What we learn about bipolar disorder from large-scale neuroimaging: Findings and future directions from theENIGMABipolar Disorder Working Group

MRI‐derived brain measures offer a link between genes, the environment and behavior and have been widely studied in bipolar disorder (BD). However, many neuroimaging studies of BD have been underpowered, leading to varied results and uncertainty regarding effects. The Enhancing Neuro Imaging Genetics through Meta‐Analysis (ENIGMA) Bipolar Disorder Working Group was formed in 2012 to empower discoveries, generate consensus findings and inform future hypothesis‐driven studies of BD. Through this effort, over 150 researchers from 20 countries and 55 institutions pool data and resources to produce the largest neuroimaging studies of BD ever conducted. The ENIGMA Bipolar Disorder Working Group applies standardized processing and analysis techniques to empower large‐scale meta‐ and mega‐analyses of multimodal brain MRI and improve the replicability of studies relating brain variation to clinical and genetic data. Initial BD Working Group studies reveal widespread patterns of lower cortical thickness, subcortical volume and disrupted white matter integrity associated with BD. Findings also include mapping brain alterations of common medications like lithium, symptom patterns and clinical risk profiles and have provided further insights into the pathophysiological mechanisms of BD. Here we discuss key findings from the BD working group, its ongoing projects and future directions for large‐scale, collaborative studies of mental illness

Vertical Signalling Involves Transmission of Hox Information from Gastrula Mesoderm to Neurectoderm

<div><p>Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of <i>Xenopus laevis</i>. Our results show that copying of positional information involves non cell autonomous autoregulation of particular <i>Hox</i> genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the <i>Hox</i> pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved <i>Hox</i> genes.</p></div

Craniofacial structures of <i>Xenopus laevis</i> tadpoles upon injection of <i>Hoxd1</i> mRNA or protein.

<p><b>a</b>: Schematic ventral view of an uninjected untreated control embryo. <b>b</b>: uninjected embryo. <b>c</b>: embryo injected with wild type GFP into the blastoecel at blastula stage. <b>d</b>: Embryo injected with <i>Hoxd1</i> mRNA at 4 cell stage. <b>e</b>: Embryo injected with recombinant HOXD1 protein into 4 cell stage embryo. <b>f</b>: injection of recombinant HOXD1 protein into the blastoecel. Please note that standard injection of Hoxd1 mRNA or its protein counterpart injection in the cytoplasm or in the extracellular matrix leads to similar phenotypes in <b>d, e</b> and <b>f</b>. The embryos are strongly posteriorised as shown by the reduction (or deletion) of anterior structures. These data strongly suggest that the Hoxd1 protein successfully crossed the cellular membranes and retained its function as it leads to a severe truncation of anterior cartilage structures. Infrarostrale (in), Meckel’s cartilage (me), palatoquadrate (pa), ceratohyale (ce), basibranchiale (ba), branchial arches (br), eye (ey), intestine (in). Each photo represents 10 identically treated embryos giving the same result.</p

Mesodermal <i>Hox</i> loss of function of a single <i>Hox</i> gene prevents neurectodermal <i>Hox</i> expression of the same <i>Hox</i> gene.

<p><b>Aa</b>, Wrap assay consists of a piece of non-organiser mesoderm (NOM) and a piece of Spemann organiser mesoderm (SO) combined between two ectodermal animal caps. All tissues are excised from early gastrulae (st. 10a) <b>Ab</b>, <b>Ba</b>, <b>Ca:</b> External views of late gastrula stage <i>Xenopus laevis</i> expressing <i>Hoxd1, Hoxb4</i> and <i>Hoxb9</i> respectively. Note that the midline of the embryo, overlying the SO, does not express any <i>Hox</i> gene. <b>Ac</b>, <b>Bb</b>, <b>Cb:</b> wraps containing only SO explants [AC(SO/SO)AC]. <b>Ad</b>, <b>Bc</b>, <b>Cc:</b> wraps containing SO and NOM treated with control morpholino (ctMO) [AC(SO/NOM+ctMO)AC]. <b>Ae</b>, <b>Bd</b>, <b>Cd</b>, wrap with NOM treated with <i>Hoxd1-, Hoxb4</i> and <i>Hoxb9</i> MO’s respectively. Please note that in each case, only the wraps containing NOM and SO show <i>Hox</i> expression in the neurectoderm (<b>Ad</b>, <b>Bc</b>, <b>Cc</b>) and those containing only SO do not show any expression in accordance with the embryo’s lack of <i>Hox</i> expression in SO (<b>Ab</b>, <b>Ba</b>, <b>Ca</b>, and <b>Ac</b>, <b>Bb</b>, <b>Cb</b>). In each case, <i>Hox</i> MO treatment of NOM mesoderm also prevents the expression of the homologous <i>Hox</i> gene in neurectoderm (<b>Ae</b>, <b>Bd</b>, <b>Cd</b>). These wraps were fixed and analysed 6–8 hrs after they were made. Each photo of two recombinates or an embryo in this figure is representative of at least 20 recombinates or embryos, all showing the same result.</p

Mesodermal ectopic expression of a single <i>Ho</i>x gene copies expression of the same <i>Hox</i> gene from mesoderm to neurectoderm.

<p><b>A– A’’’, E, F</b> Localisation of mesoderm and neurectoderm in the wrap assay shown by expression of the mesodermal markers <i>Chordin (Chd,</i> A<i>)</i> and <i>Brachyury (Bra,</i><b>A’</b><i>)</i>, and the neural marker <i>Nrp1</i> (<b>A”</b>). In <b>A’’’</b>, lineage labelling by ectopic expression of GFP in the NOM only. These recombinants were analyzed 6 to 8 h after tissue healing. These data show that there is no tissue intermingling during wrap culture. Mesodermal <i>Chd</i> (expressed in SO, <b>A</b>) and GFP (within NOM, <b>A’’’</b>) do not mix with each other within the time course of wrap culture. Consistently, <i>Bra</i>, a pan mesodermal marker, is expressed in both types of mesoderm in accordance with its expression domain in the embryo (<b>A’</b>).The neural marker <i>Nrp-1</i> is expressed in the space between mesoderm and the outermost ectodermal layer of the wrap, consistent with its known pattern of expression within the embryo (<b>A’’</b>). <b>B–B’</b>: Induction of <i>Hoxd1</i><b>B:</b> A wrap containing SO and NOM and ectoderm [AC(SO/NOM)AC] shows induction of <i>Hoxd1</i> in the neurectoderm as well as mesoderm. <b>B’, A</b> wrap containing normal SO and SO ectopically expressing <i>Hoxd1</i> also shows the induction of endogenous <i>Hoxd1</i> in the neurectoderm as well as in the mesoderm. Endogenous Hoxd1 expression was detected using a 3′UTR probe that recognizes only the endogenous messenger. <b>C</b>, ectopic <i>Hoxb4</i> in SO induces its own expression within the neurectoderm and in the mesoderm as in <b>B’. D</b>, wrap as in <b>B’</b> and <b>C</b> but with ectopic <i>Hoxc6</i> expression. This shows induction of <i>Hoxc6</i> in neurectoderm and in the mesoderm. We used 3′UTR probes to detect expression of the endogenous mRNA’s in each of these experiments. <b>E, F</b> sections showing expression of <i>Nrp1</i> (neural) and <i>Bra</i> (mesodermal) in control or standard [AC(SO/NOM)AC] recombinant. <b>E</b><i>Nrp1</i> expression is internal in the recombinant but excluded from an internal cell mass that is clearly the mesoderm. It is particularly strong around one end of the cell mass which is the neural inducing SO. Expression is also absent from the very outer layer of the recombinant, which represents the outer non neural layer of the neurectoderm. <b>F:</b><i>Bra</i> expression is in an internal cell mass (the mesoderm). Please note that the germ layer markers <i>Bra, Ch</i>, and the mesodermal lineage label GFP are confined to an internal cell mass excluding tissue intermingling and that Hox expression is detected in neurectoderm as well as the mesodermal cell mass. Each photo in this figure represents at least 20 recombinants and embryos with consistently the same results.</p

Uptake of <i>d1-HD-GFP</i> and <i>GFP</i> by <i>Drosophila</i> imaginal wing discs.

<p><i>Drosphila</i> imaginal wing discs were incubated with <i>Hoxd1-HD-GFP (d1-HD-gfp)</i> recombinant protein (<b>B</b>) or wild type GFP protein (<b>A</b>). or mutated <i>mut</i>-<i>d1-HD-GFP.</i> Recombinant <i>d1-HD-gfp</i> was taken up by the discs while wild type GFP and <i>mut-d1-HD-GFP</i> were not. Each photo in this figure represents 10 imaginal discs giving the same result. These data clearly show the cargo function of Hoxd1 homeodomain and suggest that this uptake is by a species independent mechanism.</p