Dev Dyn Research Articles

Behavioral Assays Dissecting NMDA Receptor Function in Zebrafish.

Zebrafish are a powerful system to study brain development and to dissect the activity of complex circuits. One advantage is that they display complex behaviors, including prey capture, learning, responses to photic and acoustic stimuli, and social interaction (Dreosti et al., Front Neural Circuits 9:39, 2015; Bruckner et al., PLoS Biol 20:e3001838, 2022; Zoodsma et al., Mol Autism 13:38, 2022) that can be probed to assess brain function. Many of these behaviors are easily assayed at early larval stages, offering a noninvasive and high-throughput readout of nervous system function. Additionally, larval zebrafish readily uptake small molecules dissolved in water making them ideal for behavioral-based drug screens. Together, larval zebrafish and their behavioral repertoire offer a means to rapidly dissect brain circuitry and can serve as a template for high-throughput small molecule screens.NMDA receptor subunits are highly conserved in zebrafish compared to mammals (Zoodsma et al., Mol Autism 13:38, 2022; Cox et al., Dev Dyn 234:756-766, 2005; Zoodsma et al., J Neurosci 40:3631-3645, 2020). High amino acid and domain structure homology between humans and zebrafish underlie conserved functional similarities. Here we describe a set of behavioral assays that are useful to study the NMDA receptor activity in brain function.

Cerebellar Cognitive Affective Syndrome in Mexican Pediatric Patients with Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is a disease caused by mutations in the ATM gene (11q22.3-23.1) that induce neurodegeneration Sasihuseyinoglu AS et al. Pediatr Allergy Immunol Pulmonol 31(1):9-14, 2018,Teive HAG et al. Parkinsonism Relat Disord 46:3-8, 2018. Clinically, A-T is characterized by ataxia, mucocutaneous telangiectasia, immunodeficiency, and malignancy. Movement disorders have been the most described and well-studied symptoms of A-T. Other studies have reported visuospatial processing disorders, executive function disorders and emotional regulation disorders, which are clinical manifestations that characterize cerebellar cognitive affective syndrome (CCAS)Choy KR et al. Dev Dyn 247(1):33-46, 2018.To describe the neurocognitive and emotional state of pediatric patients with ataxia-telangiectasia and to discuss whether they have cerebellar cognitive affective syndrome.This observational, cross-sectional, and descriptive study included 9 patients with A-T from May 2019 to May2021. A complete medical history was retrieved, and tests were applied to assess executive functions, visual-motor integration and abilities, language, psychological disorders, and ataxia.Six girls and 3 boys agreed to participate. The age range was 6 to 14years. The participants included five schoolchildren and four teenagers. Eight patients presented impaired executive functioning. All patients showed some type of error in copying and tracing (distortion) in the performance of visual perceptual abilities. Emotional disorders such as anxiety and depression were observed in six patients. Eight patients presented with dyslalia and impairments in word articulation, all patients presented with ataxia, and seven patients used a wheelchair.All patients presented symptoms consistent with CCAS and had variable cognitive performance.

SNED1: a Novel ECM Protein Regulating Neural Crest Cells Phenotype and Craniofacial Morphogenesis

The extracellular matrix (ECM) is a complex meshwork of proteins that provides structural and biochemical support to cells. We have recently reported that the homozygous ablation of Sned1, which encodes the novel ECM protein SNED1, caused neonatal lethality, and the few knockout (KO) survivors we obtained exhibited craniofacial malformations including shorter snouts and wider neurocrania (Figure 1). Since cranial neural crest cells (NCCs) form the facial skeleton and given the importance of the ECM in guiding NCC phenotype, we hypothesized that SNED1 influences the behavior of this cell population. To test this, we generated an NCC-specific Sned1 KO mouse. While Sned1NCC-/NCC- mice survive, they present craniofacial anomalies resembling the malformations observed in the global Sned1 KOmice (Barqué et al., Dev Dyn, 2020). In order to form craniofacial structures, cephalic NCCs first need to delaminate from the neural tube, then migrate towards the frontonasal region and the pharyngeal arches, and there differentiate into bone and cartilage. Our goal is now to determine precisely which cellular processes along the NCC path are directly controlled by SNED1. To assess this, we are first performing in-vivo assays including in-situ hybridization and immunohistochemical staining on Sned1NCC-/NCC- and control mouse embryos at different embryonic timepoints to assess NCC behaviors. In parallel, we are conducting in-vitro assays using neural tube explants excised from Sned1NCC-/NCC- and control mouse embryos and recapitulating the key steps NCCs take towards the formation of craniofacial structures. These steps include migration, proliferation, and chondrogenic and osteogenic differentiation and can be monitored by tracking the expression of specific molecular markers. Once we identify which cellular processes are controlled by SNED1, we will turn our focus to the identification of the molecular mechanisms by which SNED1 governs NCC phenotype and craniofacial morphogenesis. Sequence analysis of SNED1 revealed two consensus binding sites for integrins, which are known ECM receptors. Thus, we have developed SNED1 constructs containing mutations in the putative integrin-binding sites that we will use to elucidate whether integrins serve as SNED1 receptors. Importantly, deletion of 2q37.3, a region that comprises the SNED1 locus, has been linked to facial dysmorphism in patients. We thus propose that SNED1 contributes to the phenotype of these patients and that our novel mouse model may shed light on some of the features of this syndrome. Moreover, we previously reported SNED1 as a promoter of breast cancer metastasis (Naba et al., eLife, 2014). Since metastatic cells share common features with NCC, our studies will also allow us to better understand the role of SNED1 in metastasis.

Quantitative Analysis of Human Cancer Cell Extravasation Using Intravital Imaging.

Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation, survival in circulation, extravasation, and formation of metastatic colonies. Currently, in vitro assays are limited in their ability to investigate these intricate processes and do not faithfully reflect metastasis as it occurs in vivo. Traditional in vivo models of metastasis are limited by their ability to visualize the seemingly sporadic behavior of where and when cancer cells spread (Reymond et al., Nat Rev Cancer 13:858-870, 2013). The avian embryo model of metastasis is a powerful platform to study many of the critical steps in the metastatic cascade including the migration, extravasation, and invasion of human cancer cells in vivo (Sung et al., Nat Commun 6:7164, 2015; Leong et al., Cell Rep 8, 1558-1570, 2014; Kain et al., Dev Dyn 243:216-28, 2014; Leong et al., Nat Protoc 5:1406-17, 2010; Zijlstra et al., Cancer Cell 13:221-234, 2008; Palmer et al., J Vis Exp 51:2815, 2011). The chicken chorioallantoic membrane (CAM) is a readily accessible and well-vascularized tissue that surrounds the developing embryo. When the chicken embryo is grown in a shell-less, ex ovo environment, the nearly transparent CAM provides an ideal environment for high-resolution fluorescent microcopy approaches. In this model, the embryonic chicken vasculature and labeled cancer cells can be visualized simultaneously to investigate specific steps in the metastatic cascade including extravasation. When combined with the proper image analysis tools, the ex ovo chicken embryo model offers a cost-effective and high-throughput platform for the quantitative analysis of tumor cell metastasis in a physiologically relevant in vivo setting. Here we discuss detailed procedures to quantify cancer cell extravasation in the shell-less chicken embryo model with advanced fluorescence microscopy techniques.

The NOTCH signaling pathway in normal and malignant blood cell production.

The NOTCH pathway is an evolutionarily conserved signalling network, which is fundamental in regulating developmental processes in invertebrates and vertebrates (Gazave et al. in BMC Evol Biol 9:249, 2009). It regulates self-renewal (Butler et al. in Cell Stem Cell 6:251–264, 2010), differentiation (Auderset et al. in Curr Top Microbiol Immunol 360:115–134, 2012), proliferation (VanDussen et al. in Development 139:488–497, 2012) and apoptosis (Cao et al. in APMIS 120:441–450, 2012) of diverse cell types at various stages of their development. NOTCH signalling governs cell-cell interactions and the outcome of such responses is highly context specific. This makes it impossible to generalize about NOTCH functions as it stimulates survival and differentiation of certain cell types, whereas inhibiting these processes in others (Meier-Stiegen et al. in PLoS One 5:e11481, 2010). NOTCH was first identified in 1914 in Drosophila and was named after the indentations (notches) present in the wings of the mutant flies (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010). Homologs of NOTCH in vertebrates were initially identified in Xenopus (Coffman et al. in Science 249:1438–1441, 1990) and in humans NOTCH was first identified in T-Acute Lymphoblastic Leukaemia (T-ALL) (Ellisen et al. in Cell 66:649–61, 1991). NOTCH signalling is integral in neurogenesis (Mead and Yutzey in Dev Dyn 241:376–389, 2012), myogenesis (Schuster-Gossler et al. in Proc Natl Acad Sci U S A 104:537–542, 2007), haematopoiesis (Bigas et al. in Int J Dev Biol 54:1175–1188, 2010), oogenesis (Xu and Gridley in Genet Res Int 2012:648207, 2012), differentiation of intestinal cells (Okamoto et al. in Am J Physiol Gastrointest Liver Physiol 296:G23–35, 2009) and pancreatic cells (Apelqvist et al. in Nature 400:877–881, 1999). The current review will focus on NOTCH signalling in normal and malignant blood cell production or haematopoiesis.

Pursuing novel molecular mechanisms and treatment strategies to enhance lung growth in neonatal chronic lung disease (CLD)

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany 16-17 April 2015 This supplement has not been sponsored. Meeting abstracts Rationale Infants born prematurely or with intrauterine growth restriction (IUGR) are often afflicted with CLD, a debilitating disorder marked by diminished alveoli and lung micro-vessels. Despite advances in neonatology, CLD's incidence remains high and its prevention elusive. Mechanical ventilation (MV), high inspired O2, and IUGR can disrupt key signaling pathways, reduce cell survival and disrupt matrix elements, thus inhibiting lung growth [1–6]. Aims (1) To define the impact of MV, O2 and IUGR on lung structure and function in newborn rodents. (2) identify altered signaling pathways of neonatal lung injury, and test novel treatment strategies. (3) mechanistic studies to define how such aberrant signaling may impact cell survival in culture. Methods (1) Exposure of newborn mice to prolonged MV or hyperoxia; IUGR in rats induced by maternal low protein diet. (2) Prolonged MV-O2 in pups treated with a serine elastase inhibitor Elafin. (3) Studies with mouse alveolar epithelial (AEC) and lung micro-vascular endothelial (LMVEC) cells. Results MV, O2, and IUGR inhibit alveolar and micro-vessel formation and disrupt matrix components in lung, resulting in reduced respiratory system compliance and increased airway resistance. TGFβ signaling is disrupted after MV, O2, and IUGR which adversely affects homeostasis of AEC and LMVEC, contributing to impaired lung growth with disordered lung matrix. Moreover, MV-O2 inhibited EGF-receptor activation and reduced Kruppel-like factor 4 (Klf4), a regulator of cell differentiation and survival. We identified this novel pEGFR-Klf4 axis as a key regulator of AEC survival, and discovered that pEGFR-Klf4 signaling is protected by treatment with Elafin during MV-O2. Conclusion These results identify critical pathways that can impact lung growth arrest, as seen in CLD, and uncover novel mechanisms that could promote alveolarization, thus pointing the way toward new treatments to improve the outcomes of infants with CLD.

MicroRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Strange bedfellows (Mapping Differentiation Kinetics in the Mouse Retina Reveals an Extensive Period of Cell Cycle Protein Expression in Postmitotic Newborn Neurons by Marek Pacal and Rod Bremner, Dev Dyn 241:1525–1544) Pacal and Bremner's careful analysis of expression patterns of cell cycle and differentiation markers in the developing retina, reveal oddities within. The neuroblastic layer (NBL) harbors mitotic progenitors at its apical surface that initiate cell cycle exit as they migrate, eventually terminally differentiating at the basal surface. The authors find that a subpopulation of cells labeled with pan cell cycle markers – Pcna, Mcm6, and Ki67 – co-localizes with neuronal differentiation proteins. This finding is in contrast to expression of a number of other cell cycle and progenitor cell markers (Cyclins A, B, D, Vsx2) whose expression is mutually exclusive with differentiation markers. A particularly intriguing finding is that Ki67 expression is extinguished in the subpopulation of post-mitotic Ki67+ cells (6-7%) upon reaching the terminally differentiated ganglion cell layer (GCL). One explanation is that Ki67 is not functionally relevant in this population. Another, more provocative possibility is that postmitotic Ki67+ cells are a previously described subpopulation that can re-enter the cell cycle in the absence of Rb, a gene required for cell cycle exit, representing an intermediate state at the cusp of terminal differentiation. Further experiments will reveal whether the cells are simply an anomaly, or whether they reveal insights into the progression from proliferation to differentiation. It's not witchcraft (Lens Regenerates by Means of Similar Processes and Timeline in Adults and Larvae of the Newt Cynops pyrrhogaster by Takeshi Inoue, Ryo Inoue, Rio Tsutsumi, Kikuo Tada, Yuko Urata, Chiaki Michibayashi, Shota Takemura, and Kiyokazu Agata, Dev Dyn 241:1575–1583). There may be a good reason why witches' boiling cauldrons contain eye of newt. Urodeles such as the newt Cynops pyrrhogaster wield the seemingly supernatural power to regenerate lost body parts throughout their lifetime. The observation that juveniles replace lost body parts faster than adults have led to the hypotheses that younger cells have a greater regenerative ability, or that mechanisms for regeneration differ over time. Based on careful examination of lens regeneration in embryos and adults, Inoue and colleagues have reached an alternative conclusion. The authors performed lentectomies in embryonic, larval, and adult transient transgenic newts (F0 generation) that display red fluorescence under control of the γ-crystallin promoter, a marker of differentiation. Using immunohistochemistry, morphology, and reporter expression they find very similar rates of regeneration, and regeneration stages, suggesting conservation of cellular and molecular regeneration mechanisms at different life stages. They conclude that lens completion takes longer in adults because the adult lens must expand to a 1,000 μm in diameter vs. 300 μm in diameter in juveniles. This strikingly simple explanation raises an obvious question, does this finding hold true in other regenerative systems? Some aspects of regeneration may not be witchcraft after all.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Leveling the playing field (New Frontiers in Cell Competition by Simon de Beco, Marcello Ziosi, and Laura A. Johnston, Dev Dyn 241:831–841) In a world that is competitive by nature, even cells elbow out the weak. In cell competition, cells get the stats on the their neighbors' fitness by sensing and responding to their growth rates. “Losing”” cells undergo apoptosis, regulating overall organ homeostasis and growth, a process most intensely studied in growing Drosophila epithelia. This review details recent experiments that identify new players that make the fight possible, including the membrane protein Flower, the extracellular protein Sparc, the signaling molecule Wg, and the Hippo-Salvador-Warts pathway. Recent data support the hypothesis that cell competition also plays a physiological role in mammalian hematopoietic cells. However there is at least one key difference: “losers”” undergo senescence rather than apoptosis. Future directions include a thorough comparison between Drosophila and mammalian cell competition, and how the process fits into other pathways such as stress resistance, stem cell expansion, and normal development. Progress will come from cooperation between mammalian and Drosophila investigators—not competition. It's normal (Normalized Shape and Location of Perturbed Craniofacial Structures in the Xenopus Tadpole Reveal an Innate Ability to Achieve Correct Morphology by Laura N. Vandenberg, Dany S. Adams, and Michael Levin, Dev Dyn 241:863–878) Embryos are incredibly forgiving. At later stages, there may be no signs of an injury sustained in early development. To gain insights into this phenomenon Vandenberg et. al, used geometric morphometric analytical techniques to determine how relative positions of Xenopus anatomical landmarks change over time following induced craniofacial perturbations. They note that while all tissues observed eventually end up back in their normal locations, the morphology of some tissues (cranial neural crest derived jaw and branchial arch) are more easily repaired than others (ectodermal placode derived—eye, nose). Further, reaching a target position is a dynamic process governed by a structure's position relative to stable reference points (distance from brain and angle from midline). The authors suggest a model in which a craniofacial structure senses its position based on signals sent between it and an “organizing center.” Uncovering the precise nature of this exchange has implications for repair of birth defects, organ regeneration, and insults sustained during adulthood.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Chew on this (Asymmetric Requirement of Surface Epithelial β-Catenin During the Upper and Lower Jaw Development by Ye Sun, Ian Teng, Randi Huo, Michael G. Rosenfeld, Lorin E. Olson, Xiaokun Li, and Xue Li, Dev Dyn 241:663–674) One's identity is often linked to distinguishing facial features: a pouty mouth, a strong jaw, prominent cheekbones. Here, Sun et al., show that β-catenin, an effector of canonical Wnt signaling, sculpts some of the features that make us who we are. Pitx1/Cre was used to drive expression of loss-of-function (LOF) or gain-of-function (GOF) β-catenin alleles in surface epithelium of the first pharyngeal arch. Although many features are affected, those with the most striking phenotypes are the upper and lower jaw. In β-catenin LOF mutants, the mandibles (lower jaw) are absent and mandible skeletal structures are severely deformed, while the maxilla (upper jaw) and associated skeletal structures are unaffected. By contrast, in β-catenin GOF mutants, mandibular structures are expanded or enlarged while maxillary elements are deficient. Consistent with a central role in craniofacial development, β-catenin is genetically upstream of several central players, including shh, bmp4, wnt5a, fgf8, and their downstream effectors. The work shows that epithelial β-catenin is required for lower jaw formation, while its attenuation is necessary for proper upper jawbone development. The authors suggest β-catenin as a nodal point for evolutionary craniofacial adaptations. Turning back time (Avian Intervertebral Disc Arises From Rostral Sclerotome and Lacks a Nucleus Pulposus: Implications for Evolution of the Vertebrate Disc by Bradley J. Bruggeman, Jennifer A. Maier, Yasmin S. Mohiuddin, Rae Powers, Yinting Lo, Nuno Guimarães-Camboa, Sylvia M. Evans, and Brian D. Harfe, Dev Dyn 241:675–683) Sure, chicks and mice are both vertebrates, but here Harfe and colleagues reveal that their vertebrae are quite different. At the center of mouse intervertebral discs (IVD) is the nucleus pulposa, a jelly-like structure made of proteoglycans that is important for absorbing shock and resisting spinal compression. The structure is derived from the notochord, a transient structure during mouse embryogenesis. An artistic panel of histologically stained IVD from mammals, snakes, amphibians, reptiles, fish, and birds reveals for the first time that the nucleus pulpous is a mammalian-specific structure. Do nonmammalian notochord still contribute to the IVD? Contrary to the mouse, the chick notochord persists through embryogenesis, but fate mapping studies show it does not contribute to the vertebral column. Instead, the sclerotome (rostral somite) is a major contributor to the avian disc, a finding that leads to the discovery that sclerotome also gives rise to the mouse annulus fibrosis that surrounds the nucleus pulpous. The authors suggest that the premammalian IVD was an annulus fibrosis-like structure.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Go with the flow (Stepwise Arteriovenous Fate Acquisition During Mammalian Vasculogenesis by Diana C. Chong, Yeon Koo, Ke Xu, Stephen Fu, and Ondine Cleaver, Dev Dyn 240:2153–2165) Despite the fact that the circulatory system is essential for survival, there has only been a trickle of information regarding the timing and mechanisms of arterial and venous cell fate specification in mammals. Here, Chong and colleagues remedy this oversight, and show that vasculogenesis occurs in a stepwise manner. Angioblasts aggregate into lumenless cords, extend, form tubes, and differentiate, with arteries acquiring their fates before veins. Furthermore, markers for both arterial and venous fates are initially expressed in vessel primordia, and resolve to a classic pattern of fate specific markers (arterial or venous) over time. To test whether cell extrinsic mechanisms are required for vessel fate specification, marker expression was examined in Rasip−/− embryos, whose vessels have no lumens, and in cultured embryos with severed circulatory systems. In both cases, one arterial marker diminished while another was maintained, leading the authors to conclude that, while cell fate is specified at the molecular level, maintenance and/or full-fledged differentiation is under the control of hemodynamic flow. The work opens the floodgates: offering a new set of tools with which to study the multitude of existing mouse mutants with vascular remodeling defects. Facing change (Epigenetic Integration of the Developing Brain and Face by Trish E. Parsons, Eric J. Schmidt, Julia C. Boughner, Heather A. Jamniczky, Ralph S. Marcucio, and Benedikt Hallgrímsson, Dev Dyn 240:2233–2244) As the size of the brain increased in our hominid ancestors, so did facial shape, steering toward a relatively short, broad face. Such observations caused Trish Parsons and others in the Hallgrímsson and Marcucio labs to wonder, is there a relationship between brain and face development? They approached the question by quantifying morphological characteristics of the forebrain and facial prominences in four strains of mice at gestational day (GD) 10–10.5, the time of embryonic facial formation. Statistical analysis reveals that within mice in which forebrain and facial shapes deviate most significantly from the norm, there is a strong correlation in their morphologies. For example, in one strain a longer and taller forebrain covaries with a larger, anterior-projecting maxillary process (upper jaw) and a longer frontonasal mass (nose). These findings lend evidence for direct developmental interactions between the brain and face in a background where genetic, allometric, and environmental variations are carefully controlled. The evolutionary implications are two-fold. The integration of brain and face contributes to morphological variation. Paradoxically, brain and face integration also constrains variation. This is something that our ancestors certainly could not comprehend but that we, thanks to our enlarged brain, can. Imagine this (MicroCT for Molecular Imaging: Quantitative Visualization of Complete Three-Dimensional Distributions of Gene Products in Embryonic Limbs by Brian D. Metscher and Gerd B. Müller, Dev Dyn 240:2301–2308) In an episode of the animated sitcom The Simpsons (“Homer3”, 1995), an ordinarily two-dimensional Homer Simpson discovers that life is very different in a world with three dimensions. Here, Metscher and Müller delve into the world of 3D expression patterns, a worthy endeavor considering that embryos are, after all, three-dimensional. Compared with current 3D expression techniques based on reconstruction of stained tissue sections, which can be distorted during processing, the authors' X-ray tomography (microCT)-based technique directly images whole-mount samples. To obtain X-ray contrast of in situ hybridization or immunostaining patterns, the researcher uses commercially available kits to render enzyme-mediated silver deposition at sites of bound secondary antibody conjugate. Analyzing the resulting high-resolution images with imaging software enables quantifiable results, such as spatial measurements and relative intensity values, that are invaluable for a sophisticated understanding of related developmental mechanisms. Plus, the eye-popping, seemingly tangible microCT images make those obtained from traditional techniques seem, well, cartoonish.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Limbs made to order (Role of Paraxial Mesoderm in Limb/Flank Regionalization of the Trunk Lateral Plate by Miyuki Noro, Hiroki Yuguchi, Taeko Sato, Takanobu Tsuihiji, Sayuri Yonei-Tamura, Hitoshi Yokoyama, Yoshio Wakamatsu, and Koji Tamura, Dev Dyn 240:1639–1649) Gnathostomes (jawed vertebrates) invariably have two sets of paired appendages, but their placement and size vary by species. Noro et al. test the hypothesis that the presomitic mesoderm (PSM) regulates regional specification of the limb field within the subjacent lateral plate mesoderm (LPM). Here, they find that PSM from different positions along the anterior–posterior (A–P) axis specify different aspects of limb growth. Ablation of forelimb-level PSM results in smaller limb width along the A–P axis. By contrast ablation of PSM posterior to the forelimb, called flank PSM, results in limbs that do not protrude as far distally. In addition, flank PSM triggers apoptosis in the flank LPM at the beginning stages of limb bud outgrowth, a process that can be inhibited by implantation of an FGF8-soaked bead into the LPM. This finding is significant in light of the fact that FGF8 signaling from the apical ectodermal ridge (AER) is an initiating event in limb outgrowth. Based on these and other data, the authors present a model in which FGF-8 responsive LPM cells are eliminated by apoptosis, a mechanism that may help control limb spacing, and also limit the number of limbs formed. This work suggests that species variation in limb size and position may be controlled by differential allocation of PSM to forelimb and flank regions. Stress response (Tgfβ/Alk5 Signaling Is Required for Shear Stress Induced Klf2 Expression in Embryonic Endothelial Cells by Anastasia D. Egorova, Kim Van der Heiden, Simone Van de Pas, Peter Vennemann, Christian Poelma, Marco C. DeRuiter, Marie-Jose T.H. Goumans, Adriana C. Gittenberger-de Groot, Peter ten Dijke, Robert E. Poelmann, and Beerend P. Hierck, Dev Dyn 240:1670–1680) Snuggled within the womb, or protected by a shell, you may not think that an embryo would experience stress. But within them, an emerging bloodstream heaves biomechanical forces against pristine vessel walls. The finding that shear stress activates expression of a gene critical for endothelial function, Krüppel like factor 2 (KLF2), has helped show the importance of stress on normal development. Here, Egorova et al. dissect the molecular pathway that regulates stress-activated KLF2. After discovering that the TGFβ/Alk5 pathway, a well-studied developmental regulator, is activated in endothelial cells (ECs) lining the cardiac cushion in a chick shear stress model in vivo, they explore the pathway further in vitro. In cultured mouse embryonic endothelial cells (MEEC), they very thoroughly show that shear stress induction of KLF2 is dependent upon dose-response signaling through TGFβ/Alk5/Smad2 and then a Mek/Erk5 pathway. Of interest, the pathway is not activated by shear stress in two postnatal EC cell lines. Determining whether the embryonic shear stress pathway is activated under disease conditions that create abnormal shear stress, like hypertension, could lead to novel therapies. Understanding embryonic stress may, in the end, reduce ours. Untapped potential (Multipotential Differentiation Even After Lineage-Restricted Stages by Tsutomu Motohashi, Katsumasa Yamanaka, Kairi Chiba, Kentaro Miyajima, Hitomi Aoki, Tomohisa Hirobe, and Takahiro Kunisada, Dev Dyn 240:1681–1693) This work reveals the hidden potential in in vivo, lineage-restrictedneural crest (NC). Embryonic stem cells (ESCs) were generated by homologous recombination in which expression of the NC marker Sox10 is labeled with fluorescent protein. Following a protocol established previously, cells with NC-like characteristics were derived from the ESCs. Surprisingly, when cultured in vitro, the NC-like cells, which were expected to be lineage restricted, could differentiate as melanocytes (M), neurons (N), and glia (G). This included Sox10+/Kit+ cells, thought to represent dorosolaterally migrating, M restricted cells, and Sox10+/Kit− cells, representing ventrolaterally migrating, N and G restricted cells. The results were repeated with cultured Sox10+/Kit+ and Sox10+/Kit− NCs isolated from mice that were derived from the transgenic ESC line. It remains to be determined whether the repressed multipotentiality of in vivo lineage-restricted NCs is tapped later in development, or during homeostasis or regeneration.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Moving on (Evolution of Vertebrate Appendicular Structures: Insight From Genetic and Palaeontological Data by Amir Ali Abbasi, Dev Dyn 240:1005–1016). How fins evolved into limbs is one of the quintessential examples of morphological evolution, the study of the diversification of body form over time. In this Special Issue on limb development, Abbasi assembles information from molecular biology, genetics, comparative biology, and paleontology to illustrate key morphological and molecular events that enact this grand change. One remaining mystery is how the distal bones of a primordial fin/limb transformed into modern toes and fingers. Chipping away at the problem, he describes how certain molecular pathways (i.e., Shh, 5′Hoxd cluster) are involved in fin/limb patterning both pre- and postevolutionary digit formation. He further explains how the morphological advancement could be triggered by molecular changes in cis-regulatory elements of conserved genes that alter sites and/or timing of activity. His careful analysis of what is known helps him to pinpoint specific areas of research that could help fill the morphological gap. These insights should enable researchers to hit the ground running. Tales of the pinkie (HMGB Factors Are Required for Posterior Digit Development Through Integrating Signaling Pathway Activities by Junji Itou, Noboru Taniguchi, Isao Oishi, Hiroko Kawakami, Martin Lotz, Yasuhiko Kawakami, Dev Dyn 240:1151–1162) Hmgb1−/−;Hmbg2+/− mice have a subtle forelimb phenotype, loss of the most posterior digit (analogous to the human pinkie finger). Although the defect may seem minor, it speaks volumes about the role of these chromatin factors in limb patterning. The phenotype mimics reduced signaling of Sonic Hedgehog (Shh) in the limb bud, and Itou et. al, verify that Hmgb1−/−;Hmbg2+/− also have this molecular defect. How, then, might HMGB factors regulate limb Shh? The authors suspect the interaction is indirect. The largely redundant HMGB1 and 2 bind DNA nonsequence specifically and alter chromatin architecture, and also facilitate Wnt/β-catenin signaling. In light of the latter finding, they are surprised to find that dorsal ectoderm Wnt7a signaling, which regulates posterior Shh, is normal in Hmgb1−/−;Hmbg2+/− limbs. They do, however, find that Wnt/β-catenin targets display reduced expression in the posterior mesenchyme, suggesting the tissue is an uncharacterized region of Wnt7a signaling. Supporting this idea, Hmbg1 and 2 also facilitates Wnt/β-catenin signaling in numerous tissues in the developing zebrafish, including the pectoral fin, a forelimb homolog. In addition to a role in Wnt/β-catenin signaling, there is also evidence that HMGB factors modulate BMP signaling. The authors discuss how proper development of the little finger may hinge on integration of these pathways by HMGB 1 and 2. Golden eggs (Identification of Spontaneous Mutations Within the Long-Range Limb-Specific Sonic Hedgehog Enhancer (ZRS) That Alter Sonic Hedgehog Expression in the Chicken Limb Mutants oligozeugodactyly and Silkie Breed by Sarah A. Maas, Takayuki Suzuki, and John F. Fallon, Dev Dyn 240:1212–1222) What might be considered an oddity to an animal breeder can be a goldmine to a biologist. So it is with the spontaneous oligozeugodactyly (ozd) chicken mutant, which lacks all but one digit on each foot, and the preaxial polydactylous (PPD) Silke breed with extra digits. Because mutations in the evolutionarily conserved zone of polarizing activity (ZPA) regulatory sequence (ZRS)—which controls posterior limb Shh expression and anteroposterior limb patterning—has been implicated in human PPD, Maas et al. examined the ZRS in the two strains. Eureka! While ozd chickens lack most of the ZRS enhancer, the Silkie ZRS has a single point mutation within it. To further investigate ZRS function, the group devised the first enhancer assay that works in the developing limb bud in vivo. They find that although the ozd mutant does not express Shh, the wild-type ZRS enhancer drives expression of β-galactosidase (β-gal) in a small group of cells within the pre-ZPA, suggesting they are still ZPA competent. The Silkie ZRS sequence drives β-gal expression that is expanded to the anterior domain, mirroring the ectopic Shh expression observed in this breed. Together the results suggest the ZRS receives both positive and negative input. The next pot of gold goes to the one who determines how factors that prepattern the limb act upon the ZRS to fine-tune Shh expression.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Stem cells on demand (Facultative Stem Cells in Liver and Pancreas: Fact and Fancy by Kilangsungla Yanger and Ben Z. Stanger, Dev Dyn 240:521–529) Video on demand enables the viewer to watch what he wants (feel-good movies), when he wants (while sick in bed). Similarly facultative stem cells (FSCs)—differentiated cells that become multipotent—enable an organ to generate what it needs (stem cells) when it needs them (following injury). In a famous example, salamander limb loss triggers mature mesenchymal cells to de-differentiate and then re-differentiate into the myriad cell types needed to reconstitute a new limb. In this provocative review, Yanger and Stanger discuss the controversy surrounding FSC-based regeneration in mammals. Presented in detail are two putative FSC populations, oval cells that appear following hepatotoxin-based injury of the liver, and ductal cells in the regenerating pancreas. The authors discuss what is known about the existence, origins, and potential of these cell populations, and do not shy away from pointing out potential flaws in published results. What becomes clear is thatsettling the controversy is paramount. Defining what allows a differentiated cell to become a stem cell “on demand” could profoundly impact regeneration research and our understanding of the multipotent state. GATA get it right (Different Requirements for GATA Factors in Cardiogenesis Are Mediated by Non-Canonical Wnt Signaling by Boni A. Afouda and Stefan Hoppler, Dev Dyn 240:649–662) Most people hate to be mistaken for their sibling. After all, everyone deserves to be recognized as an individual. The same is true for the GATA family of transcription factors, collectively known for their roles in endoderm and cardiac development. To distinguish functions of GATA 4, 5, and 6 in cardiac development—a task that has proven to be difficult in vivo—Afouda and Hoppler employ animal cap explants that have been induced to undergo cardiogenesis. Expression of early and late cardiogenic markers were used to assess effects of morpholino depletion and ectopic induction of GATA 4-6 in such cultured explants. The combined results point to overlapping roles for GATA4 and 6 in early cardiogenesis and cardiomyocyte differentiation, and for GATA5 in differentiation. Their differential roles are further supported by the finding that ectopic expression of Wnt 11b, or the downstream non-canonical Wnt component Disheveled, rescues GATA4 and 6, but not GATA5 morphants. These results suggest that GATA4 and 6 uniquely function through the non-canonical Wnt11b pathway. Parsing the specific roles of GATAs, and their pathways may help to devise effective stem cell therapies. It is only when an individual's unique abilities are understood that they can be used to their full potential. Live action BMP signaling (Dynamic Analysis of BMP-Responsive Smad Activity in Live Zebrafish Embryos by Derek W. Laux, Jennifer A. Febbo, and Beth L. Roman, Dev Dyn 240:682–694; Dynamic Smad-Mediated BMP Signaling Revealed Through Transgenic Zebrafish by Ross F. Collery and Brian A. Link, Dev Dyn 240:712–722) Those investigating sites of bone morphogenetic protein (BMP) activity, an important regulator of development and disease, have traditionally been relegated to piecing together static images of fixed, stained tissues. Knowing that live action beats stop motion, two groups have generated fluorescent transgenic reporter lines that display live BMP signaling in the optically clear zebrafish. Their reporters are based on conserved BMP-responsive elements (BRE) from the mouse inhibitor of differentiation-1 (Id1) promoter, that are responsive to the BMP effectors, Smad 1/5/8. The reporters effectively replicate many well-known sites of Smad 1/5/8 activity, respond to genetic and chemical manipulations of the BMP pathway, and potentially reveal previously uncharacterized sites of BMP action. However, they also have some limitations; the authors discuss why the reporters fail to detect some known sites of BMP activity. Regardless, the dynamic reporter lines will undoubtedly prove useful in screening for small molecules that affect BMP signaling and identifying genetic modulators of the pathway.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Taste test (Epibranchial Placode-Derived Neurons Produce BDNF Required for Early Sensory Neuron Development by Danielle E. Harlow, Hui Yang, Trevor Williams, and Linda A. Barlow, Dev Dyn 240:309–323) In the developing gustatory system, brain-derived neurotrophic factor (BDNF) is best known for its “main course” role supporting the survival of gustatory neurons and proper innervation of peripheral targets. Here, Harlow et al. test whether BDNF is also a key ingredient for an appetizer that sets the stage for the entree. In support of this hypothesis, they demonstrate that BDNF-LacZ expression initiates in a subset of mouse geniculate ganglia (gVII) neurons at embryonic day 9.5, before previously reported BDNF expression. Within gVII, the BDNF receptor, TrkB, is expressed in placode-derived neurons, and BDNF itself in a subset of these neurons. To determine whether this apparent paracrine or autocrine BDNF signaling is important for gustatory system development, phenotypes of mice where bdnf is selectively eliminated from placodal neuroblasts were compared with bdnf knockout mice. In both cases, significant numbers of pre- and postmitotic neurons display a delay in maturation. These findings demonstrate the importance of BDNF in early gustatory neuronal development. Future work will determine how mother nature—a skilled chef—can use the same ingredients throughout the meal, but to different effects. Getting to know noncanonical Notch (Noncanonical Notch Function in Motor Axon Guidance Is Mediated by Rac GTPase and the GEF1 Domain of Trio by Jeong K. Song and Edward Giniger, Dev Dyn 240:324–332) Noncanonical Notch signaling makes the Notch pathway more nimble, enabling cross-talk with diverse regulators. During axon growth and guidance, Drosophila motor neurons rely on noncanonical Notch signaling to interact with key regulators of actin dynamics, the Rho GTPases. Notch associates with and antagonizes components of the Abl tyrosine kinase signaling pathway, including Trio, a guanine nucleotide exchange factor (GEF) for Rho GTPases. Here, Song and Giniger flesh out downstream steps of this context-dependent pathway. First, rescue experiments demonstrate that Trio's Rac-specific GEF1 domain, but not its Rho-specific GEF2 domain, is required for noncanonical Notch signaling. Consistent with the finding, constitutively active and dominant-negative Rac modulates Notch-mediated axon guidance, but manipulation of the Rho GTPases Cdc42 and Rho1 do not. Not for the faint of heart, the experiments demand only slight alterations to Trio or Rac levels, as tipping the balance too far produces combinatorial effects. The authors hypothesize that the Abl/Rac signaling network bridges Notch to actin cytoskeleton rearrangements that push and pull the axonal growth cone to its target. Running dry (An Obesogenic Diet Started Before Puberty Leads to Abnormal Mammary Gland Development During Pregnancy in the Rabbit by Cathy Hue-Beauvais, Pascale Chavatte-Palmer, Etienne Aujean, Michéle Dahirel, Patrice Laigre, Christine Péchoux, Stephan Bouet, Eve Devinoy, and Madia Charlier, Dev Dyn 240:347–356) Health risks to the obese—frequently caused by high fat, high sugar diets during adolescence—are well established. But the impact of obesity on the next generation is just beginning to be understood. Here, Hue-Beauvais and colleagues investigate physiological reasons behind reports that obese women are less likely to breastfeed, by examining mammary gland development in obese rabbits. At mid-pregnancy, rabbits fed an obesogenic (OD) diet before puberty display precocious development of mammary gland alveolar structures. Electron microscopy and Western analysis further show that ducts are prematurely filled with milk proteins and lipids. The data suggest that the observed premature mammary growth and differentiation potentially leads to dysregulation of post-partum lactation. Of interest, the authors also note that pups from mid-gestation OD mothers are significantly underweight, possibly due to decreased placental efficiency. Follow-up work may substantiate these effects on future generations, fueling the need for putting a stop to childhood obesity.

Abstract 3284: Preclinical and clinical evaluation of pharmacodynamic biomarkers of a novel anti-angiogenesis agent, anti-EGFL7

Abstract In this study we report the use of pharmacodynamic (PD) biomarkers to guide the clinical development of a novel antibody targeting epidermal growth factor-like domain 7 (EGFL7). EGFL7 is a secreted protein produced by endothelial cells of nascent blood vessels in tumors and other proliferating tissues, but is expressed at low levels in healthy quiescent vessels and many non-vascular cell types (Campagnolo et al. 2005; Fitch et al. 2004; Parker et al. 2004; Soncin et al. 2003). Upon secretion, EGFL7 becomes tightly associated with the perivascular extracellular matrix (ECM), and supports endothelial cell adhesion and migration (Parker et al. 2004; Schmidt et al. 2007). EGFL7 protein also protects endothelial cells from stress-induced apoptosis (Xu et al. 2008). Anti-EGFL7 is a humanized monoclonal antibody that binds to EGFL7. In preclinical models, anti-EGFL7 when combined with anti-VEGF augments the survival benefit observed from anti-VEGF alone. Anti-EGFL7 is currently being investigated in Phase I trials (in solid tumors) in combination with bevacizumab. In addition to endothelial cells, circulating CD34Hi/CD31dim progenitor cells (CPCs) express high levels of EGFL7 transcript. This population of cells can differentiate into endothelial cells in vitro, and we found that the EGFL7 protein promotes proliferation and possibly survival of the undifferentiated CPCs in vitro. In preclinical xenograft models, administration of anti-EGFL7 resulted in delayed (day 15) reduction in CPCs, which based on the time course, is unlikely to be mediated simply by clearance of antibody-bound CPCs. Hence, evaluation of CPCs was incorporated into the dose escalation phase of anti-EGFL7 clinical development as a pharmacodynamic biomarker (PD) of drug activity. Biomarker analysis of CPCs in humans correlated with preclinical findings. For example, reduction in CPCs in response to anti-EGFL7 therapy was observed at a time point similar to that in mice. The treatment dependent decrease in CPCs may serve as a pharmacodynamic biomarker that may help guide dose selection as well as represent another potential mechanism for the anti-angiogenic properties of anti-EGFL7 Campagnolo L, Leahy A, Chitnis S, Koschnick S et al. (2005) Am J Pathol 167(1):275-84 Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H. (2004) Dev Dyn. 230(2):316-24 Parker LH, Schmidt M, Jin SW et al (2004) Nature 428(6984):754-8. Schmidt M, Paes K, De Mazière A, (2007) Development 134(16):2913-23. Soncin F, Mattot V, Lionneton F (2003) EMBO J. 22(21):5700-11 Xu D, Perez RE, Ekekezie II (2008) Am J Physiol Lung Cell Mol Physiol. 294(1):L17-23 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3284. doi:10.1158/1538-7445.AM2011-3284

Abstract 3295: Inhibiting vascular morphogenesis in tumors: EGFL7 as a novel therapeutic target

Abstract EGFL7 is a vascular restricted extracellular matrix protein that is up-regulated during angiogenesis (Campagnolo et al., 2005; Fitch et al., 2004; Parker et al., 2004; Soncin et al., 2003). EGFL7 supports endothelial cell adhesion (Parker et al., 2004; Schmidt et al., 2007) and protects endothelial cells from stress-induced apoptosis (Xu et al., 2008). Inhibition of Egfl7 expression in zebrafish embryos abolished vascular lumen formation and reduced sprouting angiogenesis (Parker et al., 2004). We developed a panel of anti-EGFL7 monoclonal antibodies that block the adhesive and pro-survival activities of EGFL7. Anti-EGFL7 in combination with anti-VEGF resulted in significant tumor regression in multiple murine xenograft models, whereas anti-VEGF alone only slowed tumor growth in the same models. Anti-EGFL7 monoclonal antibodies also demonstrated prolonged survival and anti-tumor angiogenesis effects in stringent murine genetic tumor models when used as a single agent, and enhancement of anti-VEGF therapy in the combination setting. An ongoing Phase I study is being conducted to evaluate clinical safety, PK, PD and efficacy of anti-EGFL7.

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

Highlights in DD

Highlights in <i>DD</i>

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Not too late to remodel (Rapid Remodeling of Airway Vascular Architecture at Birth by Amy Ni, Erin Lashnits, Li-Chin Yao, Peter Baluk, and Donald M. McDonald, Dev Dyn 239:2354–2366) When form ceases to support function, it is time to remodel. Ni et al. discover that the mantra is not limited to kitchens and homes; tracheal vasculature also follows the same principle. Using the trachea as an easily studied representative of the airways, they document that embryonic day (E) 17.5 mice have a web-like primitive tracheal vascular plexus that is reminiscent of vascular architecture in the yolk sac. During postnatal stages P0–P3, an astounding 76% the vascular network is pruned, leaving a skeleton of vasculature that grows horizontally over the ladder-like cartilage rings lining the trachea. At P4, horizontal capillaries re-grow, and arterioles and venules begin to grow vertically between the rings, acquiring most features of adult vasculature by P7. Of interest, the vasculature of kidney, brain, and liver, all fail to undergo a similar overhaul at birth, suggesting that exposure to atmospheric oxygen may contribute to the remodeling phenomenon in the trachea. While the hypothesis remains to be tested, the authors do identify other mechanisms associated with vascular regression and re-growth. Hypoxic regions and hypoxia inducible factor (HIF1-α) correlate with vessel survival after birth, and survival of tracheal vasculature from P0 to P7 is dependent upon vascular endothelial growth factor (VEGF). Future work will focus on the roles of signaling pathways and changes in oxygen tension on vascular remodeling during postnatal development and disease. Smart screen (Essential Genes for Astroglial Development and Axon Pathfinding During Zebrafish Embryogenesis by Michael J.F. Barresi, Sean Burton, Kristina Dipietrantonio, Adam Amsterdam, Nancy Hopkins, and Rolf O. Karlstrom, Dev Dyn 239:2603–2618) Astroglia are the unsung heroes of neural development. Once believed to function solely as scaffold support for neurons, it is now understood that they play wide-ranging roles during neurogenesis such as in forming the blood–brain barrier, neural patterning, and regeneration. Despite their universal importance, little is known about astroglial development. To remedy this oversight, the authors screened an existing zebrafish mutant collection, for which the affected genes were already identified. They describe 25 mutants that are placed into one or more categories: axonal pathfinding, gliogenesis, glial patterning and neurogenesis. One important observation is that defects in glial patterning correlate with perturbed axon pathfinding, suggesting axon–glial interactions are important for axon scaffold formation. Another is the discovery of a new class of mutants affecting cell number of radial glial cells, the main source of neural stem cells in vertebrate embryos. The work also identifies potential novel roles for the affected genes, many of which are familiar signaling pathway components or transcription factors (i.e., chordin, tbx16). These intriguing preliminary findings double as jumping-off points from which to further the understanding of brain development. Green eggs and sperm (The ziwi Promoter Drives Germline-Specific Gene Expression in Zebrafish by David H. Leu and Bruce W. Draper, Dev Dyn 239:2714–2721) Here Leu and Draper unveil a tool that will be of interest to germ cell researchers and the zebrafish community at-large. They show that a 4.8-kb promoter of ziwi, which encodes an Argonaut class protein involved in Piwi-interacting RNA (piRNA) metabolism, can drive germline-specific, heterologous gene expression. Expression of the Tg(ziwi:EGFP) transgene initiates at 7 days postfertilization (dpf) in bipotential gonocytes and continues to be expressed after the onset of sexual differentiation (18–21 dpf) in all testis and ovary germ cells. Demonstrating the utility of the tool as a germ cell marker, Tg(ziwi:EGFP) aids the authors in defining new substages in stage-1A oocytes. What sets this germline-specific transgene apart from others, however, is that it is reliably expressed in premeiotic oocytes. The authors discuss the potential utility of the ziwi promoter in manipulating gene expression in the germline, producing genetic mosaic germlines, and speculate how the tool could propel the future of germ cell related technologies in zebrafish.