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The anti-neural role of BMP signaling is a consequence of its ancestral function in dorsoventral patterning [1]

['Paul Knabl', 'Department Of Neurosciences', 'Developmental Biology', 'University Of Vienna', 'Vienna', 'Vienna Doctoral School Of Ecology', 'Evolution', 'Vdsee', 'Konrad Lorenz Institute For Evolution', 'Cognition Research']

Date: 2026-05 

In Bilateria with centralized nervous systems (e.g., in vertebrates or arthropods), the minimum of the BMP signaling activity gradient defines the position of the central nervous system. BMP-dependent patterning of the secondary body axis is ancestral for Bilateria and possibly also for the bilaterian sister clade Cnidaria. However, the variety of levels of centralization of the nervous systems in Bilateria—from diffuse to fully centralized—as well as the lack of centralization of the nervous system in Cnidaria, suggest that BMP signaling cannot be perceived as a universally “anti-neural” signal. Here we use transgenic reporter lines in the anthozoan cnidarian Nematostella to show that BMP signaling is active in distinct neuronal populations. Moreover, attenuation of BMP signaling followed by RNA-Seq shows that BMP signaling is a positive regulator of many neuronal genes, including the top-tier neural progenitor marker soxB(2). Furthermore, we analyze BMP signaling activity in the cubozoan jellyfish Tripedalia and the scyphozoan jellyfish Stomolophus and prove that BMP signaling in parts of the diffuse nervous system is not an anthozoan but an ancestral cnidarian feature, shared by anthozoans and medusozoans. Finally, we show that the highly centralized ventral nervous system of the nonmodel spiralian, the chaetognath Spadella, forms out of paired BMP signaling-positive domains on the lateral sides of the embryo. Together, our data suggest that one of the ancestral roles of BMP signaling may have been in promoting neurogenesis. We propose that the “anti-neural” function of BMP signaling in vertebrates and arthropods is a consequence of its global role in the dorsoventral patterning of the ectoderm.

Funding: This research was funded by the Austrian Science Fund (FWF) grants P32705 and PAT2395824 to G.G. and the Austrian Science Fund (FWF) grant P34665 to T.W. During his time in the Genikhovich lab, P.K.‘s salary was paid from the grants to G.G. P.K. is a recipient of the Dimitrov Fellowship of the Austrian Academy of Sciences (OeAW) and of the Writing-up Fellowship of the Konrad Lorenz Institute for Evolution and Cognition Research (KLI). During her time in the Wollesen lab, J.O.’s salary was paid from the grant to T.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Newly generated sequencing data is deposited as bioprojects at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1269470 and http://www.ncbi.nlm.nih.gov/bioproject/1441074 . All code required to reproduce the computational analyses of this study can be found on https://github.com/Genikhovich-Lab/ and, additionally, at 10.5281/zenodo.19686897 and 10.5281/zenodo.19686882 . All other relevant data are within the paper and its Supporting information files.

Most of our knowledge about BMP signaling in Cnidaria comes from an anthozoan model—the sea anemone Nematostella vectensis. Unlike radially symmetric Medusozoa, Nematostella and other Anthozoa, display a bilaterally symmetric body plan with a secondary, “directive” body axis ( Fig 1A ). Similar to the bilaterian DV axis, the directive axis requires the formation of a BMP signaling gradient that is regulated by the interplay of multiple BMP pathway components, including bmp2/4, bmp5-8, gdf5-like (=gdf5-l), chordin, gremlin, rgm and zswim4-6 [ 30 , 33 – 35 ]. Active BMP signaling can be visualized by the spatial distribution of its transcriptional effector, phosphorylated SMAD1/5 (pSMAD1/5). In Nematostella, at late gastrula stage, BMP signaling forms a pSMAD1/5 gradient along the directive axis. This gradient persists while the directive axis is being patterned, but eventually disappears by 4 day planula larva stage, at which point BMP signaling activity becomes local and especially prominent in the mesenteries—the gastrodermal septa of the body wall [ 30 , 33 , 34 ]. Our recent study revealed pronounced BMP signaling activity in the mesenteries of the adult Nematostella polyp [ 36 ]. The mesenteries harbor a diversity of cell populations including epithelial, digestive, reproductive, muscular and neuronal cells. The two latter are concentrated in the “neuro-muscular” mesentery region, where the longitudinal muscles (the retractor and parietal muscle), the longitudinal neurite tracts, and sensory and ganglion mesentery neurons are located. While BMP signaling is strongest within the neuro-muscular domain, yet it was unclear if BMP signaling occurs in neuronal or muscular cell populations ( Fig 1 ). Here, we analyze the potential involvement of BMP signaling in the diffuse nervous system of the anthozoan cnidarian Nematostella. Additionally, we use two other distantly related medusozoan cnidarians, Tripedalia and Stomolophus for comparison and show a pro-neural action of BMP signaling in these models. Finally, we also demonstrate distinct BMP activity in the central nervous system of the nonmodel spiralian Spadella. Together, our findings suggest that BMP signaling was ancestrally a pro-neural factor.

(A) Simplified phylogenetic tree of early branching metazoans indicating (i) the presence or absence of BMP pathway genes, (ii) the presence of a BMP-dependent secondary body axis, (iii) involvement of BMP signaling in neurogenesis. White circle – absent, blue circle – present; half blue/half white circle – present in some; blue and white circles with question marks – possibly present or absent, respectively. (B) Schematic of the BMP signaling pathway, BMP signaling is activated by the binding of BMP dimers to the BMP receptor complex, resulting in the nuclear translocation of phosphorylated SMAD1/5 (pSMAD1/5), acting as BMP effector and regulating BMP-responsive gene expression. (C) BMP signaling dynamics during early development of Nematostella: no activity in the blastula, pSMAD1/5 activity around the blastopore in the early gastrula, pSMAD1/5 gradient at 2-day planula stage and dispersed pSMAD1/5 activity in the 4-day planula. Blue dots indicate nuclear pSMAD1/5. (D) Overview of Nematostella polyp anatomy in lateral view and as a cross-section of the body column, the neuro-muscular domain of the mesentery gastrodermis is highlighted in blue. (E) Detailed schematic of an individual mesentery as a cross-section, highlighting the localization of pSMAD1/5-positive nuclei within the neuro-muscular domain. bwe-body wall epidermis, mg-mesoglea, bwg-body wall gastrodermis, mes-mesenteries, bc-body cavity, RMR-retractor muscle region, MS-mesentery stalk, PMR-parietal muscle region.

The evolutionary sister clade to Bilateria is the phylum Cnidaria, encompassing Anthozoa (sea anemones and corals) and four classes of Medusozoa: Hydrozoa (hydroids), Staurozoa (stalked jellyfish), Scyphozoa (true jellyfish), and Cubozoa (box jellyfish) ( Fig 1A ). Cnidaria possess a full repertoire of BMP pathway genes, including BMP ligands, BMP antagonists, BMP receptors and BMP effectors [ 19 – 25 ]. Cnidarian nervous systems are organized as diffuse nerve nets with local condensations, forming nerve rings and neurite tracts, but lacking a brain-like centralization [ 26 – 29 ]). Neurons are found both in the epidermis and in the gastrodermis of cnidarians, and, strikingly, work on the anthozoan sea anemone Nematostella vectensis showed that gastrodermal neurons are “born” in the gastrodermis rather than migrate there, as is the case in Bilateria [ 28 ]. Neuronal gene expression in Nematostella commences already in the blastula, which is before pSMAD1/5 becomes first detectable [ 30 – 32 ]. Neuronal markers were not affected by the disruption of BMP signaling during early phases of neurogenesis [ 32 ]. Only at planula larva stage, the upregulation and, surprisingly, the downregulation of BMP signaling both reduced neuronal markers and the number of RFamide- and GLWamide-positive neurons [ 32 ] suggesting that neural “induction” is independent of BMP signals, while later neurogenesis may involve a combination of BMP activation and repression. Taken together, it is largely unclear if and to what extent BMP signals are required during cnidarian neurogenesis.

While the role of BMP signaling in regulating the DV patterning is clearly ancestral and, with few exceptions, present across all Bilateria [ 10 ], its strictly “anti-neural” function is contradicted by the variety in the degrees of centralization of bilaterian nervous systems—from diffuse, to being arranged into a varying number of nerve cords, to ganglionic, to fully centralized [ 11 , 12 ]. Similarly variable are the effects of BMP signaling perturbations on the nervous systems of different groups, from promoting to suppressing neurogenesis to having no effect at all [ 13 – 18 ]. Clearly, in order to understand the ancestral role of BMP signaling in neuron formation we need a wider phylogenetic sampling within and outside Bilateria, especially in animals with diffuse nervous systems and with centralized nervous systems forming in “nonstandard” locations.

The evolutionary origin of BMP signaling-dependent regulation of animal nervous systems is uncertain. Both in vertebrates and arthropods, the establishment of a BMP signaling gradient is required for patterning the dorsoventral (DV) body axis and the formation of the centralized nervous system (CNS). In these models, the position of the neuroectoderm is defined by the inhibition of BMP signaling on one side of the DV axis—dorsally in vertebrates and ventrally in arthropods [ 1 ]. Later during development, similar genetic programs regulate the mediolateral patterning of the vertebrate neural plate and the nerve cords of insects and annelid worms [ 2 , 3 ]. These observations made in the mouse, frog, zebrafish, fly, and ragworm have contributed to the idea that the CNS emerged once, early during bilaterian evolution on the side of the body, where BMP signaling was low [ 4 – 8 ]. DV axis inversion at the base of chordates made this ancestrally ventral BMP minimum dorsal in chordates [ 9 ].

Results

BMP pathway genes are expressed across neuronal subtypes in the developmental single-cell atlas of Nematostella To address the expression of BMP components in the neuroglandular and muscular repertoire of Nematostella, we utilized the updated developmental single-cell atlas of Nematostella [37]. The analysis of the neuroglandular subset by Cole and colleagues yielded 47 developmentally closely related but distinct cell states, including insulinoma (insm)-positive (N1) and insm-negative (N2) neurons, secretory cells (S) and digestive gland cells (GD). Among the N1 and N2 neurons, several gastrodermis-derived types have been identified so-far, including six N1 (N1.g1-6) and a single N2 type (N2.g). The analysis of the retractor muscle subset by Cole and colleagues revealed four distinct cell states, including the early retractor muscle state (RM.1), the late tentacle retractor muscle state (TR.2), the early tentacle retractor muscle state (TR.1), and the late retractor muscle state (MR.1). We generated expression plots of BMP pathway genes, including BMP ligands (bmp2/4, bmp5-8, gdf5-l, admp), BMP effectors (smad1/5, smad4, smad4-like), BMP receptors (alk2, alk3/6, bmprII, actrII) and intracellular, membrane-bound, and secreted BMP antagonists (rgm, smad6, chordin, gremlinA, gremlinB, noggin1, noggin2, crossveinless2 and follistatin) [36]. The expression of BMP components was detectable in N1 and N2 neuron types, while it was reduced in putative sensory neurons (N1S) and secretory cells (S) and absent in digestive gland (GD) types (Fig 2A). Notably, the expression of BMP components in N1 and N2 neurons is not limited to the gastrodermal populations, despite the fact that our previous analysis revealed that BMP signaling activity is largely limited to the mesentery gastrodermis [36]. The expression of extracellular molecules such as BMP ligands and antagonists appeared to be sparse with only small fractions of cells (<10%) in each subpopulation expressing the transcripts (Fig 2A). Moreover, single-cell analysis suggested that neither BMP pathway genes nor BMP receptor type expression is specific to discrete neuronal subpopulations. While our expression analysis of the developmental single-cell data pointed to the expression of the BMP module in neuronal populations, it was not sufficient to identify specific candidates among the neuronal subpopulations, which might display pSMAD1/5 activity. The expression of BMP pathway genes in the retractor muscle cell states was low or absent, except the upregulated expression of BMP ligands in the early tentacle retractor muscle TR.1 and of the BMP antagonist molecule RGM in the late tentacle retractor muscle state TR.2 (Fig 2B). PNG larger image

TIFF original image Download: Fig 2. Expression of BMP pathway genes in the neuronal and retractor muscle subset of the developmental single-cell atlas of Nematostella. Dot plots showing the expression of neuronal marker genes, retractor muscle marker myosin heavy chain1 (myhc1) and BMP signaling components (A) in the neuroglandular subset and the (B) retractor muscle subset of the Nematostella developmental single-cell by Cole et al. [37]. Average scaled expression of 0 or below is indicated in gray. Numerical data for the plot can be extracted by running the R scripts found here 10.5281/zenodo.19686897. https://doi.org/10.1371/journal.pbio.3003803.g002

Neural lineage markers are downregulated by BMP signaling attenuation in the Nematostella larvae Previous analyses suggested that BMP signaling has both positive and negative effects on neurogenesis in the planula larvae [32]. We searched for differentially expressed neural regulators in the BMP2/4MO RNA-seq data set, revealing multiple neuronal genes, most of which were significantly downregulated (S2 Table). We analyzed the expression patterns of selected neuronal genes upon the suppression of BMP signaling by BMP2/4 morpholino or K02288 treatment using in situ hybridization. First, we analyzed the expression of soxC and soxB(2), marking neuronal progenitors [42,43], upon BMP2/4MO knockdown, resulting in the complete loss of the pSMAD1/5 gradient, and gdf5-lMO knockdown, reducing graded pSMAD1/5 activity [33]. The expression of both soxC and soxB(2) was strongly reduced in bmp2/4MO (Fig 8C, 8D). In contrast, soxC and soxB(2) were not affected by gdf5-lMO (Fig 8D), suggesting that either their expression requires input specifically from BMP2/4-mediated and not GDF5-like-mediated signaling or that reduced BMP signaling is sufficient to maintain their normal expression. Then, we analyzed the effect of the chemical inhibition of BMP signaling using the K02288 inhibitor, spanning different time windows: i) from Blastula (16–18 hpf) to 2d planula stage (B- > 2dP), which overlaps with the time of the pSMAD1/5 gradient formation; ii) from 2d to 4d planula stage (2dP- > 4dP), when the pSMAD1/5 activity gradually switches from graded to dispersed and becomes located in the mesenteries, corresponding to the time of gastrodermal patterning and mesentery formation; and iii) from 4d to 5d (4dP- > 5dP), after the directive axis has been fully patterned and pSMAD1/5 is active in the mesenteries. For the different time windows of treatment, we again examined the expression of differentially expressed neural markers from the BMP2/4MO dataset, including soxC and soxB(2), as well as insulinoma (insm), which is expressed in neurosecretory and neuroglandular populations [50], prdm14d as marker for neural progenitors and ganglion neurons in the gastrodermis [40] and pou4 and elav1 as neuronal differentiation markers of gastrodermal and epidermal subpopulations [28,51]. K02288 treatments from blastula to 2d planula resulted in strong downregulation of all analyzed marker genes (Fig 8E), concurring with the results of the BMP2/4MO in situ and RNA-seq data. Similarly, BMP inhibition starting at 2d planula until 4d planula stage, strongly reduced neuronal marker genes (Fig 8E). In comparison, K02288 treatments from the 4d to 5d planula, which is after the directive axis has been patterned, and mesenteries have been formed, displayed only a mild reduction of neural marker expression (Fig 8E), suggesting that the neuronal regulation becomes less sensitive to BMP signaling input in the fully patterned late planula. While early studies indicated a combination of positive and negative effects of BMP signaling on neural patterning in the planula, our data support a predominantly pro-neural influence of BMP signaling in the nervous system of the Nematostella larva. PNG larger image

TIFF original image Download: Fig 8. Neuronal markers are downregulated upon BMP signaling inhibition in Nematostella planula. (A) Schematic of the BMP signaling pathway and how it is affected by BMP2/4 morpholino oligo and pharmacological inhibition of the BMP receptor with K02288. (B) Timeline of Nematostella early development and BMP signaling dynamics from fertilization (F), Blastula (B) and 2-5 day Planula larvae (2dpP-5dP). The gray/cyan-colored bar indicates the absence/presence of pSMAD1/5 activity. Duration and fixation time points of morpholino knockdown experiments (purple) and K02288 treatments (lilac). (C) Bar plot of expression changes (log2 fold, padj. < 0.05;) of selected neuronal marker genes in 2 day planula upon bmp2/4MO knockdown by RNA-seq analysis [30], see Materials and methods). (D) Expression analysis of soxC and soxB(2) expression upon ctrlMO, bmp2/4MO, and gdf5-lMO knockdown at 2 day planula stage by in situ hybridization. (E) Selected neuronal marker expression upon K02288 treatments at 2 day planula, 4 day planula and 5 day planula stage visualized by in situ hybridization. Asterisks indicate blastopore, scale bars 100μm. Numerical data for the bar plot can be found in the sel.BMPMO_neuro sheet of the S2 Table. https://doi.org/10.1371/journal.pbio.3003803.g008

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