Robust axis elongation by Nodal-dependent restriction of BMP signaling.

Embryogenesis results from the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key roles in germ layer specification and morphogenesis, yet their interplay to coordinate embryo patterning with morphogenesis is still insufficiently understood. Here, we took a reductionist approach using zebrafish embryonic explants to study the coordination of Nodal and BMP signaling for embryo patterning and morphogenesis. We show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors but also suppressing BMP signaling activity at the site of mesendoderm induction. Consistent with this, ectopic BMP signaling in the mesendoderm blocks cell alignment and oriented mesendoderm intercalations, key processes during explant elongation. Translating these ex vivo observations to the intact embryo showed that, similar to explants, Nodal signaling suppresses the effect of BMP signaling on cell intercalations in the dorsal domain, thus allowing robust embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis elongation by both inducing mesendoderm and suppressing BMP effects in the dorsal portion of the mesendoderm.

Uterine Nodal expression supports maternal immunotolerance and establishment of the FOXP3+ regulatory T cell population during the preimplantation period.

Pregnancy success is dependent on the establishment of maternal tolerance during the preimplantation period. The immunosuppressive function of regulatory T cells is critical to limit inflammation arising from implantation of the semi-allogeneic blastocyst. Insufficient maternal immune adaptations to pregnancy have been frequently associated with cases of female infertility and recurrent implantation failure. The role of Nodal, a secreted morphogen of the TGFß superfamily, was recently implicated during murine pregnancy as its conditional deletion (NodalΔ/Δ) in the female reproductive tract resulted in severe subfertility. Here, it was determined that despite normal preimplantation processes and healthy, viable embryos, NodalΔ/Δ females had a 50% implantation failure rate compared to NodalloxP/loxP controls. Prior to implantation, the expression of inflammatory cytokines MCP-1, G-CSF, IFN-γ and IL-10 was dysregulated in the NodalΔ/Δ uterus. Further analysis of the preimplantation leukocyte populations in NodalΔ/Δ uteri showed an overabundance of infiltrating, pro-inflammatory CD11bhigh Ly6C+ macrophages coupled with the absence of CD4+ FOXP3+ regulatory T cells. Therefore, it is proposed that uterine Nodal expression during the preimplantation period has a novel role in the establishment of maternal immunotolerance, and its dysregulation should be considered as a potential contributor to cases of female infertility and recurrent implantation failure.

Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFß to rotate the gut.

The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-ß (TGFß), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.

Regulation of Nodal signaling propagation by receptor interactions and positive feedback.

During vertebrate embryogenesis, the germ layers are patterned by secreted Nodal signals. In the classical model, Nodals elicit signaling by binding to a complex comprising Type I/II Activin receptors (Acvr) and the co-receptor Tdgf1. However, it is currently unclear whether receptor binding can also affect the distribution of Nodals themselves through the embryo, and it is unknown which of the putative Acvr paralogs mediate Nodal signaling in zebrafish. Here, we characterize three Type I (Acvr1) and four Type II (Acvr2) homologs and show that - except for Acvr1c - all receptor-encoding transcripts are maternally deposited and present during zebrafish embryogenesis. We generated mutants and used them together with combinatorial morpholino knockdown and CRISPR F0 knockout (KO) approaches to assess compound loss-of-function phenotypes. We discovered that the Acvr2 homologs function partly redundantly and partially independently of Nodal to pattern the early zebrafish embryo, whereas the Type I receptors Acvr1b-a and Acvr1b-b redundantly act as major mediators of Nodal signaling. By combining quantitative analyses with expression manipulations, we found that feedback-regulated Type I receptors and co-receptors can directly influence the diffusion and distribution of Nodals, providing a mechanism for the spatial restriction of Nodal signaling during germ layer patterning.

Building a body is complicated. Cells must organise themselves head-to-tail, belly-to-back, and inside-to-outside. They do this by laying down a chemical map, which is made up of gradients of molecular signals, high in some places and lower in others. The amount of signal each cell receives helps to decide which part of the body it will become. One of the essential signals in developing vertebrates is Nodal. It helps cells to tell inside from outside and left from right. Cells detect Nodal using an activin receptor and co-receptor complex, which catch hold of passing Nodal proteins and transmit developmental signals into cells. An important model to study Nodal signals is the zebrafish embryo, but the identity of the activin receptors and their exact role in this organism has been unclear. To find out more, Preiß, Kögler, Mörsdorf et al. studied the activin receptors Acvr1 and Acvr2 in zebrafish embryos. The experiments revealed that two putative Acvr1 and four Acvr2 receptors were present during early development. To better understand their roles, Preiß et al. eliminated them one at a time, and in combination. Losing single activin receptors had no effect. But losing both Acvr1 receptors together stopped Nodal signalling and changed the distribution of the Nodal gradient. Loss of all Acvr2 receptors also caused developmental problems, but they were partly independent of Nodal. This suggests that Acvr1s seem to be able to transmit signals and to shape the Nodal gradient, and that Acvr2s might have another, so far unknown, role. Nodal signals guide the development of all vertebrates. Understanding how they work in a model species like zebrafish could shed light on their role in other species, including humans. A clearer picture could help to uncover what happens at a molecular level when development goes wrong.

Establishment and interpretation of NODAL and BMP signaling gradients in early vertebrate development.

Transforming growth factor ß (TGF-ß) family ligands play crucial roles in orchestrating early embryonic development. Most significantly, two family members, NODAL and BMP form signaling gradients and indeed in fish, frogs and sea urchins these two opposing gradients are sufficient to organize a complete embryonic axis. This review focuses on how these gradients are established and interpreted during early vertebrate development. The review highlights key principles that are emerging, in particular the importance of signaling duration as well as ligand concentration in both gradient generation and their interpretation. Feedforward and feedback loops involving other signaling pathways are also essential for providing spatial and temporal information downstream of the NODAL and BMP signaling pathways. Finally, new data suggest the existence of buffering mechanisms, whereby early signaling defects can be readily corrected downstream later in development, suggesting that signaling gradients do not have to be as precise as previously thought.

Proteomic analysis of the IPF mesenchymal progenitor cell nuclear proteome identifies abnormalities in key nodal proteins that underlie their fibrogenic phenotype.

IPF is a progressive fibrotic lung disease whose pathogenesis remains incompletely understood. We have previously discovered pathologic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients. IPF MPCs display a distinct transcriptome and create sustained interstitial fibrosis in immune deficient mice. However, the precise pathologic alterations responsible for this fibrotic phenotype remain to be uncovered. Quantitative mass spectrometry and interactomics is a powerful tool that can define protein alterations in specific subcellular compartments that can be implemented to understand disease pathogenesis. We employed quantitative mass spectrometry and interactomics to define protein alterations in the nuclear compartment of IPF MPCs compared to control MPCs. We identified increased nuclear levels of PARP1, CDK1, and BACH1. Interactomics implicated PARP1, CDK1, and BACH1 as key hub proteins in the DNA damage/repair, differentiation, and apoptosis signaling pathways respectively. Loss of function and inhibitor studies demonstrated important roles for PARP1 in DNA damage/repair, CDK1 in regulating IPF MPC stemness and self-renewal, and BACH1 in regulating IPF MPC viability. Our quantitative mass spectrometry studies combined with interactomic analysis uncovered key roles for nuclear PARP1, CDK1, and BACH1 in regulating IPF MPC fibrogenicity.

Differential impact of TGFß/SMAD signaling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells.

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFß pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFß family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFß pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFß/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFß/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

Biological Role of Nodal Modulator: A Comprehensive Review of the Last Two Decades.

Nodal modulator (NOMO) is a type I transmembrane protein that is conserved in various human tissues. Humans have three highly similar NOMO proteins, namely NOMO1, NOMO2, and NOMO3. These three proteins are closely related and may have similar functions. NOMO has been identified as a part of a protein complex that mediates a wide range of biological processes such as tumor formation, bone and cartilage formation, embryo formation, facial asymmetry, and development of congenital heart disease. To date, a few studies have focused on the role of NOMO; however, the mechanism underlying its effects remains unknown. To improve our understanding regarding NOMO, we reviewed the role of NOMO in different diseases and investigated the mechanism underlying its effects.

YAP1 regulates the self-organized fate patterning of hESC-derived gastruloids.

The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.

Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair.

PCSK9 regulates the NODAL signaling pathway and cellular proliferation in hiPSCs.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein (LDL) cholesterol metabolism and the target of lipid-lowering drugs. PCSK9 is mainly expressed in hepatocytes. Here, we show that PCSK9 is highly expressed in undifferentiated human induced pluripotent stem cells (hiPSCs). PCSK9 inhibition in hiPSCs with the use of short hairpin RNA (shRNA), CRISPR/cas9-mediated knockout, or endogenous PCSK9 loss-of-function mutation R104C/V114A unveiled its new role as a potential cell cycle regulator through the NODAL signaling pathway. In fact, PCSK9 inhibition leads to a decrease of SMAD2 phosphorylation and hiPSCs proliferation. Conversely, PCSK9 overexpression stimulates hiPSCs proliferation. PCSK9 can interfere with the NODAL pathway by regulating the expression of its endogenous inhibitor DACT2, which is involved in transforming growth factor (TGF) ß-R1 lysosomal degradation. Using different PCSK9 constructs, we show that PCSK9 interacts with DACT2 through its Cys-His-rich domain (CHRD) domain. Altogether these data highlight a new role of PCSK9 in cellular proliferation and development.

Implication of rare genetic variants of NODAL and ACVR1B in congenital heart disease patients from Indian population.

NODAL signaling plays an essential role in vertebrate embryonic patterning and heart development. Accumulating evidences suggest that genetic mutations in TGF-ß/NODAL signaling pathway can cause congenital heart disease in humans. To investigate the implication of NODAL signaling in isolated cardiovascular malformation, we have screened 300 non-syndromic CHD cases and 200 controls for NODAL and ACVR1B by Sanger sequencing and identified two rare missense (c.152C > T; p.P51L and c.981 T > A; p.D327E) variants in NODAL and a novel missense variant c.1035G > A; p.M345I in ACVR1B. All these variants are absent in 200 controls. Three-dimensional protein-modelling demonstrates that both p.P51L and p.D327E variations of NODAL and p.M345I mutation of ACVR1B, affect the tertiary structure of respective proteins. Variants of NODAL (p.P51L and p.D327E) and ACVR1B (p.M345I), significantly reduce the transactivation of AR3-Luc, (CAGA)12-Luc and (SBE)4-Luc promoters. Moreover, qRT-PCR results have also deciphered a reduction in the expression of cardiac-enriched transcription factors namely Gata4, Nkx2-5, and Tbx5 in both the mutants of NODAL. Decreased expression of, Gata4, Nkx2-5, Tbx5, and lefty is observed in p.M345I mutant of ACVR1B as well. Additionally, reduced phosphorylation of SMAD2/3 in response to these variants, suggests impaired NODAL signaling and possibly responsible for defective cell fate decision and differentiation of cardiomyocytes leading to CHD phenotype.

RSV Promotes Epithelial Neuroendocrine Phenotype Differentiation through NODAL Signaling Pathway.

BACKGROUND: Respiratory syncytial virus (RSV) infects infants and children, predisposing them to development of asthma during adulthood. Epithelial neuroendocrine phenotypes may be associated with development of asthma. This study hopes to ascertain if RSV infection promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway. METHODS: The GSE6802 data set was obtained from the GEO database, and the differential genes were analyzed using the R language. An in vitro model was constructed with RSV infected human respiratory epithelial cells, and then real-time qPCR and immunofluorescence were used to detect the expression of different epithelial biomarkers and airway neuropeptides. The acute and chronic infection model of RSV infection was established by intranasal injection of RSV into guinea pigs. Immunohistochemistry and Western blot were used to detect the expression of pulmonary neuroendocrine cells markers ENO2 and neuropeptides. RESULTS: The expression levels of ENO2, SP, CGRP, and NODAL/ACTRII were significantly higher in the RSV infection group than those of the control group, which were abrogated by siRNA-NODAL. In vivo, we found that the expression levels of ENO2, SP, and CGRP were significantly higher than that of the control group. CONCLUSION: RSV promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway.

A case for revisiting Nodal signaling in human pluripotent stem cells.

Nodal is a transforming growth factor-ß (TGF-ß) superfamily member that plays a number of critical roles in mammalian embryonic development. Nodal is essential for the support of the peri-implantation epiblast in the mouse embryo and subsequently acts to specify mesendodermal fate at the time of gastrulation and, later, left-right asymmetry. Maintenance of human pluripotent stem cells (hPSCs) in vitro is dependent on Nodal signaling. Because it has proven difficult to prepare a biologically active form of recombinant Nodal protein, Activin or TGFB1 are widely used as surrogates for NODAL in hPSC culture. Nonetheless, the expression of the components of an endogenous Nodal signaling pathway in hPSC provides a potential autocrine pathway for the regulation of self-renewal in this system. Here we review recent studies that have clarified the role of Nodal signaling in pluripotent stem cell populations, highlighted spatial restrictions on Nodal signaling, and shown that Nodal functions in vivo as a heterodimer with GDF3, another TGF-ß superfamily member expressed by hPSC. We discuss the role of this pathway in the maintenance of the epiblast and hPSC in light of these new advances.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

Embryonic protein NODAL regulates the breast tumor microenvironment by reprogramming cancer-derived secretomes.

The tumor microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumorigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumor growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple-negative breast cancers and that it directly induces Cancer-associated fibroblasts phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g., CXCL1), cytokines (e.g., IL-6) and growth factors (e.g., PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.

Progress on the left-right asymmetry patterning in amphioxus.

The mechanisms underlying the establishment of left-right (L-R) asymmetry in bilaterians is one of the central enigmas in developmental biology. Amphioxus is an important model in studying the mechanisms of animal asymmetry specification due to its particular phylogenetic position, vertebrate-like embryogenesis and body plan. Recently, with the establishments of artificial breeding technology, high-efficiency microinjection method and gene knockout technology, researchers have successfully dissected the mechanisms of amphioxus L-R asymmetry development. In this review, we summarize the major progress in understanding L-R asymmetry specification in amphioxus and propose a model of regulation of L-R asymmetry in this species. Hh protein is transported dominantly to the right side by cilia movement, leading to R>L Hh signaling andCerexpression. Cer inhibits expression of Nodal, leading to the asymmetric expression of Nodal-dependent genes. The L-R differences in the propagation of the Nodal pathway result in the correct morphological L-R asymmetry development in amphioxus embryo. BMP signaling probably does not provide the asymmetric cue, but is necessary for correct expression ofCer andNodal.

CRISPR-Cas9 editing of non-coding genomic loci as a means of controlling gene expression in the sea urchin.

We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-ß signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.

E2A regulates neural ectoderm fate specification in human embryonic stem cells.

E protein transcription factors are crucial for many cell fate decisions. However, the roles of E proteins in the germ-layer specification of human embryonic stem cells (hESCs) are poorly understood. We disrupted the TCF3 gene locus to delete the E protein E2A in hESCs. E2A knockout (KO) hESCs retained key features of pluripotency, but displayed decreased neural ectoderm coupled with enhanced mesoendoderm outcomes. Genome-wide analyses showed that E2A directly regulates neural ectoderm and Nodal pathway genes. Accordingly, inhibition of Nodal or E2A overexpression partially rescued the neural ectoderm defect in E2A KO hESCs. Loss of E2A had little impact on the epigenetic landscape of hESCs, whereas E2A KO neural precursors displayed increased accessibility of the gene locus encoding the Nodal agonist CRIPTO. Double-deletion of both E2A and HEB (TCF12) resulted in a more severe neural ectoderm defect. Therefore, this study reveals critical context-dependent functions for E2A in human neural ectoderm fate specification.

Optogenetic investigation of BMP target gene expression diversity.

Signaling molecules activate distinct patterns of gene expression to coordinate embryogenesis, but how spatiotemporal expression diversity is generated is an open question. In zebrafish, a BMP signaling gradient patterns the dorsal-ventral axis. We systematically identified target genes responding to BMP and found that they have diverse spatiotemporal expression patterns. Transcriptional responses to optogenetically delivered high- and low-amplitude BMP signaling pulses indicate that spatiotemporal expression is not fully defined by different BMP signaling activation thresholds. Additionally, we observed negligible correlations between spatiotemporal expression and transcription kinetics for the majority of analyzed genes in response to BMP signaling pulses. In contrast, spatial differences between BMP target genes largely collapsed when FGF and Nodal signaling were inhibited. Our results suggest that, similar to other patterning systems, combinatorial signaling is likely to be a major driver of spatial diversity in BMP-dependent gene expression in zebrafish.