Our latest monthly trawl for developmental biology (and other cool) preprints. See June’s introductory post for background, and let us know if we missed anything

Another month, another bumper crop of preprints covering everything from calcium waves in the fly to the first mutant ant line, plant superhero genes to rat embryonic stem cells. Plus, as usual, a whole bunch of tools and resources (including a 3D printed micropipete!), and right at the end some enigmatic bacterial structures.

The preprints were hosted predominantly on bioRxiv, though we also found a handful on arXiv and PeerJ.

Use these links to get to the section you want –

Developmental biology

| Patterning & signalling

| Morphogenesis & mechanics

| Genes & genomes

| Stem cells, regeneration & ageing

Cell biology

Evo-devo & evo

Tools & resources

Research practice

Why not…

Happy preprinting!

Developmental biology

| Patterning & signalling

The Pax9/Wnt pathway regulates secondary palate formation in mice. Shihai Jia, Jing Zhou, Christopher Fanelli, Yinshen Wee, John Bonds, Pascal Schneider, Gabriele Mues, Rena N. D’Souza

Tbx1 represses Mef2c gene expression by inducing histone 3 deacetylation of the anterior heart field enhancer. Luna Simona Pane, Filomena Gabriella Fulcoli, Rosa Ferrentino, Marchesa Bilio, Antonio Baldini

Decoding temporal interpretation of the morphogen Bicoid in the early Drosophila embryo. Anqi Huang, Christopher Amourda, Shaobo Zhang, Nicholas S Tolwinski, Timothy E Saunders

Morphogen signalling patterns calcium waves in the Drosophila wing disc. Qinfeng Wu, Pavel Brodskiy, Cody Narciso, Megan Levis, Ninfamaria Arredondo-Walsh, Jeremiah James Zartman

A high-resolution mRNA expression time course of embryonic development in zebrafish. Richard J White, John E Collins, Ian M Sealy, Neha Wali, Christopher M Dooley, Zsofia Digby, Derek L Stemple, Daniel N Murphy, Thibaut Hourlier, Anja Fullgrabe, Matthew P Davis, Anton J Enright, Elisabeth M Busch-Nentwich

Multiple roles of PIWIL1 in mouse neocorticogenesis. Barbara Viljetic, Liyang Diao, Jixia Liu, Zeljka Krsnik, Sagara H. R. Wijeratne, Ryan Kristopovich, Marina Dutre-Clarke, Matthew L. Kraushar, Jimin Song, Jinchuan Xing, Kevin Chen, Mladen-Roko Rasin

Inhibitory neuron diversity originates from cardinal classes shared across germinal zones. Christian Mayer, Christoph Hafemeister, Rachel C Bandler, Robert Machold, Gord Fishell, Rahul Satija

Caenorhabditis elegans BMP Transcriptional Program Implicates Collagen Genes in Body Size Regulation. Uday Madaan, Edlira Yzeiraj, Michael Meade, Christine A. Rushlow, Cathy Savage-Dunn

Integrated growth factor signaling promotes lung epithelial progenitor cell expansion and maintenance in mice and humans. Alyssa Miller, Briana Dye, Melinda Nagy, Yu-Hwai Tsai, Sha Huang, Michael Ferguson, Jason Spence

Glycerophosphodiesterase GDE2 affects pancreas differentiation in zebrafish. Michiel van Veen, Jason van Pelt, Laurie A. Mans, Wouter H. Moolenaar, Anna-Pavlina Haramis

Developmental Downregulation of LIS1 Expression Limits Axonal Extension and Allows Axon Pruning. Hirotsune Tagawa, Kanako Kumamoto, Tokuichi Iguchi, Makoto Sato, Takuya Uemura

The reactivation of reprogramming factors within human blastocysts by using ATP contribute to human blastocyst development. Takashi Takemura, Midori Okabe

Can the location of a trophectoderm biopsy contribute to human blastocyst development? Tomoe Takano, Miyako Funabiki, Sagiri Taguchi, Fumie Saji, Namiko Amano, Kate Louise Young, Yoshitaka Nakamura

dMARCH8, a Drosophila ubiquitin ligase, regulates polymodifications of tubulin in the spermiogenic pathway. Ujwala Gosavi, Utsav Nyachhyon, Nabanita Chatterjee, Brian Pearce, Robert Harvey, Christopher W Bazinet

Short term changes in the proteome of human cerebral organoids induced by 5-methoxy-N,N-dimethyltryptamine. Vanja Dakic, Juliana M Nascimento, Rafaela C Sartore, Renata de Moraes Maciel, Draulio B de Araujo, Sidarta Ribeiro, Daniel Martins-de-Souza, Stevens K Rehen

SUPERMAN prevents stamen formation and promotes stem cell termination in the fourth whorl of the Arabidopsis flower. Nathanael Prunet, Weibing Yang, Pradeep Das, Elliot M Meyerowitz, Thomas P Jack

| Morphogenesis & mechanics

Viscoelastic dissipation stabilizes cell shape changes during tissue morphogenesis. Raphaël Clément, Claudio Collinet, Benoît Dehapiot, Thomas Lecuit, Pierre-François Lenne

Multiscale quantification of tissue behavior during amniote embryo axis elongation. Bertrand Benazeraf, Mathias Beaupeux, Martin Tchernookov, Allison Wallingford, Tasha Salisbury, Amelia Shirtz, Andrew Shirtz, Dave Huss, Olivier Pourquie, Paul Francois, Rusty Lansford

Assessment of asymmetric cell divisions in the early development of Caenorhabditis elegans. Rolf Fickentscher, Matthias Weiss

Modeling mammary organogenesis from biological first principles: Cells and their physical constraints. Maël Montévil, Lucia Speroni, Carlos Sonnenschein, Ana M. Soto

Morphological plant modeling: Unleashing geometric and topological potential within the plant sciences. Alexander Bucksch, Acheampong Atta-Boateng, Akomian Fortune Azihou, Mathilde Balduzzi, Dorjsuren Battogtokh, Aly Baumgartner, Brad Binder, Siobhan Braybrook, Cynthia Chang, Viktoriya Coneva, Thomas DeWitt, Alexander Fletcher, Malia Gehan, Diego Hernan Diaz Martinez, Lilan Hong, Anjali Iyer-Pascuzzi, Laura Klein, Samuel Leiboff, Mao Li, Jonathan Lynch, Alexis Maizel, Julin Maloof, RJ Cody Markelz, Ciera Martinez, Laura Miller, Washington Mio, Wojtek Palubicki, Hendrik Poorter, Christophe Pradal, Charles Price, Eetu Puttonen, John Reese, Ruben Rellan-Alvarez, Edgar Spalding, Erin Sparks, Chris Topp, Joseph Williams, Daniel Chitwood

Persistent homology: a tool to universally measure plant morphologies across organs and scales. Mao Li, Margaret H Frank, Viktoriya Coneva, Washington Mio, Christopher N Topp, Daniel H Chitwood

Categorical Description Of Plant Morphogenesis. I.V. Rudskiy

The depletion of MARVELD1 leads to placenta accreta via integrin β4-dependent trophoblast cell invasion. Yue Chen, Hui Zhang, Fang Han, Lei Yue, Chunxiao Qiao, Yao Zhang, Peng Dou, Weizhe Liu, Yu Li

Chromatin tethering to the nuclear envelope by nuclear actin filaments: a novel role of the actin cytoskeleton in the Xenopus blastula. Haruka Oda, Natsuki Shirai, Naoko Ura, Keita Ohsumi, Mari Iwabuchi

G protein-coupled estrogen receptor regulates heart rate and heart valve thickness in zebrafish. Shannon N Romano, Hailey E Edwards, Jaclyn Paige Souder, Xiangqin Cui, Daniel A Gorelick

| Genes & genomes

Bursting on a two states stochastic model for gene transcription in Drosophila embryos. Romain Yvinec, Luiz Guilherme S. da Silva, Guilherme N. Prata, John Reinitz, Alexandre Ferreira Ramos

Histone H3 Lysine 4 methyltransferases MLL3 and MLL4 Modulate Long-range Chromatin Interactions at Enhancers. Jian Yan, Shi-An A. Chen, Andrea Local, Tristin Liu, Yunjiang Qiu, Ah-Young Lee, Inkyung Jung, Sebastian Preissl, Chloe M Rivera, Chaocheng Wang, Haruhiko Ishii, Rongxin Fang, Zhen Ye, Kai Ge, Ming Hu, Bing Ren

Short DNA sequence patterns accurately identify broadly active human enhancers. Laura L Colbran, Ling Chen, John Anthony Capra

Novel CRISPR/Cas9 gene drive constructs in Drosophila reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations. Jackson Champer, Riona Reeves, Suh Yeon Oh, Chen Liu, Jingxian Liu, Andrew G Clark, Philipp W Messer

Genetic Architecture and Molecular Networks Underlying Leaf Thickness in Desert-Adapted Tomato Solanum pennellii. Viktoriya Coneva, Margaret H. Frank, Maria A. de Luis Balaguer, Mao Li, Rosangela Sozzani, Daniel H. Chitwood

Sequence properties underlying gene regulatory enhancers are conserved across mammals. Ling Chen, Alexandra E Fish, John Anthony Capra

| Stem cells, regeneration & ageing

Micromanagment of stem cell proliferation by the Drosophila testis stem cell niche. Olga A Puretskaia, Evgenii A Albert, Nadezhda V Terekhanova, Christian Boekel

Gene editing in rat embryonic stem cells to produce in vitro models and in vivo reporters. Yaoyao Chen, Sonia Spitzer, Sylvia Agathou, Ragnhildur Thora Karadottir, Austin Smith

Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells. Mariano Barbieri, Sheila Q Xie, Elena Torlai Triglia, Ines de Santiago, Miguel R Branco,David Rueda, Mario Nicodemi, Ana Pombo

Anoxia-Induced VEGF-Release from Rat Cardiomyocites Promotes Vascular Differentiation of Human Mesenchymal Stem Cells. Raquel Gutierrez_Lanza, Victor M Campa

Single cell transcriptome analysis of human pancreas reveals transcriptional signatures of aging and somatic mutation patterns. Martin Enge, Hatice Efsun Arda, Marco Mignardi, John Beausang, Rita Bottino, Seung K Kim, Stephen R Quake

Tex19.1 Restricts LINE-1 Mobilisation in Mouse Embryonic Stem Cells. Marie MacLennan, Marta Garcia-Canadas, Judith Reichmann, Elena Khazina, Carmen Salvador-Palomeque, Abigail Mann, Paula Peressini, Laura Sanchez, Christopher J Playfoot, David Read, Chao-Chun Hung, Ragnhild Eskeland, Richard R Meehan, Oliver Weichenrieder, Jose Luis Garcia- Perez, Ian R Adams

BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells (MSCs). Jifan Feng, Junjun Jing, Jingyuan Li, Hu Zhao, Vasu Punj, Tingwei Zhang, Jian Xu, Yang Chai

The regulation of tDNA transcription during the directed differentiation of stem cells. Jessica L Woolnough, David A Schneider, Keith E Giles

Impact of hypoxia induced VEGF and its signaling during caudal fin regeneration in Zebrafish. vivek sagayaraj, Malathi Ragunathan

Single-cell epigenomics maps the continuous regulatory landscape of human hematopoietic differentiation. Jason Daniel Buenrostro, Ryan Corces, Beijing Wu, Alicia N Schep, Caleb Lareau, Ravindra Majeti, Howard Chang, William Greenleaf

Heterogeneity of normal human breast stem and progenitor cells as revealed by transcriptional profiling. Justin Colacino, Ebrahim Azizi, Michael Brooks, Shamileh Fouladdel, Sean P McDermott, Michael Lee, David Hill, Maureen Sartor, Laura Rozek, Max Wicha

Cell biology

Super-resolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact C. elegans tissue. Simone Köhler, Michal Wojcik, Ke Xu, Abby F Dernburg

Contact inhibition of locomotion and junctional mechanics guide collective cell behavior in epithelial wound repair. Luke Coburn, Irin-Maya Schouwenaar, Hender Lopez, Alpha S. Yap, Vladimir Lobaskin, Guillermo A. Gomez

FAM83G/PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP. Gopal P Sapkota, Timothy D Cummins, Kevin Z. L. Wu, Polyxeni Bozatzi, Kevin S Dingwell, Thomas J Macartney, Nicola T Wood, Joby Varghese, Robert Gourlay, David G Campbell, Alan Prescott, Eric Griffis, James C Smith

Decoupling global biases and local interactions between cell biological variables. Assaf Zaritsky, Uri Obolski, Zhuo Gan, Carlos R Reis, Zuzana Kadlecova, Yi Du, Sandra L Schmid, Gaudenz Danuser

mTORC1 controls cytoplasmic crowding by regulating ribosome concentration. Morgan Delarue, Gregory Brittingham, Ivan Surovtsev, Kristopher John Kennedy, Ignacio Gutierrez, Jean Chung, Jay T. Groves, Christine Jacobs-Wagner, Liam J Holt

Controlling cell shape on hydrogels using lift-off patterning. Jens Moeller, Aleksandra K. Denisin, Joo Yong Sim, Robin E. Wilson, Alexandre J.S. Ribeiro, Beth L. Pruitt

Real-time chromatin dynamics at the single gene level during transcription activation. Thomas Germier, Silvia Kocanova, Nike Walther, Aurelien Bancaud, Haitham Ahmed Shaban, Hafida Sellou, Antonio Politi, Jan Ellenberg, Franck Gallardo, Kerstin Bystricky

Observation of histone nuclear import in living cells: implications in the processing of newly synthesised H3.1 & H4. Andrew James Bowman, Michael James Smith

NIMA-related kinase 1 (NEK1) regulates the localization and phosphorylation of α-Adducin (ADD1) and Myosin X (MYO10) during meiosis. Miguel Angel Brieno-Enriquez, Stefannie L Moak, J Kim Holloway, Paula Elaine Cohen

Meiotic recombination modulates the structure and dynamics of the synaptonemal complex during C. elegans meiosis. Divya Pattabiraman, Baptiste Roelens, Alexander Woglar, Anne M. Villeneuve

Astral microtubule dynamics regulate anaphase oscillation onset and set a robust final position of the C. elegans zygote spindle. Hélène Bouvrais, Laurent Chesneau, Sylvain Pastezeur, Marie Delattre, Jacques Pécréaux

High quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics. Jun Takagi, Yuta Shimamoto

Evo-devo & evo

orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants. Waring Trible, Ni-Chen Chang, Benjamin J Matthews, Sean K McKenzie, Leonora Olivos-Cisneros, Peter R Oxley, Jonathan Saragosti, Daniel JC Kronauer

Genetic basis of melanin pigmentation in butterfly wings. Linlin Zhang, Arnaud Martin, Michael W. Perry, Karin R.L. van der Burg, Yuji Matsuoka, Antonia Monteiro, Robert D Reed

Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking. Carsten Wolff, Jean-Yves Tinevez, Tobias Pietzsch, Evangelia Stamataki, Benjamin Harich, Stephan Preibisch, Spencer Shorte, Philipp J Keller, Pavel Tomancak, Anastasios Pavlopoulos

CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus. Svenia D Heinze, Tea Kohlbrenner, Domenica Ippolito, Angela Meccariello, Alexa Burger, Christian Mosimann, Giuseppe Saccone, Daniel Bopp

Tools & resources

High-throughput live-imaging of embryos in microwell arrays using a modular, inexpensive specimen mounting system. Seth Donoughe, Chiyoung Kim, Cassandra Extavour

Enzymatic production of single molecule FISH and RNA capture probes. Imre Gaspar, Frank Wippich, Anne Ephrussi

Polarity sensitive probes for super resolution STED microscopy. Erdinc Sezgin, Falk Schneider, Victoria Zilles, Esther Garcia, Dominic Waithe, Andrey S Klymchenko, Christian Eggeling

A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. Stefan Harmansa, Ilaria Alborelli, Emmanuel Caussinus,Markus Affolter

A fluorogenic nanobody array tag for prolonged single molecule imaging in live cells. Rajarshi Ghosh, Will Draper, J. Matthew Franklin, Quanming Shi, Jan Liphardt

Refractive index as an intrinsic imaging contrast for 3-D label-free live cell imaging. Doyeon Kim, SangYun Lee, Moosung Lee, JunTaek Oh, Su-A Yang, YongKeun Park

Phenotype and gene ontology enrichment as guides for disease modeling in C. elegans. David Angeles-Albores, Raymond Y Lee, Juancarlos Chan, Paul W Sternberg

Harnessing optogenetics to probe sub-cellular mechanics. Patrick W Oakes, Elizabeth Wagner, Christoph A Brand, Dimitri Probst, Marco Linke, Ulrich S Schwarz, Michael Glotzer, Margaret L Gardel

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells. Jianying Guo, Dacheng Ma, Rujin Huang, Jia Ming, Min Ye, Kehkooi Kee, Zhen Xie, Jie Na

CRISPRAnalyzeR: Interactive analysis, annotation and documentation of pooled CRISPR screens. Jan Winter, Marc Schwering, Oliver Pelz, Benedikt Rauscher, Tianzuo Zhan, Florian Heigwer, Michael Boutros

Reliable CRISPR/Cas9 genome engineering in Caenorhabiditis elegans using a single efficient sgRNA and an easily selectable phenotype. Sonia El Mouridi, Claire Lecroisey, Phlippe Tardy, Marine Mercier, Alice Leclercq-Blondel, Nora Zariohi, Thomas Boulin

An efficient FLP-based toolkit for spatiotemporal control of gene expression in Caenorhabditis elegans. Celia Maria Munoz-Jimenez, Cristina Ayuso, Agnieszka Dobrzynska, Antonio Torres, Patricia de la Cruz-Ruiz, Peter Askjaer

A cell based, high throughput assay for quantitative analysis of Hedgehog pathway activation using a Smoothened phosphorylation sensor. Evgenii A Albert, Christian Boekel

Microvessel Chaste: An Open Library for Spatial Modelling of Vascularized Tissues. James Grogan, Anthony J Connor, Bostjan Markelc, Ruth J Muschel, Philip K Maini, Helen M Byrne, Joe M Pitt-Francis

Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing. Junyue Cao, Jonathan S. Packer, Vijay Ramani, Darren A. Cusanovich, Chau Huynh, Riza Daza, Xiaojie Qiu, Choli Lee, Scott N. Furlan, Frank J. Steemers, Andrew Adey, Robert H. Waterston, Cole Trapnell, Jay Shendure

Polysome-profiling in small tissue samples. Shuo Liang, Hermano Bellato, Julie Lorent, Fernanda Lupinacci, Vincent Van Hoef, Laia Masvidal, Glaucia Hajj, Ola Larsson

Versatile Multicolor Nanodiamond Probes for Intracellular Imaging and Targeted Labeling. Olga Shimoni, Kerem Bray, Leonard Cheung, Igor Aharanovich, Stella Valenzuela

Synthetic DNA templates for the production of in situ hybridization probes. B. Rodney Jarvis, Brian G Condie

mixOmics: an R package for ‘omics feature selection and multiple data integration. Florian Rohart, Benoit Gautier, View Amrit Singh, Kim-Anh Le Cao

Novel methods of isolation and amplification of progenitor cells applied to avian primordial germ cells. Mariacruz Lopez Diaz

PHESANT: a tool for performing automated phenome scans in UK Biobank. Louise Millard, Neil M Davies, Tom Gaunt, George Davey Smith, Kate Tilling

MAGIC: A diffusion-based imputation method reveals gene-gene interactions in single-cell RNA-sequencing data. David van Dijk, Juozas Nainys, Roshan Sharma, Pooja Kathail, Ambrose J Carr, Kevin R Moon, Linas Mazutis, Guy Wolf, Smita Krishnaswamy, Dana Pe’er

EXTRACT 2.0: text-mining-assisted interactive annotation of biomedical named entities and ontology terms. Evangelos Pafilis, Rūdolfs Bērziņš, Lars Juhl Jensen

Reversed graph embedding resolves complex single-cell developmental trajectories. Xiaojie Qiu, Qi Mao, Ying Tang, Li Wang, Raghav Chawla, Hannah Pliner, Cole Trapnell

Evolinc: a comparative transcriptomics and genomics pipeline for quickly identifying sequence conserved lincRNAs for functional analysis. Andrew D. L. Nelson, Upendra K Devisetty, Kyle Palos, Asher K Haug-Baltzell, Eric Lyons, Mark A Beilstein

Open Design 3D-Printable Adjustable Micropipette that meets ISO Standard for Accuracy. Martin Brennan, Fahad Bokhari, David Eddington

Systematic analysis of cell phenotypes and cellular social networks in tissues using the multiplexed image cytometry analysis toolbox (miCAT). Denis Schapiro, Hartland Warren Jackson, Swetha Raghuraman, Vito Riccardo Tomaso Zanotelli, Jana R R Fischer,Daniel Schulz, Charlotte Giesen, Raul Catena, Zsuzsanna Varga, Bernd Bodenmiller

Functional Annotation of Chemical Libraries across Diverse Biological Processes. Jeff S Piotrowski, Sheena C Li, Raamesh Deshpande, Scott W Simpkins, Justin Nelson, Yoko Yashiroda, Jacqueline Barber, Hamid Safizadeh, Erin Wilson, Hiroki Okada, Abraham Gebre, Karen Kubo, Nikko Torres, Marissa LeBlanc, Kerry Andrusiak, Reika Okamoto, Mami Yoshimura, Eva DeRango-Adem, Jolanda van Leeuwen, Katsuhiko Shirahige, Anastasia Baryshnikova, Grant W Brown, Hiroyuki Hirano, Michael Costanzo, Brenda Andrews, Yoshikazu Ohya, Hiroyuki Osada, Minoru Yoshida, Chad L Myers, Charles Boone

The 3D Genome Browser: a web-based browser for visualizing 3D genome organization and long-range chromatin interactions. Yanli Wang, Bo Zhang, Lijun Zhang, Lin An, Jie Xu, Daofeng Li, Mayank NK Choudhary, Yun Li, Ming Hu, Ross Hardison, Ting Wang, Feng Yue

Research practice

Open Science strategies for NIH data management, sharing, and citation. Tim Clark​, Helena Cousijn, Daniel S Katz, Martin Fenner

The Rise of the Middle Author: Investigating Collaboration and Division of Labor in Biomedical Research using Partial Alphabetical Authorship. Philippe Mongeon, Elise Smith, Bruno Joyal, Vincent Lariviere

TOWARDS COORDINATED INTERNATIONAL SUPPORT OF CORE DATA RESOURCES FOR THE LIFE SCIENCES. Warwick Anderson, Rolf Apweiler, Alex Bateman, Guntram A Bauer, Helen Berman, Judith A Blake, Niklas Blomberg, Stephen K Burley, Guy Cochrane, Valentina Di Francesco, Tim Donohue, Christine Durinx, Alfred Game, Eric Green, Takashi Gojobori, Peter Goodhand, Ada Hamosh, Henning Hermjakob, Minoru Kanehisa, Robert Kiley, Johanna McEntyre, Rowan McKibbin, Satoru Miyano, Barbara Pauly, Norbert Perrimon, Mark A Ragan, Geoffrey Richards, Yik-Ying Teo, Monte Westerfield, Eric Westhof, Paul F Lasko

The impact factor fallacy. Frieder Michel Paulus, Nicole Cruz, Soeren Krach

Concern noted: A descriptive study of editorial expressions of concern in PubMed and PubMed Central. Melissa Vaught, Diana C Jordan, Hilda Bastian

Reproducibility2020: Progress and Priorities. Leonard Freedman, Gautham Venugopalan, Rosann Wisman

Improving data availability for brain image biobanking in healthy subjects: practice-based suggestions from an international multidisciplinary working group. Susan D Shenkin, Cyril Pernet, Thomas E Nichols, Jean-Baptiste Poline, Paul M Matthews, Aad van der Lugt, Clare Mackay, Lanyon Linda, Bernard Mazoyer, James P Boardman, Paul M Thompson, Nick Fox, Daniel S Marcus, Aziz Sheikh, Simon R Cox, Devasuda Anblagan, Dominic E Job, David Alexander Dickie, David Rodriguez, Joanna M Wardlaw

Why not…

Uncharacterized bacterial structures revealed by electron cryotomography. Megan J Dobro, Catherine M Oikonomou, Aidan Piper, John Cohen, Kylie Guo, Taylor Jensen, Jahan Tadayon, Joseph Donermeyer, Yeram Park, Benjamin A Solis, Andreas Kjaer, Andrew I Jewett, Alasdair W McDowall, Songye Chen, Yi-Wei Chang, Jian Shi, Poorna Subramanian, Cristina V Iancu, Zhuo Li, Ariane Briegel, Elitza I Tocheva, Martin Pilhofer, Grant J Jensen

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A postdoctoral research fellow position is available in the laboratory of Gregor Andelfinger at the CHU Sainte Justine Research Center, Montréal, Québec, Canada. Our work focuses on the genetic origins of cardiovascular diseases in the young and includes both genomics and functional approaches. In this project, the successful candidate will apply genomic and molecular biology approaches to develop disease models. Postdoctoral fellows will have a unique opportunity to do interdisciplinary research at all interfaces of translational medicine and publish their results in high impact journals.

The candidate will work in collaboration with all lab members and is expected to independently perform human and mouse genetic studies, as well as in vitro molecular analyses. Experience with a wide variety of techniques (developmental studies, histology, confocal microscopy, cell culture assays, RNAseq) is desired. Experience with genome editing techniques (CRISPR) and/or and bioinformatics analysis is an asset.

The successful candidate must have a recent Ph.D. degree in life sciences, molecular cell biology, or a related field. We are seeking highly motivated and well published researchers with excellent oral, written, and interpersonal communication skills.

Montreal is a vibrant bilingual city with a high quality of life. With a greater metropolitan area of 3.5 M, Montreal offers all scientific and cultural benefits of a large North American agglomeration with a European flair and beautiful surroundings.

Interested applicants are asked to provide the following documents to Dr. Gregor Andelfinger  (gregor.andelfinger@recherche-ste-justine.qc.ca):

• CV
• Cover letter describing previous experience and career goals
• Contact information for three references

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The Andrew Yoo lab at WUSTL is looking for a lab manager/technician.  The Yoo lab uses microRNA-mediated direct reprogramming of fibroblasts to neurons to study aging and neurodegenerative diseases.

http://yoolab.wustl.edu/ 

http://www.sciencedirect.com/science/article/pii/S0896627314009143 

Tissue culture experience desired.  For full details: https://jobs.wustl.edu/ job #35790

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PhD position opened in Switzerland for a developmental or cell biologist

Development & Evolution of structural colours in vertebrates

In the context of a multidisciplinary study combining cell biology, evolutionary developmental biology and physics, we offer one PhD student position for an outstanding, highly motivated, and creative experimental wet-lab biologist with strong skills in developmental biology or cell biology.

A major research project in Michel Milinkovitch’s group (Laboratory of Artificial & Natural Evolution; www.lanevol.org) at the University of Geneva (UNIGE) is to better understand the molecular developmental mechanisms generating a diversity of skin colours and patterns in reptiles.

One aim is to understand the development & evolution of surface gratings and intracellular nano-structures generating colours through the physical phenomenon of light interference. The successful candidates will use state-of-the-art microscopy and biochemical/genetic manipulations in both the zebrafish and chameleons to understand the development and evolution of surface gratings and intracellular photonic crystals.

Candidates must have a Master in biology or biochemistry. Skills and experience with developmental biology and/or cell biology are mandatory. The successful candidate will have a genuine interest for organismal biology and will appreciate interactions with developmental biologists, evolutionary biologists, physicists and computer scientists. The PhD student will be localised in the Milinkovitch lab (http://www.lanevol.org) and will have very regular interactions/collaborations with members of Marcos Gonzalez-Gaitan’ lab (https://cms.unige.ch/sciences/biochimie/-Marcos-Gonzalez-Gaitan-Lab-.html).

The University of Geneva is world-renowned for its research in Biology and Physics  and is among the top 1% best universities in the world.

PhD students are remunerated according to the standards of UNIGE, which are very generous when compared to other international programs.

Geneva is an international city occupying a privileged geographical situation with its beautiful lake and the close-by Alps.

Refs: Saenko et al. Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in Phelsuma lizards. BMC Biology 2013, 11: 105; Teyssier et al. Photonic Crystals Cause Active Colour Change in Chameleons. Nature Communications 6: 6368 (2015); Tzika et al. Reptilian Transcriptomes v2.0: An Extensive Resource for Sauropsida Genomics and Transcriptomics. Genome Biol. Evol.  7: 1827-1841 (2015); Ullate-Agote et al. The genome sequence of the corn snake (Pantherophis guttatus), a valuable resource for EvoDevo studies in  squamates. Int. J. Dev. Biol. 58: 881-888 (2014); Saenko et al. Amelanism in the corn snake is associated with the insertion of an LTR-retrotransposon in the OCA2 gene. Scientific Reports 5, 17118 (2015); Di-Poï & Milinkovitch. The Anatomical Placode in Reptile Scale Morphogenesis Indicates Shared Ancestry  Among Skin Appendages in Amniotes. Science Advances 2, e1600708 (2016).

Candidates must send their application — in the form of a single PDF file including a brief letter of interest, a CV, as well as contact information (not support letters) of two persons of reference — to: Prof. Michel Milinkovitch (michel.Milinkovitch@unige.ch).

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We are looking for a postdoc interested in studying neural development in tunicates, the sister group to the vertebrates. The larval nervous system of the tunicate Ciona has only 177 neurons and its entire “connectome” has been recently mapped (Ryan et al., eLife 2016). The Ciona genome is also highly compact and easily manipulated using CRISPR/Cas9 (Stolfi et al., Development 2014). This tractability offers an unprecedented opportunity to understand gene networks underlying the development of every single neuron in a chordate nervous system.

We have several potential projects centered on understanding the link between transcriptional regulation and cell behavior during the development of the Ciona nervous system. Specific cells of interest include homologs of vertebrate spinal cord neurons (Stolfi et al., Development 2011) and neural crest-derived sensory neurons (Stolfi et al., Nature 2015). Projects will involve isolating neural progenitors from transgenic Ciona embryos, profiling their transcriptome and chromatin states using next-generation sequencing, and targeting candidate regulators and effectors using CRISPR/Cas9.

Our lab is located at the Georgia Institute of Technology in Atlanta, USA. Georgia Tech offers first-rate research facilities and a highly collaborative research environment with traditional strengths in engineering, computing, and math. We are dedicated to promoting equity of under-represented minorities in academia.

Send CV and references to: stolfi@tunicates.org

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Seeking grants consultant with science PhD for new neuroscience institute at Columbia University

Location: Columbia University in the City of New York

Title:                      Grant Strategist, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University

Quick link:           http://bit.ly/2ieYImR

Position highlights: The Grant Strategist will support the productivity of Zuckerman Institute scientists by editing and consulting on grants, particularly sections on research strategy, aims, and narratives; acquire and maintain sound knowledge of the Institute and the research priorities; use that knowledge and understanding to seek and recommend grants for individual as well as multi-investigator grants within and between disciplines.

The Grant Strategist will serve as a project manager to pursue, identify and promote funding opportunities for multi-PI grants. The Grant Strategist will also consult with and keep abreast of the current granting environment to provide suggestions to individual researchers on specific grant opportunities.

Position requirements: Ph.D. in the biological sciences or related field, including extensive writing experience; or advanced degree in journalism with deep expertise in science. Minimum of four (4) years of successful science grant writing experience or related experience in science communication field is required. Extensive knowledge of scientific granting processes, particularly in the biological sciences; successful grant writing or consulting record is vital.

For the full description and to apply, go to http://bit.ly/2ieYImR

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Jamie R. Genthe and Wilson K. Clements

When blood goes bad, a replacement is often needed. Each year, thousands of patients in the US receive bone marrow transplants to treat life-threatening diseases like blood cancer.

But in some cases, the transplant itself can become deadly.

The problem is not necessarily the one most people think of: rejection of the transplanted material. Instead, the donor hematopoietic stem cells (HSCs), delivered to repopulate the whole blood system, sometimes produce immune cells that attack anything foreign. In these cases, the transplant rejects the patient. This condition, called graft-versus-host disease (GVDH), is common in transplant patients and often fatal.

Developing a reliable method to make transplantable stem cells that would avoid GVHD is of considerable interest. That’s why we and others have been interested in learning how to direct the differentiation of HSCs in vitro from pluripotent precursor cells.

Work from our group at St. Jude Children’s Research Hospital, published in the February 15 issue of Development, provides new clues on how to help solve this problem.

Intuitively, a way to make new HSCs would be to replicate the same instructions used to make them in the developing embryo. But first we have to identify the signals that make up those instructions. During embryonic development, HSCs are born from the descending aorta, the primitive vessel that carries blood to the lower half of the body. Decades of research have uncovered numerous signaling inputs that direct formation of the descending aorta, and its subsequent conversion to the first HSCs. However, we are still defining the complete set of signals, and how they are regulated and integrated.

We set out to examine this coordination and identify novel factors that might play critical roles in HSC development. We used zebrafish because their blood development is nearly identical to that of humans. We noticed a common theme: signaling pathways required for hematopoietic stem cell formation also frequently regulate vessel patterning. Specifically, well-established signaling pathways, like the Vegf, Notch, and Wnt pathways, play a role in both processes.

In the trunk of the embryo, smaller blood vessels called “intersegmental vessels” sprout from the developing descending aorta at about the same time that HSCs are born. We became intrigued by published results identifying a new signaling pathway necessary for the sprouting of intersegmental vessels from the aorta (Gore et al., 2011). This pathway involves R-spondin-1 (Rspo1), a secreted factor that augments Wnt signaling. Intriguingly, Gore et al. also showed rspo1 was expressed in the dorsal aorta during the time of HSC specification, the point when blood stem cells start to assume their future identity. We wondered if this pathway might also be necessary for HSC specification.

Our first goal was to determine if Rspo1 is required in HSC development. We knocked down Rspo1 in zebrafish embryos using antisense oligonucleotides and asked what would happen to HSCs.  Strikingly, markers of HSC development were visibly decreased at all time points we examined. We subsequently confirmed the rspo1 requirement in an established zebrafish hypomorphic mutant.

So Rspo1 is required for HSCs to form, but how does it work? To answer this question, we looked at the activity of other pathways already known to play a role in HSC specification to see if we could detect any changes in their activity. Our results identified two of these – the Wnt16 and Vegfa pathways – that showed alterations in expression of their downstream components.

Our findings had some surprises. Most notably, although vegfa expression was lower when Rspo1 was knocked down, a key target of Vegf signaling, notch1b, was unaffected. Our finding suggested that there might be a Notch1-independent role for Vegf in specifying hematopoietic stem cells.

As we were trying to define how this might work, the group of Roger Patient (Monteiro et al., 2016)  identified precisely such a pathway. We determined that Rspo1 acted via this Notch-independent pathway, and discovered that a particular Vegfa splice variant was involved.

Overall, these findings point to Rspo1 as a new master regulator of blood stem cells that controls two pathways both needed for the birth of these stem cells. In the future, we hope that understanding the full set of signals and their integration will provide the key to unlocking our ability to make fully functional hematopoietic stem cells in the lab.  Eventually we will have the tools to create designer treatments for leukemia and other blood disorders while making GVHD a thing of the past.

Comment on

R-spondin-1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways”. Development 2017, 144: 590-600.

References

Gore, A.V., Swift, M.R., Cha, Y.R., Lo, B., McKinney, M.C., Li, W., Castranova, D., Davis, A., Mukouyama, Y.S., Weinstein, B.M., 2011. Rspo1/Wnt signaling promotes angiogenesis via Vegfc/Vegfr3. Development 138, 4875-4886.

Monteiro, R., Pinheiro, P., Joseph, N., Peterkin, T., Koth, J., Repapi, E., Bonkhofer, F., Kirmizitas, A., Patient, R., 2016. Transforming Growth Factor beta Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells. Dev Cell 38, 358-370. [*Lead author wrote about this work on the Node last year*]

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Here are the highlights from the new issue of Development:

Adding a new layer of complexity to pre-eclampsia

Pre-eclampsia (PE) is a pregnancy complication associated with abnormal formation of the placenta. To date, most studies of PE have focussed on cytotrophoblasts (CTBs) within the villous placenta (the chorion frondosum); the deficient invasion of these cells into the uterine wall is thought to lead to abnormal placentation and hence PE. Here, on p. 767, Susan Fisher and colleagues reveal that CTBs within a different region of the human placenta – the smooth chorion – are implicated in severe PE. They first report that the CTB layer in the smooth chorion is expanded in severe cases of PE and is less organised. These morphological changes are accompanied by changes in the expression profiles of smooth chorion CTBs. In particular, smooth chorion CTBs exhibit enhanced expression of HLA-G, integrin α4 and E-cadherin, all of which are factors that extravillous CTBs normally modulate as they invade the uterine wall. The researchers further show that severe PE is associated with global gene expression changes in smooth chorion CTBs that are distinct from the transcriptional responses of villous and extravillous CTBs to severe PE, which they previously described. Overall, these findings suggest that smooth chorion CTBs play a greater role in placentation and pregnancy outcome than previously appreciated.

Integrins worm their way into brain regeneration

Tissue regeneration involves a number of cellular processes, including proliferation, differentiation, migration and patterning, but it is not clear how all of these processes are coordinated to allow the correct generation and assembly of cells during regeneration. Using the planarian flatworm Schmidtea mediterranea, which displays remarkable regenerative capacity and can regenerate any missing body part, two new studies reveal a key role for integrins in regulating tissue organisation during brain regeneration.

In the first study (p. 784), Nicolle Bonar and Christian Petersen use RNAi to show that β1-integrin regulates cell number and tissue organisation during regeneration following decapitation; in the absence of β1-integrin, brain tissue is disorganised and ectopic cell aggregates form in the head region. By contrast, global patterning is largely unaffected. The authors further show that β1-integrinRNAi animals exhibit an initial delay in regeneration but that this is followed by tissue overproduction. Finally, they report that integrin signalling, likely via a β1/α-2 complex, is required for the proper localisation of neoblasts and progenitor cells during regeneration. Together, these findings suggest that integrin signalling acts to recruit and localise progenitor cells following injury, thereby promoting the correct organisation of regenerating planarian tissue.

In the second paper (p. 795), Florian Seebeck, Kerstin Bartscherer and colleagues demonstrate that β1-integrin RNAi animals, as well as α-integrin-2 RNAi animals, exhibit impaired regeneration following amputation. They show that, in β1-integrin RNAi animals, the newly formed tissues – including the muscle and gut – display structural defects. The researchers also reveal that β1-integrin is required for neoblast migration towards the wound site. Finally, they report that β1-integrin RNAi causes the formation of ectopic neural spheres within the regenerating brain region that are composed of various neuronal cell types and that undergo continuous growth. Overall, these results suggest that integrins are required for the formation of organised tissues and for restricting neurogenesis during planarian regeneration.

Apoptosis: a delayed gut reaction to bacteria

The intestine is a tissue that is known to undergo regeneration, both continuously as part of tissue homeostasis and in response to damage – for example, that induced by bacterial aggression. While many studies have examined how the gut responds to large amounts of pathogenic or opportunistic bacteria, it is unclear how low levels of bacteria might influence gut homeostasis. Here, on p. 808, Armel Gallet and co-workers tackle this issue. They report that small amounts of the opportunistic Gram-positive bacterium Bacillus thuringiensis var. kurstaki induce a mild early stress response mediated by JNK signalling in the Drosophila midgut. This, in turn, induces the proliferation of intestinal stem cells and leads to the accumulation and overcrowding of differentiated intestinal cells (enterocytes). The authors further report that low amounts of ingested bacteria do not trigger apoptosis, whereas larger amounts do. However, they find that a wave of apoptosis is observed days after infection and acts to eliminate the excess enterocytes. Finally, they demonstrate that the Hippo pathway functions cell-autonomously to trigger the removal of supernumerary enterocytes. These findings lead the authors to propose that the mechanisms involved in the response to the ingestion of low amounts of opportunistic bacteria are different to those mediating the ʻregenerative cell deathʼ that occurs following a stronger aggression.

Nucleogenesis gets active

Neurons within the central nervous system can assemble into clusters, termed nuclei, that house neurons with similar synaptic inputs, outputs and function. This process of nucleogenesis, which is crucial for correct circuit formation, is poorly understood. Now, on p. 830, Sarah Guthrie and colleagues show that the correct assembly of developing motor neurons into nuclei in the chick brainstem requires interplay between spontaneous activity, type II cadherins and gap junctions. Using the genetically encoded calcium indicator GCaMP6, they first show that facial motor neurons exhibit activity patterns that change over the course of nucleogenesis. These patterns can be disrupted by perturbing the expression of the type II cadherin Cad20 or the gap junction protein Cx43. The authors further demonstrate that the inhibition of spontaneous activity (using calcium channel inhibitors) results in neuronal disaggregation, and also causes a reduction in the levels of Cad13, another type II cadherin, suggesting the presence of a feedback loop. In summary, these observations suggest that a network of interactions between cadherins, gap junctions and spontaneous activity governs nucleogenesis.

PLUS:

Creating to understand – developmental biology meets engineering in Paris

In November 2016, developmental biologists, synthetic biologists and engineers gathered in Paris for a meeting called ‘Engineering the embryo’. The participants shared an interest in exploring how synthetic systems can reveal new principles of embryonic development, and how the in vitro manipulation and modeling of development using stem cells can be used to integrate ideas and expertise from physics, developmental biology and tissue engineering. In their Meeting Review, Anna Kicheva and Nicolas Rivron provide a summary of this meeting and highlight the challenges arising at the intersection of these fields.

Krüppel-like factors in mammalian stem cells and development

Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors that regulate diverse processes such as cell proliferation, differentiation, development and regeneration. Several KLFs are also crucial for maintaining pluripotency and, hence, have been linked to reprogramming and regenerative medicine approaches. In their Primer, Agnieszka Bialkowska, Vincent Yang and Sandeep Mallipattu review key functions for KLFs in mammalian embryogenesis, stem cells and regeneration.

Plasticity in the lung: making and breaking cell identity

In recent years, lineage tracing studies have identified distinct epithelial stem and progenitor cell populations in the lung. These cells, together with their differentiated progeny, maintain a stable identity during steady state conditions, but can display remarkable lineage plasticity following injury. In their Review, Purushothama Tata and Jayaraj Rajagopal summarize our current understanding of the different cell lineages of the adult mammalian lung and discuss how these populations respond to injury.

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Reviews Editor: biomedical and life sciences journals

Based in Cambridge, UK

Applications are sought for the role of Reviews Editor for The Company of Biologists’ journals: Development, Journal of Cell Science, Journal of Experimental Biology, Disease Models & Mechanisms and Biology Open. The role will be offered for an initial term of three years.

Joining a long-established and successful publishing company, this is an exciting opportunity to make a significant contribution to our highly respected biomedical and life science journals. The role will initially focus on Disease Models & Mechanisms, a growing Open Access journal in the fast-moving field of translational research.

The front sections of the journals include commissioned reviews and poster articles, thought-provoking editorials and interviews with leaders in the field.

Applicants will have a PhD or MD in a relevant area. Post-doctoral and/or previous editorial experience is desirable, although we will provide full training. The successful candidate will work alongside an established publishing team in our Cambridge office.

Core responsibilities include:

• Commissioning, handling peer review and developmental editing of material for the front section of the journals.

• Travelling to international scientific conferences and research institutes, representing the journals, keeping abreast of the latest research and making contacts in the community.

• Spotting newsworthy articles, writing informative press releases and handling any media enquiries.

• Interviewing high-profile scientists.

• Contributing to our social media output.

• Creative involvement in the journals’ development and marketing activities.

Additional responsibilities may be provided for the right candidate.

Essential requirements for the job are enthusiasm, commitment, judgement and integrity. Candidates should have excellent interpersonal skills and confidence, and excellent oral and written communication skills. The successful candidate will have a broad interest in science, the scientific community and publishing.

The position is full time and will be based in The Company of Biologists’ attractive modern offices on the outskirts of Cambridge, UK. It offers an attractive salary and benefits.

The Company of Biologists (biologists.com) exists to support biologists and inspire advances in biology. At the heart of what we do are our five specialist journals –Development, Journal of Cell Science, Journal of Experimental Biology, Disease Models & Mechanisms and Biology Open – two of them fully open access. All are edited by expert researchers in the field, and all articles are subjected to rigorous peer review. We take great pride in the experience of our editorial team and the quality of the work we publish. We believe that the profits from publishing the hard work of biologists should support scientific discovery and help develop future scientists. Our grants help support societies, meetings and individuals. Our workshops and meetings give the opportunity to network and collaborate.

Applicants should send a CV by email to recruitment@biologists.com along with a covering letter that states their current salary and any limitations regards possible start date, and summarises their relevant experience and why they are enthusiastic about this opportunity.

Applicants should be eligible to work in the UK and should be able to travel internationally. Applications should be received by 20th March 2017.

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Comment on “Divergent Hox Coding and Evasion of Retinoid Signaling Specifies Motor Neurons Innervating Digit Muscles” Neuron 93, 1–14, February 22, 2017.

Alana I. Mendelsohn, Departments of Neuroscience and Biochemistry and Molecular Biophysics, Columbia University

Jeremy S. Dasen, Department of Neuroscience, NYU

Thomas M. Jessell, Departments of Neuroscience and Biochemistry and Molecular Biophysics, Columbia University

The ability of the nervous system to perform complex tasks depends on a diverse set of circuits that underlie specific behaviors. The nervous system generates distinct neural circuits in several ways. One strategy is to make many different types of neurons, varying individual properties such as cellular morphology, settling position, electrical conductance and pharmacological signaling. The other method of forming distinct circuits is to have neurons form selective connections with only a subset of other neurons. Given the scale of the human nervous system, where 100 billion neurons each forms an average of 7000 synaptic connections, the establishment of defined neural circuits is one of the most complex facets of organismal development. The scientific challenge of identifying mechanisms underlying neural specification and circuit formation is in large part what drew me to Tom Jessell’s lab as a graduate student. Tom’s lab works on the mammalian motor system, which carries a number of advantages for studying fundamental principles of nervous system development. Namely, the spinal cord is an excellent model system for studying development because it has well characterized and relatively stereotyped circuit anatomy, with defined sensory inputs and topographically segregated motor outputs.

The first project I worked on in graduate school looked at the formation of monosynaptic reflex circuits between sensory neurons and motor neurons, a process long-thought to be under total genetic control. Although our work showed that the development of these circuits is in fact shaped to some extent by neural activity, it also made clear that one fundamental aspect of circuit formation, the choice of synaptic partner, was made in this context solely on the basis of molecular recognition (Mendelsohn et al., 2015). With this finding in mind, I decided follow up this project by designing a transcriptional screen of different motor pools in mouse, with the intention of identifying molecular surface recognition molecules that might underlie selective circuit formation. Instead, the results of this screen led me down a very different scientific path, towards addressing how motor neurons acquire distinct subtype identities.

In initially following up the results of the screen, I was unable to identify new genes that could plausibly account for differences in sensory-motor connectivity, or even genes that could distinguish motor neurons projecting to different muscles in the leg. But to my surprise, the screen also revealed something completely unexpected: a large number of genes expressed selectively in the motor neurons that project to the intrinsic muscles of the hand and foot. Moreover, I also found that one particularly important gene, Aldh1a2, which encodes the retinoic acid synthesis enzyme Raldh2, was absent in these motor neurons. Given the historical difficulty of defining selective markers of motor pools, this was an exciting and shocking finding.

When I first showed these results to Tom, he immediately grasped that they carried profound consequences beyond the scope of developmental neurobiology. The formation of the motor system does not occur in a vacuum, as the development of motor circuits has to correspond with the formation of muscles in the limb that receive motor innervation. In fact, the formation of the limb and motor system use many similar patterning mechanisms, including Shh, FGFs, BMPs, retinoic acid and Hox genes (Cooper et al., 2011; Dasen and Jessell, 2009; Mercader et al., 2000; Roselló-Díez et al., 2011; Zakany and Duboule, 2007). Consequently, Tom realized that the divergent transcriptional profile of digit-innervating motor neurons could have something to do with the natural history of digits themselves. Thinking now in evolutionary terms, Tom quickly sent me off to read Neil Shubin’s best-selling book “Your Inner Fish,” along with a stack of academic papers on, among other things, the embryology of shark and paddlefish fins. I soon learned that evolutionary biologists have long been concerned with the question of how digits formed during the transformation of fish fins into tetropod limbs (Shubin et al., 1997).  It appears that the emergence of distal limb elements is a specialized step in limb patterning, requiring evolutionarily conserved molecular determinants (Nakamura et al., 2016; Schneider and Shubin, 2013; Woltering and Duboule, 2010). As a result, we started to wonder whether the specialized evolutionary emergence of digits might explain why we found such divergent patterns of gene expression in digit-innervating motor neurons.

In order to clarify the mechanisms underlying digit-innervating motor neuron specification, we first sought to characterize the patterns of gene expression that emerged from the screen using a combination of in situ hybridization, immunohistochemistry and retrograde anatomical tracing. This work revealed several markers, including Cpne4 and Fign, which selectively labeled digit-innervating motor neurons throughout embryonic development. It also confirmed that digit-innervating motor neurons fail to make retinoic acid from the moment of their specification. In looking at patterns of gene expression under the microscope day after day, I stumbled on another interesting way in which digit-innervating motor neurons are unique. Unlike other limb-innervating motor neurons, digit-innervating motor neurons express the signaling molecule pSMAD in a manner that is dependent on retrograde cues from the limb. Though intrigued by this finding, we weren’t sure how to proceed given that not much is known about the role of the SMAD family in motor neuron development. On the other hand, it was already well known that retinoic acid is involved in the initial generation of motor neurons, as well as their acquisition of broader divisional identities (Sockanathan et al., 2003). As a result, we decided to focus on the role of retinoic acid signaling in digit-motor neuron development.

Since digit-innervating motor neurons fail to make retinoids, we wondered whether the absence of retinoic acid was necessary for their development. To address this, we decided to force developing motor neurons to engage in retinoic acid signaling, reasoning that the non-digit innervating motor neurons that already made retinoids would be unaffected. The quickest and easiest way to test this question was to go into chick, where embryologists have long used in ovo electroporation as a technique to manipulate gene expression in developing embryos. We electroporated chicken embryos with a genetic construct in which the retinoic acid receptor RAR was fused to a motif that would render the receptor constitutively active, and found that while other limb-innervating motor neurons were unaffected, the development of digit-innervating motor neurons was selectively disrupted.

The finding that retinoid evasion was important for digit-innervating motor neuron development was satisfying for several reasons. One is that retinoic acid is responsible for the downstream regulation of hundreds of other genes, providing a potential explanation for why digit-innervating motor neurons express so many different genes from other limb-innervating motor neurons. A second reason is that the absence of retinoic acid signaling also helped to explain why intrinsic hand and intrinsic foot innervating motor neurons expressed so many of the same genes, even though each pool is located in a different part of the spinal cord and innervates separate limbs. However, we were still left with the nagging question of why digit-innervating motor neurons were failing to make retinoic acid. To address this, we turned to Hox genes.

Hox genes are involved in both limb and nervous system planning, and have been a longstanding subject of interest in Tom’s lab for their role in specifying motor neurons. Much of this work had been carried out by Jeremy Dasen when he was a post-doc in the lab, in a series of classic studies that I first became acquainted and enamored with as an undergraduate student (Dasen et al., 2003, 2005, 2008). In re-reading these studies, as well as more recent work from Jeremy’s lab at NYU, I started to hone in on the role of two Hox genes, Hoxc8 and Hoxc9. Hoxc8 is expressed at brachial levels of the spinal cord and contributes to limb-innervating motor neuron specification, whereas Hoxc9 is expressed at thoracic levels and is thought to block limb-innervating motor neuron identity. What I found interesting is that even though most limb-innervating motor neurons were unaffected by the loss of Hoxc9, there was a surprising reduction in the number of motor neurons in these mutants projecting to the distal limb (Jung et al., 2010). After initially confirming that digit-innervating motor neurons at brachial levels expressed Hoxc8 as well as low levels of Hoxc9, I started to wonder whether they might both be required for their development. This possibility was especially tantalizing given previous evidence that Hoxc9 is capable of blocking the expression of Raldh2 (Dasen et al., 2003, 2008; Jung et al., 2010), possibly serving as an explanation for why digit-innervating motor neurons fail to make retinoic acid. At this point in the project, it was clear that we would benefit from directly enlisting Jeremy’s help.

We first asked whether Hoxc8 and Hoxc9 were necessary for digit-innervating motor neurons specification, examining the expression of digit motor neuron selective markers in Hoxc8 and Hoxc9 mutants, which Jeremy’s lab provided for us (Catela et al., 2016; Jung et al., 2010). We found that whereas all caudal motor neurons, including those projecting to the hand, were lost in the Hoxc8 mutants, only digit-innervating motor neurons were lost in the Hoxc9 mutants. Moreover, when we ectopically expressed Hoxc8 and Hoxc9 together in chick embryos through electroporation, we could induce the generation of digit-innervating motor neurons, suggesting that this combination of gene expression was also sufficient to specify digit-innervating motor neurons.

Together, our Hox and retinoid manipulation studies suggested a model for the specification of digit-innervating motor neurons, implicating low levels of Hoxc9 in regulating the absence of Raldh2 in these neurons. While this work provides an important initial step to clarifying how digit-innervating motor neurons acquire their divergent identity, it raises many more questions than it answers. We still don’t understand the role of pSMAD signaling in these motor neurons or how Hox genes operate at lumbar levels of the spinal cord. We also don’t know how retinoid evasion coordinates the expression of downstream genes or the molecular mechanisms by which digit-innervating motor neurons form appropriate connections with muscle in the periphery. But what made this project so exciting and satisfying to work on was that it emerged spontaneously. I had no idea when I started my PhD that I would end up working with chick embryos or reading about the latest advances in paleontology. The freedom to pursue unforeseen and interesting questions and the joy of unexpected discovery has been the most meaningful part of my graduate work, and one that I will hopefully continue to experience throughout my scientific career.

References

Catela, C., Shin, M.M., Lee, D.H., Liu, J.P., and Dasen, J.S. (2016). Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfrα Genes. Cell Rep. 14, 1901–1915.

Cooper, K.L., Hu, J.K.-H., ten Berge, D., Fernández-Terán, M.A., Ros, M.Á., and Tabin, C.J. (2011). Initiation of proximal-distal patterning in the vertebrate limb by signals and growth. Science (80-. ). 332, 1083–1086.

Dasen, J.S., and Jessell, T.M. (2009). Hox networks and the origins of motor neuron diversity. (Elsevier Inc.).

Dasen, J.S., Liu, J.-P., and Jessell, T.M. (2003). Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature 425, 926–933.

Dasen, J.S., Tice, B.C., Brenner-Morton, S., and Jessell, T.M. (2005). A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell 123, 477–491.

Dasen, J.S., De Camilli, A., Wang, B., Tucker, P.W., and Jessell, T.M. (2008). Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell 134, 304–316.

Jung, H., Lacombe, J., Mazzoni, E.O., Liem, K.F., Grinstein, J., Mahony, S., Mukhopadhyay, D., Gifford, D.K., Young, R.A., Anderson, K. V, et al. (2010). Global control of motor neuron topography mediated by the repressive actions of a single hox gene. Neuron 67, 781–796.

Mendelsohn, A.I., Simon, C.M., Abbott, L.F., Mentis, G.Z., and Jessell, T.M. (2015). Activity Regulates the Incidence of Heteronymous Sensory-Motor Connections. Neuron 87, 111–123.

Mercader, N., Leonardo, E., Piedra, M.E., Martínez-A, C., Ros, M.A., and Torres, M. (2000). Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes. Development 127, 3961–3970.

Nakamura, T., Gehrke, A.R., Lemberg, J., Szymaszek, J., and Shubin, N.H. (2016). Digits and fin rays share common developmental histories. Nature 1–16.

Roselló-Díez, A., Ros, M.A., and Torres, M. (2011). Diffusible signals, not autonomous mechanisms, determine the main proximodistal limb subdivision. Science 332, 1086–1088.

Schneider, I., and Shubin, N.H. (2013). The origin of the tetrapod limb: from expeditions to enhancers. Trends Genet. 29, 419–426.

Shubin, N., Tabin, C., and Carroll, S. (1997). Fossils, genes and the evolution of animal limbs. Nature 388, 639–648.

Sockanathan, S., Perlmann, T., and Jessell, T.M. (2003). Retinoid receptor signaling in postmitotic motor neurons regulates rostrocaudal positional identity and axonal projection pattern. Neuron 40, 97–111.

Woltering, J.M., and Duboule, D. (2010). The origin of digits: expression patterns versus regulatory mechanisms. Dev. Cell 18, 526–532.

Zakany, J., and Duboule, D. (2007). The role of Hox genes during vertebrate limb development. Curr. Opin. Genet. Dev. 17, 359–366.

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