TBXT dose sensitivity and the decoupling of nascent mesoderm specification from EMT progression in 2D human gastruloids.

In the nascent mesoderm, TBXT expression must be precisely regulated to ensure that cells exit the primitive streak and pattern the anterior-posterior axis, but how varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dosage reduction during early human gastrulation using human induced pluripotent stem cell models of gastrulation and mesoderm differentiation. Multi-omic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprising wild-type, TBXT heterozygous or TBXT null human induced pluripotent stem cells reveal that varying TBXT dosage does not compromise the ability of a cell to differentiate into nascent mesoderm, but instead directly influences the temporal progression of the epithelial-to-mesenchymal transition with wild type transitioning first, followed by TBXT heterozygous and then TBXT null. By differentiating cells into nascent mesoderm in a monolayer format, we further illustrate that TBXT dosage directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that epithelial-to-mesenchymal transition progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

scRNA-seq analysis of cells comprising the amphioxus notochord.

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate.

The fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head.

Chimeric 3D gastruloids - a versatile tool for studies of mammalian peri-gastrulation development.

Stem cell-derived three-dimensional (3D) gastruloids show a remarkable capacity of self-organisation and recapitulate many aspects of gastrulation stage mammalian development. Gastruloids can be rapidly generated and offer several experimental advantages, such as scalability, observability and accessibility for manipulation. Here, we present approaches to further expand the experimental potency of murine 3D gastruloids by using functional genetics in mouse embryonic stem cells (mESCs) to generate chimeric gastruloids. In chimeric gastruloids, fluorescently labelled cells of different genotypes harbouring inducible gene expression or loss-of-function alleles are combined with wild-type cells. We showcase this experimental approach in chimeric gastruloids of mESCs carrying homozygous deletions of the Tbx transcription factor brachyury or inducible expression of Eomes. Resulting chimeric gastruloids recapitulate reported Eomes and brachyury functions, such as instructing cardiac fate and promoting posterior axial extension, respectively. Additionally, chimeric gastruloids revealed previously unrecognised phenotypes, such as the tissue sorting preference of brachyury deficient cells to endoderm and the cell non-autonomous effects of brachyury deficiency on Wnt3a patterning along the embryonic axis, demonstrating some of the advantages of chimeric gastruloids as an efficient tool for studies of mammalian gastrulation.

Mesoderm induction and patterning: Insights from neuromesodermal progenitors.

The discovery of mesoderm inducing signals helped usher in the era of molecular developmental biology, and today the mechanisms of mesoderm induction and patterning are still intensely studied. Mesoderm induction begins during gastrulation, but recent evidence in vertebrates shows that this process continues after gastrulation in a group of posteriorly localized cells called neuromesodermal progenitors (NMPs). NMPs reside within the post-gastrulation embryonic structure called the tailbud, where they make a lineage decision between ectoderm (spinal cord) and mesoderm. The majority of NMP-derived mesoderm generates somites, but also contributes to lateral mesoderm fates such as endothelium. The discovery of NMPs provides a new paradigm in which to study vertebrate mesoderm induction. This review will discuss mechanisms of mesoderm induction within NMPs, and how they have informed our understanding of mesoderm induction more broadly within vertebrates as well as animal species outside of the vertebrate lineage. Special focus will be given to the signaling networks underlying NMP-derived mesoderm induction and patterning, as well as emerging work on the significance of partial epithelial-mesenchymal states in coordinating cell fate and morphogenesis.

Brachyury promotes extracellular matrix synthesis through transcriptional regulation of Smad3 in nucleus pulposus.

Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury (Bry) has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.NEW & NOTEWORTHY Mechanically, in the nucleus, Bry enhances the transcription of Smad3, leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that Bry can alleviate IVDD through this mechanism.

A phase 2 study of a brachyury-targeting vaccine in combination with radiation therapy for the treatment of advanced chordoma.

BACKGROUND: This was a single-arm, phase 2 clinical trial of Bavarian Nordic (BN)-Brachyury vaccine plus radiotherapy (RT) designed to determine the objective response rate (ORR), progression-free survival (PFS), and safety of the combination in chordoma. METHODS: A total of 29 adult patients with advanced chordoma were treated with two subcutaneous priming vaccine doses of modified vaccinia Ankara-Bavarian Nordic (MVA-BN)-Brachyury and one vaccine dose of fowlpox virus (FPV)-Brachyury before RT. After RT, booster vaccinations were given with FPV-Brachyury every 4 weeks for 4 doses, then every 12 weeks (week 110). A minimum RT dose of >8 Gy in one fraction for each target was required. Response was evaluated by modified Response Evaluation Criteria in Solid Tumors 1.1 (mRECIST), where only radiated lesions were considered targets, and by standard RECIST 1.1 in a subset of patients. RESULTS: Two of 26 evaluable patients experienced durable partial response (PR) (ORR of 7.7%; 90% confidence interval [CI], 2.6-20.8]) by mRECIST 1.1. A total of 21 patients (80.8%; 90% CI, 65.4-90.3) had stable disease, and three patients (11.5%; 90% CI, 4.7-25.6) had progressive disease as best response per mRECIST 1.1. Median PFS was not reached during the study. CONCLUSIONS: This trial confirms the safety of BN-Brachyury and RT. Although the study did not meet the predefined study goal of four responses in 29 patients, we did observe two PRs and a PFS of greater than 2 years. For a vaccine-based study in chordoma, an ultra-rare disease where response rates are low, a randomized study or novel trial designs may be required to confirm activity.

A 37†kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.

The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.

Brachyury controls Ciona notochord fate as part of a feed-forward network.

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator.

Identification of in vivo Hox13-binding sites reveals an essential locus controlling zebrafish brachyury expression.

During early embryogenesis, the vertebrate embryo extends from anterior to posterior because of the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for NMps to retain mesodermal potential and, hence, normal axis development. We recently showed that Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor-binding sites, we identified more than 500 potential Hox13 target genes in zebrafish. Importantly, we found two highly conserved Hox13-binding elements far from the tbxta transcription start site that also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis-stage expression to a tbxta promoter that, on its own, only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.

Brachyury positively regulates extracellular matrix synthesis via directly promoting aggrecan transcription in nucleus pulposus.

Nucleus pulposus (NP) degeneration is characterized by the decreased cellularity of nucleus pulposus cells (NPCs) and diminished content of hydrophilic extracellular matrix (ECM). Overexpression of brachyury has been reported to reverse the degenerated NPCs into healthy phenotypes. However, the direct correlation between brachyury and ECM has not been fully elucidated. This study revealed that brachyury expression decreased in human degenerated NP tissues and Lipopolysaccharide (LPS)-induced degenerated rat NPCs model. In vitro and in vivo experiments further showed that brachyury deficiency suppressed the synthesis of aggrecan and collagen II in NP. Mechanistically, ChIP-qPCR assays demonstrated that brachyury bound to the promoter region of aggrecan in NPCs. Furthermore, luciferase reporter assays revealed that brachyury transcriptionally activated aggrecan expression through binding with a novel specific motif. In rat in vivo model, brachyury overexpression partially reversed the degenerative phenotype. In conclusion, brachyury positively regulated ECM synthesis via directly promoting aggrecan transcription in NPCs. Accordingly, it may be helpful to be developed into a promising therapeutic target for NP degeneration.

Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Development of a Small Molecule Downmodulator for the Transcription Factor Brachyury.

Brachyury is an oncogenic transcription factor whose overexpression drives chordoma growth. The downmodulation of brachyury in chordoma cells has demonstrated therapeutic potential, however, as a transcription factor it is classically deemed "undruggable". Given that direct pharmacological intervention against brachyury has proven difficult, attempts at intervention have instead targeted upstream kinases. Recently, afatinib, an FDA-approved kinase inhibitor, has been shown to modulate brachyury levels in multiple chordoma cell lines. Herein, we use afatinib as a lead to undertake a structure-based drug design approach, aided by mass-spectrometry and X-ray crystallography, to develop DHC-156, a small molecule that more selectively binds brachyury and downmodulates it as potently as afatinib. We eliminated kinase-inhibition from this novel scaffold while demonstrating that DHC-156 induces the post-translational downmodulation of brachyury that results in an irreversible impairment of chordoma tumor cell growth. In doing so, we demonstrate the feasibility of direct brachyury modulation, which may further be developed into more potent tool compounds and therapies.

Ectopic expression of T in the paraxial mesoderm disrupts somite maturation in the mouse.

T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T, which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1, known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

Syntaxin4, P-cadherin, and CCAAT enhancer binding protein ß as signaling elements in the novel differentiation pathway for cultured embryonic stem cells.

Pluripotent stem cells possess the potential to differentiate into all three germ layers. However, upon removal of the stemness factors, pluripotent stem cells, such as embryonic stem cells (ESCs), exhibit EMT-like cell behavior and lose stemness signatures. This process involves the membrane translocation of the t-SNARE protein syntaxin4 (Stx4) and the expression of the intercellular adhesion molecule P-cadherin. The forced expression of either of these elements induces the emergence of such phenotypes even in the presence of stemness factors. Interestingly, extracellular Stx4, but not P-cadherin, appears to induce a significant upregulation of the gastrulation-related gene brachyury, along with a slight upregulation of the smooth muscle cell-related gene ACTA2 in ESCs. Furthermore, our findings reveal that extracellular Stx4 plays a role in preventing the elimination of CCAAT enhancer binding protein ß (C/EBPß). Notably, the forced overexpression of C/EBPß led to the downregulation of brachyury and a significant upregulation of ACTA2 in ESCs. These observations suggest that extracellular Stx4 contributes to early mesoderm induction while simultaneously activating an element that alters the differentiation state. The fact that a single differentiation cue can elicit multiple differentiation responses may reflect the challenges associated with achieving sensitive and directed differentiation in cultured stem cells.

Brachyury-targeted immunotherapy combined with gemcitabine against head and neck cancer.

Brachyury is a transcription factor belonging to the T-box gene family and is involved in the posterior formation of the mesoderm and differentiation of chordates. As the overexpression of Brachyury is a poor prognostic factor in a variety of cancers, the establishment of Brachyury-targeted therapy would be beneficial for the treatment of aggressive tumors. Because transcription factors are difficult to treat with a therapeutic antibody, peptide vaccines are a feasible approach for targeting Brachyury. In this study, we identified Brachyury-derived epitopes that elicit antigen-specific and tumor-reactive CD4+ T cells that directly kill tumors. T cells recognizing Brachyury epitopes were present in patients with head and neck squamous cell carcinoma. Next, we focused on gemcitabine (GEM) as an immunoadjuvant to augment the efficacy of antitumor responses by T cells. Interestingly, GEM upregulated HLA class I and HLA-DR expression in tumor, followed by the upregulation of anti-tumor T cell responses. As tumoral PD-L1 expression was also augmented by GEM, PD-1/PD-L1 blockade and GEM synergistically enhanced the tumor-reactivity of Brachyury-reactive T cells. The synergy between the PD-1/PD-L1 blockade and GEM was also confirmed in a mouse model of head and neck squamous cell carcinoma. These results suggest that the combined treatment of Brachyury peptide with GEM and immune checkpoint blockade could be a promising immunotherapy against head and neck cancer.

Hox13 genes are required for mesoderm formation and axis elongation during early zebrafish development.

The early vertebrate embryo extends from anterior to posterior due to the addition of neural and mesodermal cells from a neuromesodermal progenitor (NMp) population located at the most posterior end of the embryo. In order to produce mesoderm throughout this time, the NMps produce their own niche, which is high in Wnt and low in retinoic acid. Using a loss-of-function approach, we demonstrate here that the two most abundant Hox13 genes in zebrafish have a novel role in providing robustness to the NMp niche by working in concert with the niche-establishing factor Brachyury to allow mesoderm formation. Mutants lacking both hoxa13b and hoxd13a in combination with reduced Brachyury activity have synergistic posterior body defects, in the strongest case producing embryos with severe mesodermal defects that phenocopy brachyury null mutants. Our results provide a new way of understanding the essential role of the Hox13 genes in early vertebrate development.This article has an associated 'The people behind the papers' interview.