Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways

The transcriptional effector SMAD4 is a core component of the TGF-β family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-β family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-β family ligands, which has implications for diseases where Smad4 is mutated or deleted.

Structure of the unique tetrameric STENOFOLIA homeodomain bound with target promoter DNA

Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants. In plants, the WUSCHEL-related homeobox (WOX) family of transcription factors function as central organizers of several developmental programs ranging from embryo patterning to meristematic stem-cell maintenance through transcriptional activation and repression mechanisms. The Medicago truncatula STENOFOLIA (STF) gene is a master regulator of leaf-blade lateral development. Here, the crystal structure of the homeodomain (HD) of STF (STF-HD) in complex with its promoter DNA is reported at 2.1 Å resolution. STF-HD binds DNA as a tetramer, enclosing nearly the entire bound DNA surface. The STF-HD tetramer is partially stabilized by docking of the C-terminal tail of one protomer onto a conserved hydrophobic surface on the head of another protomer in a head-to-tail manner. STF-HD specifically binds TGA motifs, although the promoter sequence also contains TAAT motifs. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides DNA binding in the minor groove through basic residues located at its C-terminus. The structural and functional data in planta reported here provide new insights into the DNA-binding mechanisms of plant-specific HDs from the WOX family of transcription factors.

Similarities and Differences in the GFP Movement in the Zygotic and Somatic Embryos of Arabidopsis

Intercellular signaling during embryo patterning is not well understood and the role of symplasmic communication has been poorly considered. The correlation between the symplasmic domains and the development of the embryo organs/tissues during zygotic embryogenesis has only been described for a few examples, including Arabidopsis. How this process occurs during the development of somatic embryos (SEs) is still unknown. The aim of these studies was to answer the question: do SEs have a restriction in symplasmic transport depending on the developmental stage that is similar to their zygotic counterparts? The studies included an analysis of the GFP distribution pattern as expressed under diverse promoters in zygotic embryos (ZEs) and SEs. The results of the GFP distribution in the ZEs and SEs showed that 1/the symplasmic domains between the embryo organs and tissues in the SEs was similar to those in the ZEs and 2/the restriction in symplasmic transport in the SEs was correlated with the developmental stage and was similar to the one in their zygotic counterparts, however, with the spatio-temporal differences and different PDs SEL value between these two types of embryos.

An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.

Conserved, divergent and heterochronic gene expression duringBrachypodiumandArabidopsisembryo development

Embryogenesis, transforming the zygote into the mature embryo, represents a fundamental process for all flowering plants. Current knowledge of cell specification and differentiation during plant embryogenesis is largely based on studies of the dicot model plantArabidopsis thaliana. However, the major crops are monocots and the transcriptional programs associated with the differentiation processes during embryogenesis in this clade were largely unknown. Here, we combined analysis of cell division patterns with development of a temporal transcriptomic resource during embryogenesis of the monocot model plantBrachypodium distachyon. We found that early divisions of theBrachypodiumembryo were highly regular, while later stages were marked by less stereotypic patterns. Comparative transcriptomic analysis betweenBrachypodiumandArabidopsisrevealed that the early and late embryogenesis shared a common transcriptional program, whereas mid-embryogenesis was divergent between species. Analysis of orthology groups revealed widespread heterochronic expression of potential developmental regulators between the species. Interestingly,Brachypodiumgenes tend to be expressed at earlier stages thanArabidopsiscounterparts, which suggests that embryo patterning may occur early duringBrachypodiumembryogenesis. Detailed investigation of auxin-related genes shows that the capacity to synthesize, transport, and respond to auxin is established early in the embryo. However, while early PIN1 polarity could be confirmed, it is unclear if an active response is mounted. This study presents a resource for studyingBrachypodiumand grass embryogenesis, and shows that divergent angiosperms share a conserved genetic program that is marked by heterochronic gene expression.Key messageDevelopmental and transcriptomic analysis ofBrachypodiumembryogenesis, and comparison withArabidopsis, identifies conserved and divergent phases of embryogenesis, and reveals widespread heterochrony of developmental gene expression.

Suboptimal Intermediates Underlie Evolution of the Bicoid Homeodomain

Changes in regulatory networks generate materials for evolution to create phenotypic diversity. For transcription networks, multiple studies have shown that alterations in binding sites of cis-regulatory elements correlate well with the gain or loss of specific features of the body plan. Less is known about alterations in the amino acid sequences of the transcription factors (TFs) that bind these elements. Here we study the evolution of Bicoid (Bcd), a homeodomain (HD) protein that is critical for anterior embryo patterning in Drosophila. The ancestor of Bcd (AncBcd) emerged after a duplication of a Zerknullt (Zen)-like ancestral protein (AncZB) in a suborder of flies. AncBcd diverged from AncZB, gaining novel transcriptional and translational activities. We focus on the evolution of the HD of AncBcd, which binds to DNA and RNA, and is comprised of four subdomains: an N-terminal arm (NT) and three helices; H1, H2, and Recognition Helix (RH). Using chimeras of subdomains and gene rescue assays in Drosophila, we show that robust patterning activity of the Bcd HD (high frequency rescue to adulthood) is achieved only when amino acid substitutions in three separate subdomains (NT, H1, and RH) are combined. Other combinations of subdomains also yield full rescue, but with lower penetrance, suggesting alternative suboptimal activities. Our results suggest a multi-step pathway for the evolution of the Bcd HD that involved intermediate HD sequences with suboptimal activities, which constrained and enabled further evolutionary changes. They also demonstrate critical epistatic forces that contribute to the robust function of a DNA-binding domain.

Lyl-1 marks and regulates primitive macrophages and microglia development

During ontogeny, resident macrophages (MΦs) of the nervous system emerge from haematopoietic stem cell-independent progenitors originating in the Yolk Sac (YS), so that factors impairing YS MΦ development may lead to neurodevelopmental disorders resulting from defective brain resident MΦ.Here we show that Lyl-1, a bHLH transcription factor related to Scl/Tal-1, marks primitive macrophage (MΦPrim) progenitors in the YS. Transcriptomic analysis of YS MΦ progenitors indicated that MΦPrim progenitors present at embryonic day (E) 9 are clearly distinct from those present at E10. Lyl-1 bHLH disruption led to an increased production and a defective differentiation of MΦPrim progenitors. These differentiation defects were associated with profound modifications of the expression of genes involved in embryonic patterning and neurodevelopment. They also induced a reduced production of mature MΦ/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the new-born.We thus identify Lyl-1 as a critical regulator of MΦPrim and microglia development, which disruption may impair organogenesis, including neurodevelopment processes.Key pointsLyl-1 expression marks yolk sac macrophages and brain macrophage/microglia/BAM.Lyl-1 deficiency impairs primitive macrophage development and leads to the up-regulation of genes involved in embryo patterning.Lyl-1-expressing primitive macrophages have an immuno-modulatory phenotype.Lyl-1 deficiency impairs microglia development and the expression of genes involved in neuro-development.

Suboptimal intermediates underlie evolution of the Bicoid homeodomain

Changes in regulatory networks generate materials for evolution to create phenotypic diversity. For transcription networks, multiple studies have shown that alterations in binding sites of cis-regulatory elements correlate well with the gain or loss of specific features of the body plan. Less is known about alterations in the amino acid sequences of the transcription factors (TFs) that bind these elements. Here we study the evolution of Bicoid (Bcd), a homeodomain (HD) protein that is critical for anterior embryo patterning in Drosophila. The ancestor of Bcd (AncBcd) emerged after a duplication of a Zerknullt (Zen)-like ancestral protein (AncZB) in a suborder of flies. AncBcd diverged from AncZB, gaining novel transcriptional and translational activities. We focus on the evolution of the HD of AncBcd, which binds DNA and RNA, and is comprised of four subdomains: an N-terminal arm (NT) and three helices; H1, H2, and Recognition Helix (RH). Using chimeras of subdomains and gene rescue assays in Drosophila, we show that robust patterning activity of the Bcd HD (high frequency rescue to adulthood) is achieved only when amino acid substitutions in three separate subdomains (NT, H1, and RH) are combined. Other combinations of subdomains also yield full rescue, but with lower penetrance, suggesting alternative suboptimal activities. Our results suggest a multi-step pathway for the evolution of the Bcd HD that involved intermediate HD sequences with suboptimal activities, which constrained and enabled further evolutionary changes. They also demonstrate critical epistatic forces that contribute to the robust function of a DNA-binding domain.

Structure of the unique tetrameric STENOFOLIA homeodomain bound with DNA

Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants1. In plants, the WUSCHEL-related homeobox (WOX) family transcription factors function as central organizers of several developmental programs from embryo patterning to meristematic stem cell maintenance through transcriptional activation and repression2-4. The structure of WOX Homeodomain (HD) and the molecular mechanism of its interaction with DNA are unknown. Here, we report the 2.1 Å crystal structure of the STENOFOLIA (STF) HD from Medicago truncatula in complex with DNA. STF binds DNA as a novel cooperative tetramer, enclosing nearly entire bound DNA surface. The STF tetramer is partially stabilized by docking of the C-terminal tail from one protomer onto a conserved hydrophobic surface on the head of another in a head-to-tail manner. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides extensive binding to DNA in the minor groove. Our structural and functional data reveal that STF specifically targets ‘TGA’ sequence and the cooperative tetrameric binding with DNA is key to transcriptional repression in plants. Our data reveal an unprecedented HD:DNA recognition mechanism, representing the first plant HD structure from WOX family of transcription factors.