Mathematical model of morphogen electrophoresis through gap junctions.

Gap junctional communication is important for embryonic morphogenesis. However, the factors regulating the spatial properties of small molecule signal flows through gap junctions remain poorly understood. Recent data on gap junctions, ion transporters, and serotonin during left-right patterning suggest a specific model: the net unidirectional transfer of small molecules through long-range gap junctional paths driven by an electrophoretic mechanism. However, this concept has only been discussed qualitatively, and it is not known whether such a mechanism can actually establish a gradient within physiological constraints. We review the existing functional data and develop a mathematical model of the flow of serotonin through the early Xenopus embryo under an electrophoretic force generated by ion pumps. Through computer simulation of this process using realistic parameters, we explored quantitatively the dynamics of morphogen movement through gap junctions, confirming the plausibility of the proposed electrophoretic mechanism, which generates a considerable gradient in the available time frame. The model made several testable predictions and revealed properties of robustness, cellular gradients of serotonin, and the dependence of the gradient on several developmental constants. This work quantitatively supports the plausibility of electrophoretic control of morphogen movement through gap junctions during early left-right patterning. This conceptual framework for modeling gap junctional signaling -- an epigenetic patterning mechanism of wide relevance in biological regulation -- suggests numerous experimental approaches in other patterning systems.     

Gap junctions relay FGF8‐mediated right‐sided repression of Nodal in rabbit

In vertebrate gastrula/neurula embryos, a cilia‐driven leftward flow asymmetrically activates the Nodal cascade in the left lateral plate mesoderm (LPM). In frog embryos left–right axis formation was postulated to depend on gap junctions (GJs) during cleavage. Here, we show that GJs cooperate with fibroblast growth factor‐8 (FGF8) to specify asymmetric Nodal in the rabbit embryo at gastrula/neurula. GJs and FGF signaling were manipulated in whole embryo and explant cultures of rabbit blastodiscs. These experiments demonstrate that right‐sided inhibition of Nodal by FGF8 depended on intercellular communication by means of GJs, and that left‐sided induction of Nodal required attenuation of gap junctional communication (GJC). Before flow, the left and right side were equally competent but actively prevented from Nodal induction through FGF8/GJ. Our data suggest that flow unilaterally attenuates FGF8/GJ‐mediated repression of Nodal on the left side, integrating GJC and FGF8 into the flow‐based mechanism of symmetry breakage in the vertebrate embryo. Developmental Dynamics 237:3516–3527, 2008. © 2008 Wiley‐Liss, Inc.     

Using total internal reflection fluorescence (TIRF) microscopy to visualize cortical actin and microtubules in the Drosophila syncytial embryo

The Drosophila syncytial embryo is a powerful developmental model system for studying dynamic coordinated cytoskeletal rearrangements. Confocal microscopy has begun to reveal more about the cytoskeletal changes that occur during embryogenesis. Total internal reflection fluorescence (TIRF) microscopy provides a promising new approach for the visualization of cortical events with heightened axial resolution. We have applied TIRF microscopy to the Drosophila embryo to visualize cortical microtubule and actin dynamics in the syncytial blastoderm. Here, we describe the details of this technique, and report qualitative assessments of cortical microtubules and actin in the Drosophila syncytial embryo. In addition, we identified a peak of cortical microtubules during anaphase of each nuclear cycle in the syncytial blastoderm, and using images generated by TIRF microscopy, we quantitatively analyzed microtubule dynamics during this time. Developmental Dynamics 238:2622–2632, 2009. © 2009 Wiley‐Liss, Inc.     

Two-dimensional morphogen gradient in Xenopus: boundary formation and real-time transduction response.

Morphogen gradients play an important role in pattern formation in embryo. However, the interpretation of position in a morphogen gradient is not well understood. Because it is hard to analyze morphogen gradients especially in opaque embryos such as those of Xenopus, it is necessary to fix and section the embryo, thereby eliminating the possibility of real-time observation, and making more difficult the interpretation of events that take place in three dimensions. We describe here a two-dimensional preparation of cells from a Xenopus blastula animal cap, in which an activin concentration gradient appears to be formed and interpreted at the same rate and in the same way as in normal embryos. We use two-dimensional preparations of this kind to contribute the following new information about gradient formation and interpretation in embryo. We determine the dynamics of formation of an activin activity gradient in real time. We demonstrate that this gradient is established by diffusion of activin through intercellular space and does not require internalization of receptor or ligand. We also show that the generation of a boundary of gene expression depends on the interpretation, rather than a change of composition, of the concentration gradient.     

Morphogen gradients, positional information, and Xenopus: interplay of theory and experiment.

The idea of morphogen gradients has long been an important one in developmental biology. Studies with amphibians and with Xenopus in particular have made significant contributions to demonstrating the existence, identity, and mechanisms of action of morphogens. Mesoderm induction and patterning by activin, nodals, bone morphogenetic proteins, and fibroblast growth factors have been analyzed thoroughly and reveal recurrent and combinatorial roles for these protein growth factor morphogens and their antagonists. The dynamics of nodal-type signaling and the intersection of VegT and beta-catenin intracellular gradients reveal detailed steps in early long-range patterning. Interpretation of gradients requires sophisticated mechanisms for sharpening thresholds, and the activin-Xbra-Gsc system provides an example of this. The understanding of growth factor signal transduction has elucidated growth factor morphogen action and provided tools for dissecting their direct long-range action and distribution. The physical mechanisms of morphogen gradient establishment are the focus of new interest at both the experimental and theoretical level. General themes and emerging trends in morphogen gradient studies are discussed.     

Mathematical modelling of digit specification by a sonic hedgehog gradient

Background: The three chick wing digits represent a classical example of a pattern specified by a morphogen gradient. Here we have investigated whether a mathematical model of a Shh gradient can describe the specification of the identities of the three chick wing digits and if it can be applied to limbs with more digits. Results: We have produced a mathematical model for specification of chick wing digit identities by a Shh gradient that can be extended to the four digits of the chick leg with Shh‐producing cells forming a digit. This model cannot be extended to specify the five digits of the mouse limb. Conclusions: Our data suggest that the parameters of a classical‐type morphogen gradient are sufficient to specify the identities of three different digits. However, to specify more digit identities, this core mechanism has to be coupled to alternative processes, one being that in the chick leg and mouse limb, Shh‐producing cells give rise to digits; another that in the mouse limb, the cellular response to the Shh gradient adapts over time so that digit specification does not depend simply on Shh concentration. Developmental Dynamics 243:290–298, 2014. © 2013 Wiley Periodicals, Inc.     

Morphogen gradient interpretation by a regulated trafficking step during ligand-receptor transduction

Morphogen gradients are important in early development, but how cells recognize their position in such a gradient is not well understood. Cells need to correctly interpret a morphogen concentration when the morphogen is no longer present in the extracellular medium. This memory of morphogen exposure is necessary for correct cell fate decisions in the changing morphogen gradient concentration in an embryo. Our results demonstrate that a previously unrecognized step in gradient interpretation is a temporal stop that arrests the progression of a ligand-receptor complex between internalization and lysosomal destruction. Signaling continues during this arrested progression, which constitutes the basis of memory of morphogen concentration. We show that prolonged signaling requires Dynamin-dependent internalization of the complex. Rab5QL- and Rab7QL-mediated increases in the speed of the endo-lysosomal progression do not affect memory. In contrast, memory is abolished by increasing the targeting of receptors to the lysosome through expression of the Smad7/Smurf2 ubiquitin ligase. We conclude that the basis for memory is the long-lasting residence of a signaling complex in the endo-lysosomal pathway. The regulated duration of this step helps to determine the choice of gene expression resulting from gradient interpretation.     

Inhibitory morphogens and monopodial branching of the embryonic chicken lung

Background: Branching morphogenesis generates a diverse array of epithelial patterns, including dichotomous and monopodial geometries. Dichotomous branching can be instructed by concentration gradients of epithelial‐derived inhibitory morphogens, including transforming growth factor‐β (TGFβ), which is responsible for ramification of the pubertal mammary gland. Here, we investigated the role of autocrine inhibitory morphogens in monopodial branching morphogenesis of the embryonic chicken lung. Results: Computational modeling and experiments using cultured organ explants each separately revealed that monopodial branching patterns cannot be specified by a single epithelial‐derived autocrine morphogen gradient. Instead, signaling by means of TGFβ1 and bone morphogenetic protein‐4 (BMP4) differentially affect the rates of branching and growth of the airways. Allometric analysis revealed that development of the epithelial tree obeys power‐law dynamics; TGFβ1 and BMP4 have distinct but reversible effects on the scaling coefficient of the power law. Conclusions: These data suggest that although autocrine inhibition cannot specify monopodial branching, inhibitory morphogens define the dynamics of lung morphogenesis. Developmental Dynamics 241:852–862, 2012. © 2012 Wiley Periodicals, Inc.     

Microenvironmental control of stem cell fate in intestinal homeostasis and disease

The conventional model of intestinal epithelial architecture describes a unidirectional tissue organizational hierarchy with stem cells situated at the crypt base and daughter cells proliferating and terminally differentiating as they progress along the vertical (crypt–luminal) axis. In this model, the fate of a cell that has left the niche is determined and its lifespan limited. Evidence is accumulating to suggest that stem cell control and daughter cell fate determination is not solely an intrinsic, cell autonomous property but is heavily influenced by the microenvironment including paracrine, mesenchymal, and endogenous epithelial morphogen gradients. Recent research suggests that in intestinal homeostasis, stem cells transit reversibly between states of variable competence in the niche. Furthermore, selective pressures that disrupt the homeostatic balance, such as intestinal inflammation or morphogen dysregulation, can cause committed progenitor cells and even some differentiated cells to regain stem cell properties. Importantly, it has been recently shown that this disruption of cell fate determination can lead to somatic mutation and neoplastic transformation of cells situated outside the crypt base stem cell niche. This paper reviews the exciting developments in the study of stem cell dynamics in homeostasis, intestinal regeneration, and carcinogenesis, and explores the implications for human disease and cancer therapies. © 2015 Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Cilia play a role in breaking left–right symmetry of the sea urchin embryo

Left–right asymmetry of bilaterian animals is established during early development. In mice, frogs and fishes, the ciliated left–right organizer plays an essential role in establishing bilateral asymmetry, and leftward flow of extracellular fluid generated by ciliary motion results in Nodal activity on the left side. However, H⁺/K⁺‐ATPase activity is also involved in the determination of left–right asymmetry in a variety of animals, and it has been thought to be an ancestral mechanism in deuterostomes. In sea urchin, the determination of the left–right asymmetry based on H⁺/K⁺‐ATPase activity was already clarified, but it remains to be uncovered whether ciliary motion is involved in the left–right asymmetry of the embryo. Here, we show evidence that ciliary motion is involved in the establishment of left–right asymmetry of sea urchin embryo. Furthermore, we show that the initial cilia generated on small micromeres during the early stage of embryogenesis may be involved in this process. These results suggest that the cilia‐mediated mechanism for the determination of left–right asymmetry may be acquired at the base of the deuterostomes.     

Embryonic development of the self‐fertilizing mangrove killifish Kryptolebias marmoratus

Developing embryos of the self‐fertilising mangrove killifish Kryptolebias marmoratus. PTU‐treated embryo (left) lacking melanin pigmentation and a normal embryo (right), 10 days post‐fertilisation. Due to its ability to self‐fertilise, this species proves to be a highly useful embryological model with unique genetics. From Mourabit et al., Developmental Dynamics 240:1694–1704, 2011.     

Left cardiac isomerism in the Sonic hedgehog null mouse

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left–right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant‐negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.     

Rotation of Organizer Tissue Contributes to Left–Right Asymmetry

Current hypotheses regarding vertebrate left‐right asymmetry patterns are based on the presumption that genetic regulatory networks specify sidedness via extracellular morphogens and/or ciliary activity. We show empirical time‐lapse evidence for an asymmetric rotation of epiblastic nodal tissue in avian embryos. This rotation spans the interval when initial symmetric expression of Shh and Fgf8 becomes asymmetrical with respect to the midline. Anat Rec, 292:557–561, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of spatial distribution of BMP ligands for pattern formation

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, have been shown to contribute to embryogenesis and organogenesis during animal development. Relevant studies provide support for the following concepts: (a) BMP signals are evolutionarily highly conserved as a genetic toolkit; (b) spatiotemporal distributions of BMP signals are precisely controlled at the post-translational level; and (c) the BMP signaling network has been co-opted to adapt to diversified animal development. These concepts originated from the historical findings of the Spemann-Mangold organizer and the subsequent studies about how this organizer functions at the molecular level. In this Commentary, we focus on two topics. First, we review how the BMP morphogen gradient is formed to sustain larval wing imaginal disc and early embryo growth and patterning in Drosophila. Second, we discuss how BMP signal is tightly controlled in a context-dependent manner, and how the signal and tissue dynamics are coupled to facilitate complex tissue structure formation. Finally, we argue how these concepts might be developed in the future for further understanding the significance of BMP signaling in animal development.     

Transcriptional regulation of a pair-rule stripe in Drosophila

The periodic, seven-stripe pattern of the primary pair-rule gene even-skipped (eve) is initiated by crude, overlapping gradients of maternal and gap gene proteins in the early Drosophila embryo. Previous genetic studies suggest that one of the stripes, stripe 2, is initiated by the maternal morphogen bicoid (bcd) and the gap protein hunchback (hb), while the borders of the stripe are formed by selective repression, involving the gap protein giant (gt) in anterior regions and the Krüppel (Kr) protein in posterior regions. Here, we present several lines of evidence that are consistent with this model for stripe 2 expression, including in vitro DNA-binding experiments and transient cotransfection assays in cultured cells. These experiments suggest that repression involves a competition or short-range quenching mechanism, whereby the binding of gt and Kr interferes with the binding or activity of bcd and hb activators at overlapping or neighboring sites within the eve stripe 2 promoter element. Such short-range repression could reflect a general property of promoters composed of multiple, but autonomous regulatory elements.     

Androgenic regulation of ventral epithelial bud number and pattern in mouse urogenital sinus

The ventral urogenital sinus (UGS) of control male mice has two rows of 3–4 prostatic buds at birth, but how androgens regulate ventral bud (VB) number and patterning is unclear. VBs in both sexes appeared to be a mixture of prostatic and urethral buds. UGSs from Tfm male and antiandrogen (flutamide)‐exposed mice had small VBs, suggesting that initiation of some VBs is androgen independent. Tfm male mice are widely considered completely androgen insensitive yet their UGSs were 5α‐dihydrotestosterone (DHT)‐ responsive. VBs (6–8) were generally distributed bimodally on the left‐right axis at both minimal and normal male androgen signaling. Yet control females and DHT‐exposed Tfm males had 13–14 VBs, whose left‐right distribution was fairly uniform. These results suggest that VB number and distribution respond biphasically as androgen signaling increases from minimal, and that androgens regulate bud specification. Complete VB agenesis by the selective budding inhibitor 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) required high androgen signaling. Developmental Dynamics 239:373–385, 2010. © 2009 Wiley‐Liss, Inc.     

Dissecting the role of Fgf signaling during gastrulation and left‐right axis formation in mouse embryos using chemical inhibitors

Fgf signaling plays pivotal roles in mouse gastrulation and left‐right axis formation. However, although genetic analyses have revealed important aspects of Fgf signaling in these processes, the temporal resolution of genetic studies is low. Here, we combined whole‐embryo culture with application of chemical compounds to inhibit Fgf signaling at specific time points. We found that sodium chlorate and PD173074 are potent inhibitors of Fgf signaling in early mouse embryos. Fgf signaling is required for the epithelial‐to‐mesenchymal transition of the primitive streak before the onset of gastrulation. Once gastrulation begins, Fgf signaling specifies mesodermal fates via the Ras/MAPK downstream cascade. Finally, Fgf signaling on the posterior side of the embryo during gastrulation induces Nodal expression in the node via Tbx6‐Dll1, the initial event required for Nodal expression in the left lateral plate mesoderm. Developmental Dynamics 239:1768–1778, 2010. © 2010 Wiley‐Liss, Inc.