Cell lineage-guided mass spectrometry reveals increased energy metabolism and reactive oxygen species in the vertebrate organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

ZSWIM4 regulates embryonic patterning and BMP signaling by promoting nuclear Smad1 degradation.

The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning. Zinc Finger SWIM-Type Containing 4 (zswim4) is expressed in the Spemann-Mangold organizer at the onset of Xenopus gastrulation and is then enriched in the developing neuroectoderm at the mid-gastrula stages. Knockdown or knockout of zswim4 causes ventralization. Overexpression of zswim4 decreases, whereas knockdown of zswim4 increases the expression levels of ventrolateral mesoderm marker genes. Mechanistically, ZSWIM4 attenuates the BMP signal by reducing the protein stability of SMAD1 in the nucleus. Stable isotope labeling by amino acids in cell culture (SILAC) identifies Elongin B (ELOB) and Elongin C (ELOC) as the interaction partners of ZSWIM4. Accordingly, ZSWIM4 forms a complex with the Cul2-RING ubiquitin ligase and ELOB and ELOC, promoting the ubiquitination and degradation of SMAD1 in the nucleus. Our study identifies a novel mechanism that restricts BMP signaling in the nucleus.

The Spemann-Mangold organizer discovery and society.

This paper analyzes the influence of Hans Spemann and Hilde Mangold's discovery of the embryonic organizer on society outside the narrower confines of embryology. It begins by overviewing how in the 1920s-1930s, the organizer discovery fit well into the agenda of both right-wing and left-wing political camps, as it supported holistic theories of biological development that aligned with their ideological views. The paper then explores the organizer's influence on the formation and interdisciplinary discussions of the 1930s Cambridge Theoretical Biology Club. Club member C.H. Waddington's concept of the epigenetic landscape, inspired in part by Spemann and Mangold's work, went on to impact diverse fields including mathematics, psychology, anthropology, architecture, and art. Examples include mathematician René Thom's catastrophe landscapes and the field of ekistics (the science of human settlements). Spemann and Mangold's creation of an embryo chimera by combining tissues from different newt species also helped shape philosophers' and artists' understanding of the relationship between nature and technology as an integrated whole rather than separate entities. Additionally, Spemann's embryonic field concept helped influence field theories in other areas of science, and thinkers have pointed out metaphorical similarities between the organizer experiment and philosophical dialectics, film editing, or historical events. However, several factors likely limited the long-term societal impact of Spemann's holistic perspective, notably the rise of molecular biology and DNA-centric reductionist views of biology in the mid-late 20th century. While such reductionism still dominates public perceptions of biology, there seems to be a renewed openness to holistic perspectives reminiscent of Spemann's views.

Spemann-Mangold organizer and mesoderm induction.

The discovery of the Spemann-Mangold organizer strongly influenced subsequent research on embryonic induction, with research aiming to elucidate the molecular characteristics of organizer activity being currently underway. Herein, we review the history of research on embryonic induction, and describe how the mechanisms of induction phenomena and developmental processes have been investigated. Classical experiments investigating the differentiation capacity and inductive activity of various embryonic regions were conducted by many researchers, and important theories of region-specific induction and the concept for chain of induction were proposed. The transition from experimental embryology to developmental biology has enabled us to understand the mechanisms of embryonic induction at the molecular level. Consequently, many inducing substances and molecules such as transcriptional factors and peptide growth factors involved in the organizer formation were identified. One of peptide growth factors, activin, acts as a mesoderm- and endoderm-inducing substance. Activin induces several tissues and organs from the undifferentiated cell mass of amphibian embryos in a concentration-dependent manner. We review the extent to which we can control in vitro organogenesis from undifferentiated cells, and discuss the application to stem cell-based regenerative medicine based on insights gained from animal experiments, such as in amphibians.

"And it was the worst possible result, because it actually worked:" An interview with Richard Harland.

One hundred years ago, Hilde Mangold and Hans Spemann published their seminal paper on what came to be known as The Organizer, but seven decades would pass before the molecular basis of this remarkable phenomenon was revealed. Richard Harland and his laboratory played a key role in that discovery, and in this interview he discusses not just the science and the people but also other important factors like mental health and luck.

Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus.

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F1Fo ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when ß-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling.

LIM homeodomain proteins and associated partners: Then and now.

The field of molecular embryology started around 1990 by identifying new genes and analyzing their functions in early vertebrate embryogenesis. Those genes encode transcription factors, signaling molecules, their regulators, etc. Most of those genes are relatively highly expressed in specific regions or exhibit dramatic phenotypes when ectopically expressed or mutated. This review focuses on one of those genes, Lim1/Lhx1, which encodes a transcription factor. Lim1/Lhx1 is a member of the LIM homeodomain (LIM-HD) protein family, and its intimate partner, Ldb1/NLI, binds to two tandem LIM domains of LIM-HDs. The most ancient LIM-HD protein and its partnership with Ldb1 were innovated in the metazoan ancestor by gene fusion combining LIM domains and a homeodomain and by creating the LIM domain-interacting domain (LID) in ancestral Ldb, respectively. The LIM domain has multiple interacting interphases, and Ldb1 has a dimerization domain (DD), the LID, and other interacting domains that bind to Ssbp2/3/4 and the boundary factor, CTCF. By means of these domains, LIM-HD-Ldb1 functions as a hub protein complex, enabling more intricate and elaborate gene regulation. The common, ancestral role of LIM-HD proteins is neuron cell-type specification. Additionally, Lim1/Lhx1 serves crucial roles in the gastrula organizer and in kidney development. Recent studies using Xenopus embryos have revealed Lim1/Lhx1 functions and regulatory mechanisms during development and regeneration, providing insight into evolutionary developmental biology, functional genomics, gene regulatory networks, and regenerative medicine. In this review, we also discuss recent progress at unraveling participation of Ldb1, Ssbp, and CTCF in enhanceosomes, long-distance enhancer-promoter interactions, and trans-interactions between chromosomes.

Evolution of cis-regulatory modules for the head organizer gene goosecoid in chordates: comparisons between Branchiostoma and Xenopus.

BACKGROUND: In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated. RESULTS: To address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5' upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5' upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5' region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos. CONCLUSIONS: On the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5' upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate.

Organizers in Development.

An "organizer" is formally defined as a region, or group of cells in an embryo that can both induce (change the fate) and pattern (generate an organized set of structures) adjacent embryonic cells. To date, about four such regions have been demonstrated: the primary or Spemann organizer (Hensen's node in amniotes), the notochord, the zone of polarizing activity of the limb bud, and the mid-hindbrain boundary. Here we review the evidence for these and compare them with a few other regions which have been proposed to represent other organizers and we speculate on why so few such regions have been discovered.

Gene expression patterns that support novel developmental stress buffering in embryos of the annual killifish Austrofundulus limnaeus.

BACKGROUND: The cellular signaling mechanisms and morphogenic movements involved in axis formation and gastrulation are well conserved between vertebrates. In nearly all described fish, gastrulation and the initial patterning of the embryonic axis occur concurrently with epiboly. However, annual killifish may be an exception to this norm. Annual killifish inhabit ephemeral ponds in South America and Africa and permanent populations persist by the production of stress-tolerant eggs. Early development of annual killifish is unique among vertebrates because their embryonic blastomeres disperse randomly across the yolk during epiboly and reaggregate several days later to form the embryo proper. In addition, annual killifish are able to arrest embryonic development in one to three stages, known as diapause I, II, and III. Little is known about how the highly conserved developmental signaling mechanisms associated with early vertebrate development may have shifted in order to promote the annual killifish phenotype. One of the most well-characterized and conserved transcription factors, oct4 (Pou5f1), may have a role in maintaining pluripotency. In contrast, BMP-antagonists such as chordin, noggin, and follistatin, have been previously shown to establish dorsal-ventral asymmetry during axis formation. Transcription factors from the SOXB1 group, such as sox2 and sox3, likely work to induce neural specification. Here, we determine the temporal expression of these developmental factors during embryonic development in the annual killifish Austrofundulus limnaeus using quantitative PCR and compare these patterns to other vertebrates. RESULTS: Partial transcript sequences to oct4, sox2, sox3, chordin, noggin-1, noggin-2, and follistatin were cloned, sequenced, and identified in A. limnaeus. We found oct4, sox3, chordin, and noggin-1 transcripts to likely be maternally inherited. Expression of sox2, follistatin, and noggin-2 transcripts were highest in stages following a visible embryonic axis. CONCLUSIONS: Our data suggest that embryonic cells acquire their germ layer identity following embryonic blastomere reaggregation in A. limnaeus. This process of cellular differentiation and axis formation may involve similar conserved signaling mechanisms to other vertebrates. We propose that the undifferentiated state is prolonged during blastomere dispersal, thus functioning as a developmental stress buffer prior to the establishment of embryonic asymmetry and positional identity among the embryonic cells.