNA and/or MO wasinjected per embryo.

In situ hybridisation, immunofluorescence, and movies

The following antisense probes were used: mRFP (Wu et al.,2011), dharma (Koos and Ho, 1998), noggin1, chordin (Miller-Bertoglio et al., 1997), goosecoid (Stachel et al., 1993), bmp2b(Kishimoto et al., 1997), bmp4 (Ramel, 2005), and bmp7a (Schmidet al., 2000). The standard ISH protocol was essentially asdescribed (Jowett, 2001) with the addition of 5% dextran sulphateto the hybridisation buffer (Lauter et al., 2011a). The doublefluorescent ISH (DFISH) using DIG- and DNP-labelled probes andrevealed with fluorescein tyramide and Fast Blue substrates wasmodified from Lauter et al. (2011b) (detailed protocol availableupon request). For p-Smad1/5 (Cell Signalling 9511, 1:100) immu-nofluorescence (IF), the protocol was as described (Tucker et al.,2008). The BMP2B (Tebu-Bio 55707, 1:500) and pMAPK (SigmaM8159, 1:500) IF protocols required an antigen retrieval step(Inoue and Wittbrodt, 2011). For ISH combined with IF, ISH wasperformed first. After ISH substrate development (Cy5 tyramide),embryos were washed in 1X PBS to stop the colourimetricreaction, then immersed in IF blocking solution before proceedingwith IF. Embryos that had undergone colourimetric ISH wereequilibrated in 80% glycerol and photographed using a LeicaDFC420C camera mounted on a Leica MZ-FLIII dissecting micro-scope. DFISH, immunostained and/or BRE-mRFP samples werecounterstained with DAPI (Roche 10 236 276 001), Hoechst33342 (Invitrogen H1399), or DRAQ5 (Biostatus DR50200),mounted in 0.8% low melt agarose (Sigma-Aldrich A9045) inglass-bottom microwell dishes (MatTek P35G-1.5–14-C) andimaged using a Zeiss LSM710 inverted confocal microscope.Movies were acquired using a Zeiss LSM710 inverted or a ZeissLSM700 upright confocal microscope and live embryos weremounted in low-melt agarose in fish water supplemented withanaesthetic. Supplementary movie 4 was obtained using a NikonECLIPSE TE2000-E inverted wide field microscope and an AndoriXonEMþ DU-888 back-illuminated EMCCD scientific camera.Images and movies were acquired and/or processed with thefollowing software packages: Leica Application Suite, Zeiss Zen,

M.-C. Ramel, C.S. Hill / Developmental Biology 378 (2013) 170–182172

Zeiss LSM Image Browser, Metamorph, Image J and AdobePhotoshop.

Protein preparation and Western blotting

Embryonic protein extracts were prepared as described (Wuet al., 2011). The following antibodies were used: rabbit anti-mRFP(Invitrogen R10367) (Fig. S3), mouse anti-mRFP (SignalChem R46-61M-100) (Fig. S2), rabbit anti-pSmad2 (a mixture of MilliporeAB3849 and 04953), mouse anti-pMAPK (Sigma M8159), mouseanti-Actin (Sigma 3853) and goat anti-MCM6 (Santa Cruz sc-9843).

Chemical inhibitor treatments

LDN-193189 (gift from Paul Yu), SU5402 (Calbiochem 572631),and SB-505124 (Tocris Bioscience 3263) stocks were made inDMSO. These inhibitors were diluted in fish water to obtain thedesired final concentration. 8- to 16-cell stage embryos wereimmersed in fish water supplemented with either DMSO (control)or the chemical inhibitors.

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Results

Characterisation of mRFP expression in BRE-mRFP embryos

To study BMP transcriptional activity and gradient formationduring zebrafish development, we used our previously generatedBRE-mRFP reporter transgenic line (Wu et al., 2011), employingboth ISH for mRFP and direct observation of mRFP fluorescence inthis line to assess BMP/GDF activity.

To observe early BMP signalling, we visualised mRFP mRNA,which gives a very sensitive and dynamic readout. mRFP RNA isexpressed ubiquitously in 8-cell stage embryos (Fig. 1A), suggest-ing that some mRFP mRNA is maternally deposited. mRFP mRNA isalso ubiquitous during blastula stages before and after the onset ofzygotic transcription, at the 512-cell and sphere stages, respec-tively (Fig. 1B). During gastrulation, mRFP transcripts are stronglyreduced dorsally and are enriched ventrally in both the mesodermand ectoderm, and thus transgenic embryos display a clear ventralto dorsal BMP activity gradient (Fig. 1C). During somitogenesis,mRFP expression is highly dynamic (Fig. 1D, S1). Most notably,mRFP mRNA is detected in the newly formed somites, the devel-oping eyes, tailbud and presomitic mesoderm, consistent withstudies implicating BMP activity in these tissues (Esterberg et al.,

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Video S3. Supplementary material related to this article can be found online athttp://dx.doi.org/10.1016/j.ydbio.2013.03.003.


2008; French et al., 2009; Patterson et al., 2010; Pyati et al., 2006).Finally, at 28 hpf,mRFPmRNA is found in additional tissues such asthe pineal gland, the developing heart, and the pectoral fin buds(Fig. 1E).

In BRE-mRFP embryos, mRFP fluorescence is only first visiblearound the 6-somite stage, when a strong signal is seen in the tailbud and presomitic mesoderm (Fig. S2A). This delay in detection isdue to insufficient mRFP protein being synthesised during gas-trulation for direct mRFP fluorescence visualisation, as full-lengthmRFP protein is virtually undetectable before the 6-somite stageusing Western blotting (Fig. S2B). During somitogenesis, thestrongest mRFP protein expression domains are the tail bud andthe presomitic mesoderm (Fig. S2A; Videos S1,S2), thereby con-firming the in situ results. Most notably, at 24 hpf, strong mRFPfluorescence is detected in the dorsal retina, the epiphysis/pinealgland, the epidermis, the cloaca, and the somites (Fig S2A andVideo S3) (Alexander et al., 2011; Collery and Link, 2011; Frenchet al., 2009; Laux et al., 2011; Patterson et al., 2010; Pyati et al.,2006; Row and Kimelman, 2009). In addition, mRFP fluorescenceis found in the cells of the midbrain–hindbrain border and thetrigeminal ganglia (Fig. S2A) (data not shown). Finally, mRFPfluorescence is detected in the atrioventricular canal of 50 hpftransgenic embryos (Video S4), consistent with reported BMPactivity during mammalian early heart valve formation in theBRE-GFP transgenic mouse (Monteiro et al., 2008).


The BRE-mRFP transgenic zebrafish is a bona fide in vivo reporterfor BMP/GDF activity.

To establish unequivocally that mRFP expression reflects BMP/GDF signalling, we manipulated BMP signalling at different timesduring embryogenesis and assessed its effect on mRFP mRNA orprotein levels.



Stimulation of BMP signalling using overexpression of BMP2B(a zygotic BMP ligand) (Nguyen et al., 1998) and the constitutively-active BMP receptor CA-ALK8 (Bauer et al., 2001) or knockdown ofthe BMP inhibitor Chordin (Schulte-Merker et al., 1997) resulted inincreased mRFP expression and expansion of the mRFP domain tothe dorsal side (Fig. 2A). Conversely, overexpression of Chordinand the BMP antagonist Noggin (Zimmerman et al., 1996) com-pletely abolished mRFP expression at 70–80% epiboly (Fig. 2A) (Wuet al., 2011). Thus, changes in BMP activity are reflected in mRFPexpression along the D/V axis of the gastrulating embryo.

We also used a genetic approach to inhibit zygotic BMP activity,investigating mRFP expression in a bmp2b-deficient background(Fig. 2B). At 70% epiboly, mRFP expression is strongly reduced inhomozygous swrTA72A (bmp2b−) embryos. From bud stage/earlysomitogenesis, swrTA72A/swrTA72A embryos display a striking elon-gated morphology (Mullins et al., 1996) unlike their swrTA72A/þand wild-type siblings. In 4-somite stage swrTA72A homozygousembryos, mRFP expression is also strongly reduced. No differencein mRFP expression between wild-type and swrTA72A heterozygoussiblings was detected (data not shown).

BMP activity is important at the end of gastrulation to specifyventro-posterior fates, including formation of the cloaca, thecommon opening of the urogenital and digestive tracts in zebra-fish (Pyati et al., 2006, 2005). BMP4 is thought to be the criticalzygotic ligand for the specification of cloacal fates as severebmp4ST72 homozygous zebrafish mutants do not form a propercloaca (Stickney et al., 2007). Injection of a splice-blocking MO thattargets bmp4 mRNA (Chocron et al., 2007) results in severelyreduced mRFP expression in the cloaca of 28 hpf embryos, butstrikingly, has no effect on mRFP expression in the somites whereother BMP ligands must contribute to mRFP transcription (Fig. 2C).

The dorsal retina is another region of high mRFP expression(Fig. 1E, S1, S2A). The GDF6A ligand is required for p-Smad1/5activation in the dorsal retina (French et al., 2009), suggesting thatit might be the ligand responsible for mRFP expression in thistissue. Injection of a gdf6a splice-blocking MO results in reduced

http://dx.doi.org/10.1016/j.ydbio.2013.03.003http://dx.doi.org/10.1016/j.ydbio.2013.03.003http://dx.doi.org/10.1016/j.ydbio.2013.03.003http://dx.doi.org/10.1016/j.ydbio.2013.03.003

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Fig. 2. mRFP expression in BRE-mRFP embryos is a bona fide readout for BMP and GDF activities (A) mRFP ISH in BRE-mRFP embryos injected with chordin, bmp2b or CA-alk8 mRNAs or chordin MO. (B) BRE-mRFP expression in swrTA72A/swrTA72A homozygous embryos compared with þ/þ or swrTA72A/þ siblings. The embryos shown resultedfrom a cross between swrTA72A/þ heterozygotes that contained the BRE-mRFP transgene. (C) mRFP ISH in BRE-mRFP embryos injected with a control or bmp4 MO. Arrowspoint to the cloaca. (D) BRE-mRFP embryos were injected with a control or gdf6aMO and mRFP expression in the dorsal retina was analysed (fluorescence and ISH). pg, pinealgland; s, somites. In (A) and (B), lateral views are shown; V, ventral; D, dorsal; A, anterior; P, posterior. In all cases, stages are indicated and the number of embryos out of thetotal analysed that showed the presented staining pattern is given. For (B), this number represents the number of homozygous mutant embryos out of the total number ofembryos analysed.


dorsal retina mRFP mRNA expression as well as reduced mRFPfluorescence in 26 hpf embryos (Fig. 2D), confirming that the BRE-mRFP transgenic line is also sensitive to changes in signalling byligands from the GDF subfamily. We were unable to detect anyeffect of GDF6A knockdown on mRFP expression in other tissuessuch as the somites, despite a recent study implicating GDF6A as afactor required for somitic BMP activity during myogenesis(Nguyen-Chi et al., 2012).

Finally, we decreased BMP/GDF signalling globally infish embryos using the chemical inhibitors Dorsomorphin andLDN-193189, which target the type I BMP/GDF receptors, and leadto a dorsalized phenotype (Cannon et al., 2010; Yu et al., 2008)(Fig. S3). The chemical inhibitors are autofluorescent, resulting inconsiderable background when mRFP fluorescence is visualised.However, inhibition of mRFP expression is readily detected bymRFP ISH and Western blotting for mRFP protein (Fig. S3).

Taking all the results in this section together, we conclude thatmRFP expression in the BRE-mRFP transgenic embryos is anaccurate readout of dynamic BMP/GDF activity at all stages ofdevelopment examined.

The initial ventral to dorsal BMP transcriptional activity gradient isestablished between 30% and 40% epiboly

The ability to visualise the ventral to dorsal BMP activitygradient in the BRE-mRFP embryos by monitoring levels of mRFPmRNA gave us a unique opportunity to investigate in detail howthis gradient forms.

From the 8-cell to the 30% epiboly stage, mRFP expression isuniform (Fig. 3). The mRFP transcripts detected at the 8-, 256-, and512-cell stage must be deposited maternally. In contrast, specificnuclear p-Smad1/5 staining is only apparent from the 512-cell

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Fig. 3. Establishment of the BMP activity gradient in zebrafish embryos. p-Smad1/5 IF and mRFP ISH in early stages BRE-mRFP embryos. Stages and views are indicated. V,ventral; D, dorsal. Arrows indicate the extent of mRFP staining. Note that the DAPI staining at the 8-cell stage did not resolve to single nuclei as the cells are dividing veryquickly.


stage onwards (Fig. 3) and must occur as a result of maternal BMPsignalling initially (Goutel et al., 2000; Sidi et al., 2003), then as aresult of zygotic BMP signalling. At 40% epiboly, mRFP mRNA isclearly enriched ventrally and expressed in a graded fashion inBRE-mRFP embryos (Fig. 3). Comparison of the p-Smad1/5 stainingwith the mRFP expression pattern revealed that decrease ofp-Smad1/5 from the dorsal side at 30% epiboly fractionally pre-ceded loss of mRFP expression (Fig. 3). Hence, the BMP transcrip-tional activity gradient (mRFP) is established between 30% and 40%epiboly and occurs minutes after ventral enrichment of p-Smad1/5. Notably, clearance of mRFP transcripts dorsally occurs in a veryshort time frame (~20 min), confirming that the mRFP mRNA half-life is very short. Thus, observing mRFP transcripts at these earlystages provides a very dynamic readout of the BMP activitygradient. Moreover, this comparative experiment shows that inthe case of early zebrafish embryogenesis, it is also possible to usep-Smad1/5 immunostaining as a readout for BMP pathwayactivation.

The BMP transcriptional activity gradient is established in a domainof graded ligand transcription

Having determined exactly when the BMP activity gradient isinitially formed, we explored the mechanism underlying it. Theexpression of zygotic bmp ligands is thought to be set up in twokey steps. First, after the mid-blastula transition (MBT; ~3.25 hpf),a combination of the maternal BMP ligand GDF6A (possibly actingthrough maternal ALK8 and Smad5) and the transcription factorPOU2 induce the ubiquitous expression of zygotic bmp2b andbmp7a, which is shortly followed by bmp4 expression (Langdonand Mullins, 2011). Second, the sequential action of dorsally-expressed negative regulators contributes to the increase inventral expression of the BMP ligands. As early as sphere stage(~4 hpf), Dharma, a transcriptional repressor that is expressed inthe dorsal-most blastomeres, directly represses bmp2b transcrip-tion (Koos and Ho, 1999; Leung et al., 2003). Subsequently, acombination of the dorsally-secreted BMP antagonists Noggin1

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Fig. 4. The establishment of the BMP activity gradient occurs in a pre-established gradient of BMP ligand expression. (A) BRE-mRFP or wild-type embryos stained for mRFP,dharma, noggin1, chordin, bmp2b, bmp7a or bmp4 mRNA by ISH or immunostained for pMAPK. Arrows indicate the extent of the domain of staining for a particular mRNA.The number of embryos out of the total analysed that showed the presented staining pattern is given. (B) DFISH for mRFP and bmp2b in blastula stages BRE-mRFP embryos.The fluorescent substrates used were fluorescein tyramide (mRFP) and Fast Blue (bmp2b, pseudocoloured in red). The images are the projection of a 10–12 picture stack takenat the margin of the blastoderm to avoid fluorescence from the yolk. V, ventral; D, dorsal.


and Chordin acts to inhibit BMP signalling, with Chordin beingessential for BMP signalling repression (Dal-Pra et al., 2006). It isnot clear though when Chordin begins to confer its effect on BMPligand expression. In addition, there is evidence that the FGFsignalling pathway acts on the dorsal side to repress bmp tran-scription (Furthauer et al., 2004). As a result of all these interac-tions bmp ligand expression is clearly graded by the end of theblastula stage (Furthauer et al., 2004; Sidi et al., 2003). However, itis not known how the expression domain of the ligands relates tothe BMP activity gradient or to transcriptional output during thesecritical stages.

To establish the sequence of events between dome stage and40% epiboly, we compared the timing and pattern of mRFP mRNAexpression with that of the three zygotic BMP ligands: BMP2B and

BMP7A, which act as obligate heterodimers (Little and Mullins,2009), and BMP4. We also investigated expression at the level ofmRNA of the BMP antagonists expressed dorsally at blastulastages, Chordin and Noggin1, the transcriptional repressorDharma, as well as dorsal FGF activity. At dome stage, dharma,noggin1 and chordin are all expressed dorsally, and in the case ofnoggin1 and chordin, expression increases at 30% and 40% epiboly(Fig. 4A). We monitored FGF activity by immunostaining for di-phosphorylated MAPK (pMAPK) (Jurynec and Grunwald, 2010),and demonstrated that FGF is active at all stages examined, withstronger activity detected on the dorsal side. This is especiallyclear for pMAPK staining at dome stage (Fig. 4A). bmp2b andbmp7a transcripts are expressed uniformly throughout most of theembryo at dome stage, except on the most dorsal side, where a


clear gap in staining is visible (Fig. 4A). At 30% and 40% epiboly,bmp2b and bmp7a show a clear asymmetric pattern, being stron-ger on the ventral side and weaker on the dorsal side (Fig. 4A), inagreement with reports that bmp2b and bmp7a are expresseduniformly upon activation of zygotic transcription, then becomeenriched ventrally (Hammerschmidt and Mullins, 2002; Leunget al., 2003). bmp4 expression, in contrast, is detected ubiquitouslyat dome stage (Fig. 4A) and only becomes ventrally enriched laterat 30% and 40% epiboly (Fig. 4A) (Langdon and Mullins, 2011).Importantly, despite bmp ligands being expressed in a gradedmanner from 30% epiboly, mRFP transcripts remain uniform in themajority of embryos examined. In contrast, at 40% epiboly, thegradient of mRFP, reflecting BMP transcriptional activity, wasevident, and strikingly, it appears very similar to the expressionpatterns of the ligands (Fig. 4A).

To confirm that the gradient of BMP ligand expression was thesame as the gradient of BMP activity, we used DFISH for bmp2band mRFP (Fig. 4B). As DFISH caused substantial autofluorescencefrom the yolk we visualised mRFP and bmp2b mRNAs at theblastoderm margin only. As expected, bmp2b is expressed in aventral to dorsal gradient at both 30% and 40% epiboly andconsistent with the single ISH results (Figs 3 and 4A), mRFP isonly expressed in a gradient from 40% epiboly. DFISH shows a verygood overlap of bmp2b and mRFP transcripts at 40% epiboly,confirming that at this stage the BMP transcriptional activitygradient occurs in a pre-established domain of graded ligandtranscription.

Graded bmp transcription results in a BMP protein gradient with littleor no transcriptional activity at a distance from BMP protein-expressing cells

Our observations that the transcripts of the three major BMPligands are expressed in a gradient as early as 30% epiboly does notnecessarily mean that the BMP proteins are also expressed in agradient at that time. Indeed, post-transcriptional processing,diffusion, transport and other regulatory mechanisms could resultin BMP protein expression differing from that of the bmp tran-scripts. As no data are available for endogenous BMP proteinexpression during early stages of zebrafish development, we useda new commercial zebrafish specific BMP2B antibody to visualiseBMP2B protein expression.

Analysis of BMP2B protein expression revealed a graded stain-ing pattern at all stages examined, from 30% to 75% epiboly(Fig. 5A and data not shown). At 30% epiboly, strong BMP2Bprotein expression is detected on one side of the embryo, whileBMP2B protein levels are much lower on the opposite side. At 60–70% epiboly, a clear gradient of BMP2B protein expression isdetected in both the mesoderm and ectoderm. To confirm thatthe enrichment of BMP2B protein is indeed ventral, we comparedBMP2B with pMAPK expression and found that strong BMP2B andpMAPK staining was obtained on opposite sides of the embryo at30% epiboly (Fig. S4A). Using combined ISH for the dorsal markerchordin and IF for BMP2B, it was also evident that BMP2B proteinsare enriched ventrally and expressed in a gradient during gastru-lation (Fig. S4B). Close analysis of wild-type embryos injected withmembrane mRFP mRNA revealed that the BMP2B protein isexpressed in punctate cytoplasmic structures (Fig. S4C). Since theantibody used was raised against the prodomain region of zebra-fish BMP2B (Tebu-Bio/Anaspec, personal communication), it isexpected to detect pre-processed BMP2B. As a further check forspecificity we performed IF in embryos overexpressing BMP2B andthe nuclear marker Cherry H2B in a mosaic fashion and found thatincreased BMP2B immunoreactivity correlates with cells over-expressing BMP2B/Cherry H2B (Fig. S4D). We also injectedembryos with a translation blocking bmp2b MO (Imai and Talbot,

2001) and examined BMP2B expression in the blastoderm at the30% epiboly stage. BMP2B expression was reduced in the majorityof the bmp2b morphants compared with controls (Fig. 5B).

We have shown at 40% epiboly that the gradient of bmp2bmRNA expression matches the BMP activity gradient read out bymRFP mRNA (Fig. 4B). In contrast to what has been reported forDpp in the Drosophila wing (Entchev et al., 2000; Gibson et al.,2002; Teleman and Cohen, 2000), this result strongly suggests thatactive BMP ligands do not diffuse significantly beyond the cellsthat synthesise them in the zebrafish embryo. Our ability to detectboth an endogenous BMP protein and BMP transcriptional activityin the same embryo allowed us to determine whether transcrip-tional activity can be detected at a distance from BMP proteinexpressing cells. Using combined ISH for mRFP with IF for BMP2Bin 40% and 70% epiboly BRE-mRFP embryos, we found that theexpression of mRFP mRNA and BMP2B protein strongly overlap(Fig. 5C). Our antibody detects pre-processed BMP2B, and inprinciple, processed mature BMP2B, which we cannot detect,may diffuse further than the BMP2B-expressing cells. However,the fact that we do not detect any BMP transcriptional activity(mRFP) beyond cells expressing BMP2B suggests that processedBMP2B is not significantly diffusible.

For the first time therefore, we have shown the proteinexpression pattern of an endogenous BMP ligand in zebrafish.BMP2B protein is expressed in a ventral to dorsal gradient in theearly zebrafish embryo, which recapitulates both the bmp2bmRNAexpression, and the activity gradient read out by the mRFPreporter. Thus, taken together, our results provide no evidencethat extensive diffusion of BMP2B protein occurs to generate theBMP transcriptional activity gradient detected with mRFP.

Graded bmp ligand transcription, established by dharma and FGFsignalling and maintained through auto-regulation, determines theBMP activity gradient

Since we have shown that graded bmp transcription precedesthe formation of the BMP activity gradient, we next investigatedhow graded bmp transcription is established. Transcription of theBMP ligands is known to be under autoregulation from about 60%epiboly (Kishimoto et al., 1997; Nguyen et al., 1998; Schmid et al.,2000). However, this does not appear to occur during blastulastages (Furthauer et al., 2004). To inhibit BMP signalling duringblastula stages, we used LDN-193189 treatment from the 8–16 cellstage onwards (Fig. S3) (Cannon et al., 2010) and found that mostembryos still retained graded bmp2b transcript expression at 30%epiboly (Fig. 6A), despite a clear effect on overall transcript levels,probably due to reduced maternal BMP signalling. This resultdemonstrates that graded bmp2b transcription at 30% epiboly isnot dependent on zygotic BMP activity. Consistent with this view,Chordin knockdown, which should lead to increased BMP signal-ling, did not result in expanded bmp2b expression at 30% epiboly(Fig. 6B) (Furthauer et al., 2004).

Having ruled out positive feedback by autocrine BMP signallingas important for establishing graded bmp2b expression at 30%epiboly, we tested the involvement of Dharma and FGF signalling(Furthauer et al., 2004; Koos and Ho, 1999; Leung et al., 2003). Thelack of bmp2b and bmp7a transcripts dorsally at dome stage is astrong indication of localised transcriptional repression by Dharma(Fig. 4A), and this was confirmed by the observation that injectionof a dharma MO resulted in expanded bmp2b transcription at 30%epiboly in the majority of embryos examined (Fig. 6B). Notably, wefound that ectopic bmp2b is only detected at the margin, whereDharma is expressed (Fig. 4A), but not in the dorsal ectoderm (seewhite dotted lines in Fig. 6B). Consistent with dorsal bmp2b beingdetected upon Dharma knockdown, we found that dharma mor-phants display increased mRFP expression dorsally at the margin

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Fig. 5. Graded expression of endogenous BMP2B protein results in graded transcriptional activity. (A) IF for BMP2B in wild-type embryos. (B) IF for BMP2B in control andbmp2b morphants. (C) Combined IF for BMP2B and ISH for mRFP in BRE-mRFP embryos. Note that there is some background staining for BMP2B that is due toautofluorescence from the yolk. Stages and views are indicated. The fluorescent substrate used for mRFP in (C) was Cy5 tyramide (pseudocoloured in red). In (A) and(C, bottom row), the pictures are the projection of a 25–30 stack of images taken from the edge of the embryo until about half way. In (B) and (C, top row), only theblastoderm margin was imaged. The white dotted line in (C, bottom row) shows the outline of the embryo. V, ventral; D, dorsal.


in 40% epiboly embryos (Fig. 6C). Expression of pMAPK at domeand 30% epiboly stages indicates active FGF signalling in a dorsal toventral gradient, with stronger activity occurring at dome stage(Fig. 4A). Using the FGF receptor inhibitor SU5402 (Mohammadiet al., 1997), we demonstrated that FGF signalling inhibition resultsin expansion of the domain of bmp2b expression to the dorsal sideat 30% epiboly (Fig. 6A) (Furthauer et al., 2004). Interestingly,SU5402 treatment also results in reduced chordin expression atthis stage (Fig. S5). Thus, Dharma and FGF signalling are the criticalfactors for establishing the graded bmp transcription that ispresent at 30% epiboly, rather than auto-regulation via BMPsignalling.

Chronologically, it is generally assumed that activity gradientsare established as a direct consequence of morphogen diffusion/transport and that therefore these gradients are formed de novo.However, our mRFP ISH results show that BMP activity in blastulastage embryos is initially ubiquitous, then graded (Fig. 3). Hencethere must be a molecular mechanism that results in clearance ofmRFP transcripts dorsally (Fig. 6D). Indeed, our data indicate thatat 40% epiboly, when the gradient of BMP activity is established, itfaithfully reflects the gradient of BMP ligand transcription (Fig. 4B).However, our mRFP ISHs and p-Smad1/5 staining indicates thatBMP activity is initially ubiquitous and is cleared from the dorsalside between 30% and 40% epiboly (Fig. 3) (Tucker et al., 2008).

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Fig. 6. Regulation of graded bmp ligand transcription by Dharma, FGF and BMP signalling and consequences for the transcriptional activity of the BMP pathway. (A) bmp2bISH in 30% epiboly wild-type embryos treated with DMSO, LDN-193189 (LDN) or SU5402. (B) bmp2b ISH in 30% epiboly wild-type embryos injected with either control,dharma, or chordin MOs. Ectopic bmp2b expression is detected at the margin upon Dharma knockdown but not in the dorsal ectoderm (white dotted lines). (C) mRFP ISH inBRE-mRFP embryos injected with either control, dharma, or chordin MOs. The fluorescent substrate used in (C) was Fast Blue (pseudocoloured in red). Note that the imagesare projection of a 10–12 picture stack taken at the margin of the blastoderm to avoid fluorescence from the yolk. (D) Graphical representation of the levels of mRFP, chordin,and bmp2b mRNAs, as well as BMP and p-Smad1/5 proteins across the D/V axis at 30% and 40% epiboly. Between these two stages, Chordin acts to antagonise residual BMPactivity dorsally, resulting in a gradient of mRFP at 40% epiboly that reflects the gradient of BMP ligand transcription. For all panels in ((A)–(C)), stages and/or views areindicated. Arrows indicate the limit of the expression domain of the transcripts analysed. V, ventral; D, dorsal.


This occurs after the bmp transcripts disappear dorsally, suggestingthat this might occur at the level of BMP protein inhibition(residual dorsal BMP protein), if BMP receptors act to constantlymonitor ligand levels. This inhibition of signalling is most likelymediated by Chordin. To test this idea we knocked down Chordinwith a MO and investigated the effect on mRFP mRNA levels at 40%epiboly when they are normally graded. We could clearly demon-strate that in chordin morphants the BMP activity gradient wasabolished and mRFP expression was uniform (Fig. 6C). This occursdespite the fact that auto-regulation of bmp2b transcription onlyoccurs after that stage (Fig. S6). We therefore conclude thatbetween 30% and 40% epiboly, expression of Chordin on the dorsalside of the embryo must contribute to a loss of dorsal BMP activity,and that after 40–50% epiboly the BMP activity gradient becomesmaintained by the gradient of ligand transcription.

Discussion

Using ISH for mRFP in our BRE-mRFP transgenic line, which is ahighly sensitive reporter for BMP/GDF signalling, we can visualisethe dynamics of BMP signalling in early zebrafish embryos. Wehave shown that the BMP transcriptional activity gradient isestablished by 40% epiboly and is preceded by graded expressionof BMP ligands (mRNA and protein). For the first time, we are ableto detect the expression of an endogenous BMP protein, BMP2B,and found that it is expressed in a gradient during blastula andgastrula stages and that no extensive diffusion of BMP2B occurs togenerate transcriptional activity at a distance. Our analysisstrongly supports the notion that the BMP activity gradient inzebrafish occurs as a result of graded BMP expression and does notfit the classical models of gradient formation, where ligands

I- INDUCTION OF BMPS AND MAIN BMP ANTAGONISTS LIGAND ACTIVITY

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Fig. 7. Sequence of signalling events leading to graded BMP ligand transcription and hence a gradient of BMP activity. While the dorsal determinants are actively induced(simplified representation; cross-regulation is not shown), the transcription of zygotic BMPs is initially ubiquitous. The symmetry-breaking event for bmp expression is thetranscriptional repression of bmp2b (and possibly bmp7a) by Dharma. FGF signalling is critical for establishing graded bmp expression. Both graded bmp ligand expressionand the BMP activity gradient are subsequently mostly maintained through auto-regulation of bmp expression. For details, see text.


synthesised from a local source would diffuse away in order toproduce a signalling gradient.

A powerful tool to visualise BMP/GDF activity

We confirmed using various experimental approaches thatmRFP expression in our BRE-mRFP transgenic fish is a faithfulreadout of BMP/GDF activity. We found that mRFP fluorescence isonly detected from early somitogenesis stages, which is mostlikely due to insufficient amounts of mRFP protein being produced.A similar lack of fluorescence at early stages has been reported forother transgenic reporter lines such as the Wnt reporter lineTOPdGFP (Dorsky et al., 2002), the Hedgehog pathway reporterline Gli-d:mCherry (Schwend et al., 2010) and the other zebrafishBMP reporter lines (Alexander et al., 2011; Collery and Link, 2011;Laux et al., 2011). However, mRFP ISH in our strong BRE-mRFP linesvisualised with either colourimetric or fluorescent substratesprovides an excellent method to detect highly dynamic BMPtranscriptional activity.

Timing and mechanisms of BMP activity gradient formation duringblastula stages

We found that the ventral to dorsal BMP activity gradient,revealed with mRFP ISH, is set up by 40% epiboly during the

blastula period. The appearance of this gradient is preceded by theestablishment of graded expression of BMP ligands which occursbetween dome stage and 30% epiboly, and graded pathwayactivation read out by p-Smad1/5 levels occurs fractionally beforethe appearance of the mRFP gradient.

Using our results, combined with those from other studies(Furthauer et al., 2004; Langdon and Mullins, 2011; Maegawaet al., 2006), we propose a model for BMP activity gradientformation that features the temporal and spatial requirement forspecific signalling events (Fig. 7). First, immediately after MBT,bmps are induced ubiquitously in the embryo. In contrast, thevarious BMP antagonists are locally induced by maternal β-catenin2 on the future dorsal side of the embryo. At dome stage,Dharma acts dorsally to locally repress bmp2b transcription. Thissymmetry-breaking event is followed by the action of FGF signal-ling at around 30% epiboly, which appears to act in a dorsal toventral graded fashion to inhibit bmp ligand transcription in adose-dependent manner. It is not yet clear how FGF signallingachieves this. As a result, by 30% epiboly, all three bmp transcriptsare expressed in a graded manner. However, at this stage, ourexperiments suggest that there must be some residual BMPprotein present on the dorsal side and that the BMP proteingradient lags behind the bmp transcript gradient (Fig. 6D). Oneclear indication of residual dorsal BMP protein is that p-Smad1/5 isstill detected dorsally at 30% epiboly, albeit at slightly lower levels,


and that mRFP transcripts are also strongly detected there (Fig. 3).Between 30% and 40% epiboly, inhibition of residual BMP proteinfunction dorsally, via Chordin, rapidly leads to reduction inp-Smad1/5 levels and the disappearance of mRFP transcripts.Coupled with fast mRFP mRNA turnover, this contributes to thedetection of the BMP transcriptional activity gradient at 40%epiboly. Interestingly, the rapid clearance of mRFP transcriptsdorsally and the fact that Chordin knockdown inhibits this, suggestthat BMP receptors must be able to monitor ligand activity at thecell surface and thus respond to rapid changes in signalling. Wehypothesise that from 40% to 50% epiboly onwards, the BMPactivity gradient becomes proportional to the amount of BMPtranscripts and proteins, the expression of which is maintainedthrough auto-regulation, combined with pathway inhibition bydorsally-expressed Chordin (Fig. 7; Fig. S6). One important featureof this model is that there is a complex level of interactionbetween all the molecular players. One example is that Chordinexpression in the blastula is partially under the control of FGF andNodal signaling (Fig. S5) and that optimal repression of bmp2bexpression by FGF signalling also appears to require Chordinfunction (Maegawa et al., 2006).

The zebrafish ventral to dorsal BMP activity gradient: An alternativemodel for the formation of BMP gradients

BMPs have long been assumed to be morphogens in theclassical sense; that is that they are expressed from a local sourceand diffuse in the extracellular space to generate an activitygradient that is proportional to the amount of extracellularproteins. Not only is the issue of BMP extracellular diffusion stillquestionable, but the ways in which the activity gradients areformed in various developmental systems also show that the BMPsignalling activity does not necessarily correlate with the amountof signalling BMP proteins (Wolpert, 2011).

Our understanding of how BMP gradients first form comespredominantly from studies in Drosophila, where two differentmechanisms have been uncovered (reviewed by Ramel and Hill,2012). To generate the Dpp activity gradient that determines A/Ppatterning of the Drosophila wing, the Dpp ligand is secreted froma central stripe of cells in the wing imaginal disc and acts at adistance to form a concentration gradient both anteriorly andposteriorly (Kicheva and Gonzalez-Gaitan, 2008; Raftery andUmulis, 2012). In contrast, the D/V Dpp activity gradient thatforms in the embryonic Drosophila blastoderm results from redis-tribution of ligand within a uniform dpp expression domain. This isachieved by binding and transport of Dpp and a related ligandScrew with the ligand antagonists Twisted Gastrulation and ShortGastrulation (Drosophila Chordin) (O'Connor et al., 2006). Ananalogous ligand shuttling mechanismwas proposed to be respon-sible for the D/V BMP activity gradient in Xenopus, and waspreferred over an alternative inhibition-based mechanism wherean inhibition gradient of diffusible inhibitors like Chordin iscreated over a uniform field of activators (Ben-Zvi et al., 2008).However, there is little evidence for this mechanism in theXenopus embryo and this issue is very controversial (Francoiset al., 2009).

We have shown that the early D/V BMP activity gradient isformed in a very different manner in zebrafish. Our studydemonstrates that the BMP activity gradient is actually createdin a tissue where activity is initially ubiquitous. Therefore thisparticular gradient is not created de novo. We also found that thecontrol of BMP ligand transcription is the crucial determinant forgenerating a BMP activity gradient that is robust and sustainable -rather than extensive diffusion or redistribution of BMP proteins.Indeed, consistent with published data on the diffusion of BMP4 inXenopus (Jones et al., 1996) and cell transplantation experiments in

zebrafish (Nikaido et al., 1999), we find no evidence that zebrafishBMP proteins diffuse to generate signalling activity at a distance.Our data highlights the fact that each BMP gradient is unique inthe way it is established and maintained.

Acknowledgements

We would like to thank Darren Martin, Chris Sergeant, and PhilTaylor for fish maintenance and care, as well as the LondonResearch Institute light microscopy facility and equipment park.We thank Sabine Reichert for help with the fluorescent in situhybridisation, and Paul Yu, Holger Bielen, James Dutko, KoichiKawakami, Ray Keller, Arne Lekven, Nancy Papalopulu, and MaryMullins for reagents and zebrafish lines. We thank Nic Tapon andmembers of the Hill laboratory for important discussions andcritical reading of the manuscript. The work was supported byCancer Research UK and the European Commission Network ofExcellence EpiGeneSys (HEALTH-F4-2010-257082).

Appendix. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/http://dx.doi.org/10.1016/j.ydbio.2013.03.003.

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The ventral to dorsal BMP activity gradient in the early zebrafish embryo is determined by graded expression of BMP ligandsIntroductionMaterials and methodsFish maintenance, screening, strains and crossesPlasmids, mRNAs, morpholinos and injectionsIn situ hybridisation, immunofluorescence, and moviesProtein preparation and Western blottingChemical inhibitor treatments

ResultsCharacterisation of mRFP expression in BRE-mRFP embryosThe BRE-mRFP transgenic zebrafish is a bona fide in vivo reporter for BMP/GDF activity.The initial ventral to dorsal BMP transcriptional activity gradient is established between 30% and 40% epibolyThe BMP transcriptional activity gradient is established in a domain of graded ligand transcriptionGraded bmp transcription results in a BMP protein gradient with little or no transcriptional activity at a distance from...Graded bmp ligand transcription, established by dharma and FGF signalling and maintained through auto-regulation,...

DiscussionA powerful tool to visualise BMP/GDF activityTiming and mechanisms of BMP activity gradient formation during blastula stagesThe zebrafish ventral to dorsal BMP activity gradient: An alternative model for the formation of BMP gradients

AcknowledgementsSupporting informationReferences

The ventral to dorsal BMP activity gradient in the early ...The ventral to dorsal BMP activity...

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