Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

The people behind the papers - Fay Cooper and Anestis Tsakiridis.

Neuromesodermal progenitor (NMPs) give rise to neural and mesodermal tissues during axis elongation. In their study, Fay Cooper, Anestis Tsakiridis and colleagues reveal the role of Notch signalling in NMP differentiation and its role in Hox gene expression. To learn more about their work, we spoke to first and co-corresponding author, Fay Cooper, and to co-corresponding author Anestis Tsakiridis, Group Leader at the University of Sheffield, UK.

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.

Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator.

BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.

R-Spondin 2 governs Xenopus left-right body axis formation by establishing an FGF signaling gradient.

Establishment of the left-right (LR, sinistral, dextral) body axis in many vertebrate embryos relies on cilia-driven leftward fluid flow within an LR organizer (LRO). A cardinal question is how leftward flow triggers symmetry breakage. The chemosensation model posits that ciliary flow enriches a signaling molecule on the left side of the LRO that promotes sinistral cell fate. However, the nature of this sinistralizing signal has remained elusive. In the Xenopus LRO, we identified the stem cell growth factor R-Spondin 2 (Rspo2) as a symmetrically expressed, sinistralizing signal. As predicted for a flow-mediated signal, Rspo2 operates downstream of leftward flow but upstream of the asymmetrically expressed gene dand5. Unexpectedly, in LR patterning, Rspo2 acts as an FGF receptor antagonist: Rspo2 via its TSP1 domain binds Fgfr4 and promotes its membrane clearance by Znrf3-mediated endocytosis. Concordantly, we find that at flow-stage, FGF signaling is dextralizing and forms a gradient across the LRO, high on the dextral- and low on the sinistral side. Rspo2 gain- and loss-of function equalize this FGF signaling gradient and sinistralize and dextralize development, respectively. We propose that leftward flow of Rspo2 produces an FGF signaling gradient that governs LR-symmetry breakage.

Long-range formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo.

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.

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.

Morphoelastic models discriminate between different mechanisms of left-right asymmetric stomach morphogenesis.

The mechanisms by which the vertebrate stomach undergoes its evolutionarily conserved leftward bending remain incompletely understood. Although the left and right sides of the organ are known to possess different gene expression patterns and undergo distinct morphogenetic events, the physical mechanisms by which these differences generate morphological asymmetry remain unclear. Here, we develop a continuum model of asymmetric stomach morphogenesis. Using a morphoelastic framework, we investigate the morphogenetic implications of a variety of hypothetical, tissue-level growth differences between the left and right sides of a simplified tubular organ. Simulations reveal that, of the various differential growth mechanisms tested, only one category is consistent with the leftward stomach curvature observed in wild-type embryos: equal left and right volumetric growth rates, coupled with transversely isotropic tissue thinning on the left side. Simulating this mechanism in a defined region of the model over a longer period of growth leads to mature stomach-like curvatures.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors.

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.

Self-organized BMP signaling dynamics underlie the development and evolution of digit segmentation patterns in birds and mammals.

Repeating patterns of synovial joints are a highly conserved feature of articulated digits, with variations in joint number and location resulting in diverse digit morphologies and limb functions across the tetrapod clade. During the development of the amniote limb, joints form iteratively within the growing digit ray, as a population of distal progenitors alternately specifies joint and phalanx cell fates to segment the digit into distinct elements. While numerous molecular pathways have been implicated in this fate choice, it remains unclear how they give rise to a repeating pattern. Here, using single-cell RNA sequencing and spatial gene expression profiling, we investigate the transcriptional dynamics of interphalangeal joint specification in vivo. Combined with mathematical modeling, we predict that interactions within the BMP signaling pathway-between the ligand GDF5, the inhibitor NOGGIN, and the intracellular effector pSMAD-result in a self-organizing Turing system that forms periodic joint patterns. Our model is able to recapitulate the spatiotemporal gene expression dynamics observed in vivo, as well as phenocopy digit malformations caused by BMP pathway perturbations. By contrasting in silico simulations with in vivo morphometrics of two morphologically distinct digits, we show how changes in signaling parameters and growth dynamics can result in variations in the size and number of phalanges. Together, our results reveal a self-organizing mechanism that underpins amniote digit segmentation and its evolvability and, more broadly, illustrate how Turing systems based on a single molecular pathway may generate complex repetitive patterns in a wide variety of organisms.

Revisiting bicoid function: complete inactivation reveals an additional fundamental role in Drosophila egg geometry specification.

INTRODUCTION: The bicoid (bcd) gene in Drosophila has served as a paradigm for a morphogen in textbooks for decades. Discovered in 1986 as a mutation affecting anterior development in the embryo, its expression pattern as a protein gradient later confirmed the prediction from transplantation experiments. These experiments suggested that the protein fulfills the criteria of a true morphogen, with the existence of a homeodomain crucial for activation of genes along the anterior-posterior axis, based on the concentration of the morphogen. The bcd gene undergoes alternative splicing, resulting in, among other isoforms, a small and often neglected isoform with low abundance, which lacks the homeodomain, termed small bicoid (smbcd). Most importantly, all known classical strong bcd alleles used in the past to determine bcd function apparently do not affect the function of this isoform. RESULTS: To overcome the uncertainty regarding which isoform regulates what, I removed the bcd locus entirely using CRISPR technology. bcdCRISPR eggs exhibited a short and round appearance. The phenotype could be ascribed to smbcd because all bcd alleles affecting the function of the major transcript, termed large bicoid (lgbcd) showed normally sized eggs. Several patterning genes for the embryo showed expression in the oocyte, and their expression patterns were altered in bcdCRISPR oocytes. In bcdCRISPR embryos, all downstream segmentation genes showed altered expression patterns, consistent with the expression patterns in "classical" alleles; however, due to the altered egg geometry resulting in fewer blastoderm nuclei, additional constraints came into play, further affecting their expression patterns. CONCLUSIONS: This study unveils a novel and fundamental role of bcd in shaping the egg's geometry. This discovery demands a comprehensive revision of our understanding of this important patterning gene and prompts a reevaluation of past experiments conducted under the assumption that bcd mutants were bcdnull-mutants.

Evolution of patterning.

Developing tissues are patterned in space and time; this enables them to differentiate their cell types and form complex structures to support different body plans. Although space and time are two independent entities, there are many examples of spatial patterns that originate from temporal ones. The most prominent example is the expression of the genes hunchback, Krüppel, pdm, and castor, which are expressed temporally in the neural stem cells of the Drosophila ventral nerve cord and spatially along the anteroposterior axis of the blastoderm stage embryo. In this Viewpoint, we investigate the relationship between space and time in specific examples of spatial and temporal patterns with the aim of gaining insight into the evolutionary history of patterning.

DLX gene expression in the developing chick pharyngeal arches and relationship to endothelin signaling and avian jaw patterning.

BACKGROUND: A hinged jaw that articulates with the skull base is a striking feature of the vertebrate head and has been greatly modified between, and within, vertebrate classes. Genes belonging to the DLX homeobox family are conserved mediators of local signaling pathways that distinguish the dorsal and ventral aspects of the first pharyngeal arch. Specifically, a subset of DLX genes are expressed in the cranial neural crest-derived mandibular ectomesenchyme in response to ventral endothelin signaling, an important step that confers the first arch with maxillary and mandibular identities. Downstream targets of DLX genes then execute the morphogenetic processes that lead to functional jaws. Identifying lineage-specific variations in DLX gene expression and the regulatory networks downstream of DLX action is necessary to understand how different kinds of jaws evolved. RESULTS: Here, we describe and compare the expression of all six DLX genes in the chick pharyngeal arches, focusing on the period of active patterning in the first arch. Disruption of endothelin signaling results in the down-regulation of ventral-specific DLX genes and confirms their functional role in avian jaw patterning. CONCLUSIONS: This expression resource will be important for comparative embryology and for identifying synexpression groups of DLX-regulated genes in the chick.

Cytoglobin regulates NO-dependent cilia motility and organ laterality during development.

Cytoglobin is a heme protein with unresolved physiological function. Genetic deletion of zebrafish cytoglobin (cygb2) causes developmental defects in left-right cardiac determination, which in humans is associated with defects in ciliary function and low airway epithelial nitric oxide production. Here we show that Cygb2 co-localizes with cilia and with the nitric oxide synthase Nos2b in the zebrafish Kupffer's vesicle, and that cilia structure and function are disrupted in cygb2 mutants. Abnormal ciliary function and organ laterality defects are phenocopied by depletion of nos2b and of gucy1a, the soluble guanylate cyclase homolog in fish. The defects are rescued by exposing cygb2 mutant embryos to a nitric oxide donor or a soluble guanylate cyclase stimulator, or with over-expression of nos2b. Cytoglobin knockout mice also show impaired airway epithelial cilia structure and reduced nitric oxide levels. Altogether, our data suggest that cytoglobin is a positive regulator of a signaling axis composed of nitric oxide synthase-soluble guanylate cyclase-cyclic GMP that is necessary for normal cilia motility and left-right patterning.

Molecular evidence of anteroposterior patterning in adult echinoderms.

The origin of the pentaradial body plan of echinoderms from a bilateral ancestor is one of the most enduring zoological puzzles1,2. Because echinoderms are defined by morphological novelty, even the most basic axial comparisons with their bilaterian relatives are problematic. To revisit this classical question, we used conserved anteroposterior axial molecular markers to determine whether the highly derived adult body plan of echinoderms masks underlying patterning similarities with other deuterostomes. We investigated the expression of a suite of conserved transcription factors with well-established roles in the establishment of anteroposterior polarity in deuterostomes3-5 and other bilaterians6-8 using RNA tomography and in situ hybridization in the sea star Patiria miniata. The relative spatial expression of these markers in P. miniata ambulacral ectoderm shows similarity with other deuterostomes, with the midline of each ray representing the most anterior territory and the most lateral parts exhibiting a more posterior identity. Strikingly, there is no ectodermal territory in the sea star that expresses the characteristic bilaterian trunk genetic patterning programme. This finding suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals and provides a developmental rationale for the re-evaluation of the events that led to the evolution of the derived adult body plan of echinoderms.

Neofunctionalization of Toll Signaling in Insects: From Immunity to Dorsoventral Patterning.

Toll signaling plays a crucial role in pathogen defense throughout the animal kingdom. It was discovered, however, for its function in dorsoventral (DV) axis formation in Drosophila. In all other insects studied so far, but not outside the insects, Toll is also required for DV patterning. However, in insects more distantly related to Drosophila, Toll's patterning role is frequently reduced and substituted by an expanded influence of BMP signaling, the pathway implicated in DV axis formation in all major metazoan lineages. This suggests that Toll was integrated into an ancestral BMP-based patterning system at the base of the insects or during insect evolution. The observation that Toll signaling has an immune function in the extraembryonic serosa, an early differentiating tissue of most insect embryos, suggests a scenario of how Toll was co-opted from an ancestral immune function for its new role in axis formation.

Modifying gastruloids to dissect mechanisms of tissue-specific induction.

How functional organisms arise from a single cell is a fundamental question in biology with direct relevance to understanding developmental defects and diseases. Dissecting developmental processes provides the basic, critical framework for understanding disease progression and treatment. Bottom-up approaches to recapitulate formation of various components of the embryo have been effective to probe symmetry-breaking, self-organisation, tissue patterning and morphogenesis. However, these studies have been mostly concerned with axial patterning, which is essentially longitudinal. Can these models generate the appendicular axes? If so, how far can self-organisation take these? Will experimentally induced organisers be required? This short review explores these questions, highlighting how minimal models are essential for understanding patterning and morphogenetic processes.

Stochastic gene expression and environmental stressors trigger variable somite segmentation phenotypes.

Mutations of several genes cause incomplete penetrance and variable expressivity of phenotypes, which are usually attributed to modifier genes or gene-environment interactions. Here, we show stochastic gene expression underlies the variability of somite segmentation defects in embryos mutant for segmentation clock genes her1 or her7. Phenotypic strength is further augmented by low temperature and hypoxia. By performing live imaging of the segmentation clock reporters, we further show that groups of cells with higher oscillation amplitudes successfully form somites while those with lower amplitudes fail to do so. In unfavorable environments, the number of cycles with high amplitude oscillations and the number of successful segmentations proportionally decrease. These results suggest that individual oscillation cycles stochastically fail to pass a threshold amplitude, resulting in segmentation defects in mutants. Our quantitative methodology is adaptable to investigate variable phenotypes of mutant genes in different tissues.