A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.     

Extracellular modulation of BMP activity in patterning the dorsoventral axis

Signaling via bone morphogenetic proteins (BMPs) regulates a vast array of diverse biological processes in the developing embryo and in postembryonic life. Many insights into BMP signaling derive from studies of the BMP signaling gradients that pattern cell fates along the embryonic dorsal-ventral (DV) axis of both vertebrates and invertebrates. This review examines recent developments in the field of DV patterning by BMP signaling, focusing on extracellular modulation as a key mechanism in the formation of BMP signaling gradients in Drosophila, Xenopus, and zebrafish.     

The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis

Patterning of the vertebrate anteroposterior (AP) axis proceeds temporally from anterior to posterior. How dorsoventral (DV) axial patterning relates to AP temporal patterning is unknown. We examined the temporal activity of BMP signaling in patterning ventrolateral cell fates along the AP axis, using transgenes that rapidly turn "off" or "on" BMP signaling. We show that BMP signaling patterns rostral DV cell fates at the onset of gastrulation, whereas progressively more caudal DV cell fates are patterned at progressively later intervals during gastrulation. Increased BMP signal duration is not required to pattern more caudal DV cell fates; rather, distinct temporal intervals of signaling are required. This progressive action is regulated downstream of, or in parallel to, BMP signal transduction at the level of Smad1/5 phosphorylation. We propose that a temporal cue regulates a cell's competence to respond to BMP signaling, allowing the acquisition of a cell's DV and AP identity simultaneously.     

The BMP signaling gradient is interpreted through concentration thresholds in dorsal–ventral axial patterning

Bone Morphogenetic Protein (BMP) patterns the dorsal–ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.

This study tested three models of how a BMP morphogen gradient is translated into differential gene activation that specifies distinct cell fates, finding that BMP signal concentration thresholds, not gradient shape or signal duration, position three distinct gene activation domains.     

Zebrafish bmp4 functions during late gastrulation to specify ventroposterior cell fates

Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling.     

Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton

Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains.     

Temporal and spatial action of tolloid (mini fin) and chordin to pattern tail tissues

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.     

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.     

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.     

Tailbud-derived Bmp4 drives proliferation and inhibits maturation of zebrafish chordamesoderm

In zebrafish, BMP signaling establishes cell identity along the dorsoventral (DV) axis during gastrulation. Owing to the early requirements of BMP activity in DV patterning, it has been difficult to assign later roles in cell fate specification to specific BMP ligands. In this study, we have taken advantage of two follistatin-like genes (fstl1 and fstl2), as well as a transgenic zebrafish line carrying an inducible truncated form of the BMP-type 1 receptor to study the role of Bmp4 outside of the context of DV specification. Characterization of fstl1/2 suggests that they exert a redundant role as BMP antagonists during late gastrulation, regulating BMP activity in axial mesoderm. Maintenance of appropriate levels of BMP signaling is crucial for the proper development of chordamesoderm, a subset of axial mesoderm that gives rise to the notochord, but not prechordal mesoderm, which gives rise to the prechordal plate. Bmp4 activity in particular is required during a crucial window beginning at late gastrulation and lasting through early somitogenesis to promote chordamesoderm proliferation. In the absence of Bmp4, the notochord precursor pool is depleted, and the notochord differentiates prematurely. Our results illustrate a role for Bmp4 in the proliferation and timely differentiation of axial tissue after DV axis specification.     

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.     

Zili antagonizes Bmp signaling to regulate dorsal-ventral patterning during zebrafish early embryogenesis

Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-βsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.     

Computational analysis of BMP gradients in dorsal-ventral patterning of the zebrafish embryo

The genetic network controlling early dorsal-ventral (DV) patterning has been extensively studied and modeled in the fruit fly Drosophila. This patterning is driven by signals coming from bone morphogenetic proteins (BMPs), and regulated by interactions of BMPs with secreted factors such as the antagonist short gastrulation (Sog). Experimental studies suggest that the DV patterning of vertebrates is controlled by a similar network of BMPs and antagonists (such as Chordin, a homologue of Sog), but differences exist in how the two systems are organized, and a quantitative comparison of pattern formation in them has not been made. Here, we develop a computational model in three dimensions of the zebrafish embryo and use it to study molecular interactions in the formation of BMP morphogen gradients in early DV patterning. Simulation results are presented on the dynamics BMP gradient formation, the cooperative action of two feedback loops from BMP signaling to BMP and Chordin synthesis, and pattern sensitivity with respect to BMP and Chordin dosage. Computational analysis shows that, unlike the case in Drosophila, synergy of the two feedback loops in the zygotic control of BMP and Chordin expression, along with early initiation of localized Chordin expression, is critical for establishment and maintenance of a stable and appropriate BMP gradient in the zebrafish embryo.     

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.     

Dorsoventral differences in cAMP levels and correlated changes in the subcellular distribution of the PKA catalytic domain,provide further evidence that PKA signaling coordinates dorsoventral patterning of the otocyst

Dorsoventral (DV) patterning of the otocyst gives rise to formation of the morphologically and functionally complex membranous labyrinth composed of unique dorsal and ventral sensory organs. DV patterning results from extracellular signaling by secreted growth factors, which presumably form reciprocal concentration gradients across the DV axis of the otocyst. Previous work suggested a model in which two important growth factors, bone morphogenetic protein (BMP) and SHH, undergo crosstalk through an intersecting pathway to coordinate DV patterning. cAMP‐dependent protein kinase A (PKA) lies at the heart of this pathway. Here, we provide further evidence that PKA signaling coordinates DV patterning, showing that both BMPs and SHH regulate cAMP levels, with BMPs increasing levels in the dorsal otocyst and SHH decreasing levels in the ventral otocyst. This, in turn, results in regional changes in the subcellular distribution of the catalytic domain of PKA, as well as DV regulation of PKA activity, increasing it dorsally and decreasing it ventrally. These new results fill an important gap in our previous understanding of how ligand signaling acts intracellularly during otocyst DV patterning and early morphogenesis, thereby initiating the series of events leading to formation of the inner ear sensory organs that function in balance and hearing.     

Twisted gastrulation is required for forebrain specification and cooperates with Chordin to inhibit BMP signaling during X. tropicalis gastrulation

In the developing vertebrate embryo, proper dorsal-ventral patterning relies on BMP antagonists secreted by the organizer during gastrulation. The BMP antagonist chordin has a complex interaction with BMPs that is governed in part by its interaction with the secreted protein twisted gastrulation (tsg). In different contexts, tsg has activity as either a BMP agonist or as a BMP antagonist. Using morpholino oligonucleotides in Xenopus tropicalis, we show that reducing tsg gene product results in a ventralized embryo, and that tsg morphants specifically lack a forebrain. We provide new evidence that tsg acts as a BMP antagonist during X. tropicalis gastrulation since the tsg depletion phenotype can be rescued in two ways: by chordin overexpression and by BMP depletion. We conclude that tsg acts as a BMP antagonist in the context of the frog gastrula, and that it acts cooperatively with chordin to establish dorsal structures and particularly forebrain tissue during development.