Dev Dyn Research Articles

A Nucleolar Weapon in Our Fight for Regenerating Adult Hearts

See related article, pages 89–97 Ischemic diseases and heart failure remain the major causes of morbidity and mortality in the industrialized world. Several new therapeutic modalities have been developed to interfere with the response to injury and to improve cardiac function after ischemia or cardiomyopathy. Experimental and clinical studies demonstrated that the transplantation of bone marrow–derived or tissue-resident stem/progenitor cells in diseased hearts improves neovascularization and functional recovery.1 However, the recent discovery of cardiac stem cells (CSCs) in the adult heart poses the question whether activation of the endogenous pool of these resident stem cells may compensate for the loss of cardiac tissue after injury and improve functional recovery. Indeed, a genetic fate-mapping study elegantly demonstrated that stem cells or precursor cells significantly contributed to the replacement of adult mammalian cardiomyocytes after injury.2 Although it remains unclear to what extent cardiac regeneration in this genetic model was mediated by CSCs or circulating progenitor cell populations being attracted to the heart after injury, mounting evidence suggests that injury-associated signals activate CSCs in situ, which subsequently contribute to the refreshment of the injured heart.3 Obviously, to play a significant role in endogenous cardiac regeneration, the relatively small number of endogenous CSCs needs to be expanded after injury before differentiating into cardiac myocytes. However, the molecular mechanisms underlying the activation, expansion, and recruitment of resident CSCs after injury remain unclear. In the current issue of Circulation Research , Siddiqi et al provide an intriguing concept, which might help to mechanistically explain how proliferation of CSCs is regulated in response to injury.4 The authors investigated the cardiac expression of stem cell specific genes, involved in self-renewal and proliferation, during cardiomyopathic injuries and found increased expression of the nucleolar protein nucleostemin—recently discovered in embryonic and adult stem cells as well …

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. Growth Spurts (Dev Dyn 236:2668–2674) Just when you thought that Generation Y was primarily concerned with iPods, cell phones, and Facebook, a star like 17-year-old Isha Jain comes shining through. Jain is first author of the paper presented here, which helped her earn Grand Prize in the prestigious Siemens Competition in Math, Science, and Technology for US high school students. Zebrafish fins grow by the sequential addition of bony segments at the distal end, but their manner of growth has been under dispute. In collaboration with her colleagues, and mentor M. Kathryn Iovine, PhD, Jain shows that segment growth is achieved by multiple cell proliferation saltations, or pulses. The number of bromodeoxyuridine (S-phase) and H3P (mitosis) -positive mesenchymal cells per segment were counted in 12- to 14-week-old fish, and their distance from the distal end measured. The resulting graphs clearly show jumps in cell numbers at approximately 60-am intervals, a finding supported by statistical analysis. Of interest, cells across segments express the gene-based growth marker fa93e10, suggesting that even periods yielding few dividing cells are part of an overall growth cycle, and saltations are subject to extracellular cues. Jain will be one to watch, particularly if she is in the midst of an intellectual growth cycle. Beware, Cellular Mimicry (Dev Dyn 236:3199–3207) Any ecologist knows that when an individual from one species appears like that of another species, it is called mimicry. In this eye-opening review from the Stem Cells Special Issue, Angie Rizzino, PhD, says it is imperative that developmental biologists also become familiar with the concept. Before a cell can transdifferentiate into a cell type from another developmental lineage, it must overcome several hurdles. The proper battery of genes must be expressed at appropriate levels, while others are silenced or epigenetically “poised” for expression. Rizzino argues that, in many reported studies, rather than bona fide transdifferentiation, it is more likely that cells undergo cellular mimicry: they have the appearance of a desired cell type, but their molecular signature is mosaic. Indeed, identities of “transdifferentiated” cells are rarely verified with comprehensive analysis, including extensive gene and epigenetic profiling, and in vivo functional testing. Whether cells truly transdifferentiate in certain circumstances is a question that must be resolved before they can be used in a therapeutic context. Quantum Dot Mechanics (Dev Dyn 236:3393–3401) Although the term Quantum Dots (QDs) sounds like something out of Star Trek, they are today's reality. The inorganic fluorophores are tiny (b25 nm), exceptionally photostable, have a broad absorption and narrow emission spectra, and can be functionalized by cross-linking bioactive molecules. Slotkin et al. thought this skill set made QDs an ideal candidate for labeling mammalian progenitor cells in vivo, but methods for introducing them directly into the body's stem cells remained elusive. To solve this problem, the authors used two techniques to effectively deliver QDs to neural stem and progenitor cells (NSPCs) in the early mouse embryo in utero: ultrasound guided biomicroscopy (UBM) injection, and electroporation. QD-labeled NSPCs appear to migrate and differentiate normally as wild-type. In vitro experiments demonstrate that cell survival and oxidative stress are also normal. Remarkably they also show that, when embryos were labeled at the one-to two-cell stage, apparently all descendants remain labeled until at least embryonic day 13.5. Methods using QDs, like fluorescence resonance energy transfer (FRET), cell lineage tracing, or delivery of therapeutic molecules will follow on the heels of their successes, bringing mammalian embryology to the 21st (or 23rd?) century.

234. Hedgehog signalling components in adult rat testis spermatogonial cells

In normal tissues, Hedgehog-induced progenitor cell proliferation is transient and tightly regulated, preventing continuous regeneration. However, activation of constitutive Hedgehog signalling results in unregulated self-renewal of progenitor cells in association with several human cancers. Although the contribution of Hedgehog signalling to cancers is widely accepted, its impact on spermatogonial stem cells and impact on male fertility are unknown. In this study, we aimed to clarify the possible role of Hh signalling on normal spermatogenesis in the adult rat and in adult testicular stem cells in the irradiated model {1}. Adult male rats were obtained from Monash University Central Animal Service and killed by cervical dislocation before tissue removal and fixation in Bouins for routine histochemical procedures. For studies on irradiated testes, adult LBNF1 male rats (hybrids between Lewis and Brown–Norway) were purchased from Harlan Sprague–Dawley, Inc. (Indianapolis, IN, USA). Testes were irradiated with 6 Gy to deplete all maturing germ cell types. At 15 weeks after irradiation the animals were injected simultaneously with 1.5 mg each of Cetrorelix pamoate and Cetrorelix acetate. Testes were collected 1, 2 or 4 weeks after injection. In situ hybridisation combined with immunohistochemistry was performed using DIG-labelled cRNA probes to identify the cells in which Hedgehog signalling components are made {2}. Signals for mRNAs encoding t he transmembrane receptors Ptc2 and Smo are most intensely detected in spermatogonia and spermatocytes and are much less intense in the round spermatids. The mRNA for the cytoplamic regulator, Fused, is restricted to the earliest germ cell types, whereas expression of the negative cytoplasmic regulator, SuFu, only begins in the round spermatids and persists in elongating spermatids. Gli1 and Gli3 are expressed from spermatogonia through to round spermatids, whereas Gli2 is restricted to spermatogonia and spermatocytes. This pattern mimics what was reported for mouse {2}. Examination of the irradiated rat testis model revealed that Hedgehog signalling machinery is produced by resting spermatogonial stem cells but is turned off when they differentiate in response to hormones. This matches the emerging understanding of Hedgehog signals in cancer stem cells and provides the first demonstration that Hedgehog signalling may influence stem cells in the adult testis. (1) Shuttlesworth G.A. et al. 2000. Endocrinology. 141: 37–49 (2) Szczepny A. et al. 2006. Dev Dyn. 235:3063–3070.

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. A coherent hypothesis (Dev Dyn 236:2039–2049) Like oil and water, cells with differential intercellular adhesion properties do not mix. This concept is exemplified in animal pancreatic islets, which are composed of an aggregate of insulin-secreting β-cells surrounded by other endocrine cells. Until now, the presumed biophysical properties underlying this cellular arrangement had not been tested. Presented here, work from Foty and colleagues support the differential adhesion hypothesis (DAH), which predicts that β-cells have greater surface tension and, therefore, greater intercellular adhesiveness than surrounding cells. By measuring the binding energy among cells in pseudoislets assembled in vitro, they find that β-cells have greater surface tension than surrounding cells. Consistent with this result, β-cells also express higher levels of the adhesion molecule E-cadherin. Finally, when surrounding cells overexpress an exogenous adhesion molecule, P-cadherin, they “pull away” from the β-cell core. This finding shows that changing the adhesive relationship between cell types can influence their relative positions. DAH validation is an important step toward bioengineering islet cell substitutes, a potential means to control diabetes. Boning up on osteoclastogenesis (Dev Dyn 236:2181–2197) Although the skeletal system might appear to be permanent, it undergoes constant remodeling during an animal's lifetime. The dynamic process is mediated in part by osteoclasts, which remove and resorb bone. Aurora and colleagues suspected that many gene networks underlying development of these industrious cells had yet to be discovered. In their search, they used microarrays comparing gene expression in primary osteoclast precursors induced to undergo osteoclastogenesis, to mock-treated precursors. Crucial to their data analysis was generation of a coexpression network. Clusters of genes that show the same expression profile over multiple time points are thought to function together to regulate a common biological outcome. By identifying coexpressing clusters, processes never before attributed to osteoclasts were identified, including angiogenesis, reciprocal T-cell/osteoclast signaling, cholesterol regulation, and apoptosis. Potential roles for the pathways in osteoclastogenesis are discussed. Networks identified in developing osteoclasts may also predict gene interactions in other cell types. Cracking the Hox code (Dev Dyn 236:2454–2463) Although it will never achieve the notoriety of The Da Vinci code, the Hox code has been controversial in its own right. How do Hox, a cadre of genes with overlapping expression and redundant functions, pattern the vertebrate axial skeleton? In this review, Wellik presents supporting evidence that is used to build an up-to-date interpretation of how the Hox code operates in vertebrates. Of note is the observation that the same Hox genes use different mechanisms to pattern lateral plate and somitic derived axial skeletal elements. In addition, she presents evidence that debunks the “posterior prevalence model,” which states that the most posteriorly expressed Hox gene imparts relevant patterning information at a particular axial level. The data are key to her argument that coexpressed, active Hox proteins work together to pattern skeletal morphologies. In closing, Wellik discusses what additional experimental evidence must be gathered before the code can be definitively cracked. Where are Tom Hanks and Audrey Tautou when you need them?

Lung impedance measurements are/are not more useful than simpler measurements of lung function in animal models of pulmonary disease

The following letters are in response to Point:Counterpoint: “Lung impedance measurements are/are not more useful than simpler measurements of lung function in animal models of pulmonary disease” that appears in this issue. To the Editor : Mitzner argues that constant phase modeling is not more

Left Right Asymmetry, the Pulmonary Vein, and A-Fib

See related article, pages 902–909 Atrial fibrillation (AF), a common adult cardiac arrhythmia, involves abnormal atrial contractions. As every medical student is taught, it is easily diagnosed as an irregularly irregular pulse and loss of organized atrial activity on an ECG. Clinical interest in AF is considerable because AF is the most common adult arrhythmia that increases in prevalence with age, with 5% of the over 65 population having AF. Moreover, patients with AF have a significantly increased risk of stroke.1 Electrical impulses, critical for a coordinated and physiologic heartbeat, are normally initiated in the sinoatrial (SA) node. However, in AF the disorganized atrial activity overrides normal SA node function resulting in irregular conduction of impulses to the ventricles. In the majority of cases, ectopic electrical activity originates in the pulmonary veins.1 In this issue of Circulation Research , Mommersteeg and colleagues2 make a significant advance in our understanding of pulmonary vein development by making a strong connection between AF and embryonic axis determination. Moreover, their work fits nicely with exciting new data from human genetic studies implicating the same genetic pathways in familial AF.3 Mommersteeg and colleagues make a number of fundamental observations regarding pulmonary vein development. Previous work suggested that pulmonary myocardium derives from atrial myocardium.4 Using lineage tracing, the authors convincingly rule out this possibility. Moreover, they show other data revealing that pulmonary myocardium is derived from Isl1 -positive second heart field and has a distinct origin from the systemic venous circulation.5 Together, these findings suggest that the pulmonary vein myocardium forms by differentiation of mesenchyme around the pulmonary vein. To address this hypothesis, the authors use a Pitx2 mutant mouse model that had been shown to have defects in pulmonary vein development.6 The Pitx (Pituitary homeobox) family of …

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. Absolutely FABAlous (Dev Dyn 236:1790–1805) The next generation of staging tables has arrived. Studies using the basal chordate Ciona intestinalis promise to shed light on the evolutionary origins of vertebrates. However, lack of standardized embryonic staging has made interpretation of Ciona research difficult. Here, Hotta et al. present a Ciona staging table based on morphological characteristics with accompanying text that describes developmental milestones in detail. An interactive, Internet-based atlas, called Four-dimensional Ascidian Body Atlas (FABA) (http://chordate.bpni.bio.keio.ac.jp/faba/top.html), richly supplements the table. The atlas displays confocal laser scanning microscopy images of embryos stained with fluorescently labeled phalloidin, a molecule that labels cell membranes and other structures. The viewer can rotate three-dimensional images of a representative embryo from each developmental stage, and scroll through z-sections, allowing visualization of external and internal cellular arrangements. The planned integration of FABA with Ciona intestinalis Protein Database (CIPRO) will be a valuable enhancement. Now Hear This (Dev Dyn 236:1905–1917) Sometimes it is worthwhile to re-examine old data. Over a decade ago, inner ear defects in Fgfr3—/— mice were characterized by morphological analysis. Since then, molecular markers have been identified that distinguish cell types in the organ of Corti, the sensory epithelium of the inner ear. With these tools in hand, Puligilla and colleagues set out to better define the role of fibroblast growth factor receptor-3 (FGFR3) in this organ. They find that, in Fgfr3—/— cochlea, a reduction in inner pillar cell (IPC) number is accompanied by an increase in outer hair cell (OHC) and Deiters' cell numbers. However, cell death and overall proliferation are unaffected, suggesting that Fgfr3 commits precursor cells to the IPC fate. Bone morphogenetic protein-4 (BMP4), a known FGFR3 target, is up-regulated in Fgfr3—/— cochlea, and treatment of wild-type cochlear explant cultures with BMP4-soaked beads induces extra OHCs. These data support a model whereby Fgfr3 negatively regulates Bmp4, an interaction that mediates OHC fate choice. In agreement, treatment of Fgfr3—/— cochlear explants with Noggin, a BMP inhibitor, rescues the OHC phenotype. Additional Fgfr3—/— cochlea defects, including cochlear defects including aberrant innervation, and OHC and Dieter's cell differentiation are also described in careful detail. Why Ascidians? (Dev Dyn 236:1732–1747) Aside from having a good excuse to go to the beach, why use sea squirts as a model for developmental biology? This review is one of several in the Ciona Special Focus Issue that presents compelling answers to this question. The ascidian Ciona uses a small set of nonredundant genes to develop into a tadpole composed of approximately 2,500 cells. With a pared down chordate body plan, Ciona and other ascidians are accessible models useful for gaining a foothold on complex developmental events observed in other organisms. Kumano and Nishida's review highlights how these animals have furthered our understanding of cell fate specification. They begin by thoroughly covering the basics of ascidian embryology, including morphogenesis and cell lineage. Equipped with this knowledge, one is prepared for the next section, which compares and contrasts vertebrate and ascidian molecular mechanisms that govern cell fate specification. Many familiar players make appearances (β-catenin, Fgf, Notch, Fox, Gata, Ets), promising to keep the reader engaged.

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. Quieting the Noise (Dev Dyn 236:1416–1421) Without a conductor, musicians drift out of synch, their song fragmenting into a disorganized clatter of notes. Similarly, adjacent cells in the posterior presomitic mesoderm (PSM) rely on oscillators to coordinate the rhythmic cycling of segmentation gene transcription, translation, and transcriptional repression. One challenge the segmentation clock faces is that 10–15% of PSM cells are in M-phase, during which time transcription and translation are quiescent, meaning initiation of their segmentation cycles are staggered. In this review, the authors propose a model for how synchrony is achieved. PSM cells have an intracellular oscillator led by hairy, which in a direct autoinhibitory feedback loop represses its own expression, and also represses Delta expression. Delta, in turn, is an intercellular oscillator that binds the Notch receptor on adjacent cells, and activates hairy expression. In vivo and mathematical modeling experiments support their model that Delta reduces biological noise by coupling oscillator cycling in neighboring cells. Profiling Endoderm (Dev Dyn 236:1633–1649) If you research molecular mechanisms of endoderm development, then this paper is a must-see. Endoderm, which gives rise to lung, stomach, esophagus, pancreas, liver, duodenum, gallbladder, and intestine, is arguably the least studied of the three primary germ layers. In search of genes that regulate development of these organs, Han and colleagues performed microarray analysis on Xenopus endoderm at three crucial stages: the onset of regional specification, organogenesis, and differentiation of organ-specific cell types. Anterior and posterior endoderm were compared at each stage to enrich for organspecific genes. Their painstaking approach yielded 915 candidates that are differentially expressed by at least threefold. Of these, 104 were selected for in situ hybridization. A table detailing gene expression in individual organs reveals some intriguing patterns. Interested in the liver? You may want to investigate transcription factors Tbx2 and MafB. Curious about the lung? Look into signaling molecules Semaphorin6D and Frizzled-7. The information is there for the taking. Recipe for Mature Chondrocytes (Dev Dyn 236:1954–1962) Science is a lot like cooking. Mix the ingredients, incubate, and voila! Your product is ready. Presented here is a recipe for making mature chondrocytes. The transcription factor Runx2 drives the chondrocyte maturation step, chondrocyte hypertrophy, and regulates osteoblast development. Surprisingly, retroviral overexpression of Runx2 in presomitic mesoderm (PSM) explants fails to activate expression of the chondrocyte hypertrophic marker Collagen X. However, if PSM explants are first treated with Shh then BMP4, signals surrounding developing axial chondrocytes in vivo, then ectopic Runx2 can activate chondrocyte hypertrophy markers. Forced expression of Sox9 or Nkx3.2, transcription factors expressed in immature chondrocytes, can substitute for Shh. In other conditions, Runx2 overexpressing explants instead activate osteopontin, an osteoblast marker, suggesting that Runx2 only activates Collagen X when in the right cellular milieu. In agreement, overexpression of Runx2 in combination with a transcription factor that transduces bone morphogenetic protein signals, Smad1, activates a Collagen X reporter in hypertrophic chondrocytes, but not in fibroblasts or immature chondrocyte primary cultures. What is the permissive factor necessary for chondrocyte maturation? Now that contexts in which it functions are known, it will not be long until the secret ingredient is revealed.

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. Remodeling tools (Dev Dyn 236:671–683) For a good remodel, just the right tools are needed to disassemble the old to make room for the new. During cardiac remodeling, myocardium of the outflow tract (OFT) is lost and replaced with smooth muscle before becoming arterial tissue. C.B. Kern et al., set out to determine how the job is done. Here, they show that versican, an extracellular matrix chondroitin sulfate proteoglycan is a multifunctional tool in the OFT. Adenoviral-mediated expression of an uncleavable form of versican in vivo and in vitro shows that the intact protein promotes cell–cell association, stabilizing the myocardial layer. Just before remodeling, myocardial versican is proteolytically cleaved to generate a fragment, DPEAAE. Ectopic expression of a DPEAAE-like fragment augments detachment of myocardial cells from the myocardial layer, facilitating formation of the mature OFT. These results suggest that versican fortifies OFT walls, and its DPEAAE proteolytic fragment functions to break the walls down, clearing the way for their smooth muscle replacement. Addressing the Numb conundrum (Dev Dyn 236:696–705) The so-called “Numb conundrum” is based on seemingly contradictory results from mouse Numb mutants. Some observed phenotypes support the widely held belief that Numb antagonizes Notch's role in maintaining neural progenitors, while others substantiate the opposite conclusions. This study reveals that the key to solving this puzzle may lie in understanding how Numb mutants affect the four Numb isoforms. Numb1 and 3 contain an insertion in their proline-rich region and are predominantly expressed early in development, whereas numb2 and 4 are mainly expressed postnatally and into adulthood. Consistent with expression data, numb1 and 3 suppress differentiation of primary neural progenitors and p19 cells, whereas numb2 and 4 enhance differentiation. Despite differing biological roles, all isoforms inhibit Notch in vitro, illustrating that they likely also modulate other signaling pathways. Cell assassin (Dev Dyn 236:1025–1035) Looking to kill some cells? Pick your poison, perhaps diphtheria toxin? Maybe brute force is more your style—how about laser ablation? You say you want a genetic approach to temporarily remove certain cell populations for regeneration studies? Luckily, there is a new method that is just for you. First, you need transgenic fish with the Escherichia coli enzyme Nitroreductase (NTR) under a tissue specific promoter. Next, quietly slip Metronidazole (Mtz) in the fish water. In the presence of NADH or NADPH, NTR reduces Mtz, making it a DNA cross-linking agent. This cytotoxic drug effectively triggers cell death in both proliferating and quiescent cells. Wash away the Mtz, and not only is the killing spree over, but any incriminating evidence is gone.

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. A star is born (Dev Dyn 236:335–352) Botryllus schlosseri, commonly called the star ascidian, earns its name from the pretty, stellate colony formed by a group of asexually reproducing animals, or zooids. The organism has proven a useful model for morphogenesis, regeneration, allorecognition, and apoptosis. One confounding problem facing B. schlosseri biologists is lack of a common staging system. Here, Manni et al. present staging of the colonial life cycle in relation to important developmental events. Staging is supported by histology, diagrams, detailed descriptions of morphogenesis, and comparisons to other staging methods. The authors also explain the particular usefulness of B. schlosseri in studying a variety of biological phenomenon. Just as Kimmel et al. and Hamburger and Hamilton are indispensable for zebrafish and chicken staging, surely B. schlosseri biologists will come to depend on Manni et al. When good cells go bad (Dev Dyn 236:364–373) Tightly packed side-by-side, polarized epithelial cells form protective barriers lining the body's cavities, lumens, and surfaces. In their study, Szafranski and Goode use the Drosophila oocyte as a model to investigate why, in certain circumstances, the protectors lose their integrity and become invaders. They report that loss of components in basolateral junctions (BLJ), but not other epithelial junctions, induces follicular epithelial cells to invade neighboring germ cells. Events before induced invasion mirror natural events as both BLJ mutants and wild-type follicular border cells (BCs) undergo “membrane lateralization,” a loss of some junctional proteins and redistribution of others. Membrane lateralization is necessary, but not sufficient for invasion. The authors also show that overexpression of dominant-negative Rac1, a G-protein required for BC motility, suppresses migration of BLJ mutant cells. Together their data suggest that BLJ proteins work together as a supermolecular complex to prevent epithelial cell invasion by suppressing membrane lateralization and Rac motility pathways. Parallels to tumor invasion in human carcinomas are discussed. Profiling Fetal Alcohol Syndrome (Dev Dyn 236:613–631) Fetal alcohol syndrome (FAS), a tragic consequence of a mother's overindulgence, can cause craniofacial defects, mental retardation, and stunted growth. In their study, Knudsen and colleagues provide evidence that changes in gene expression within specific molecular pathways are the bases for induced phenotypes. Microarray analysis of mouse cranial neural folds shows that pathways involved in cytoskeletal reorganization, namely tight junction, adherens junction, and focal adhesion pathways, are significantly up-regulated in response to alcohol. Of interest, defects are less severe in the C57BL/6 substrain B6N than in B6J. Furthermore, PK11195, a ligand for the mitochondrial 18-kDa translocator protein (TSPO), counteracts alcohol affects in B6J, but exacerbates them in B6N. The different responses may be attributed to genetic variation. Indeed, additional alcohol-induced changes are found in B6N alone, namely downregulation of ribosomal and proteosome, and up-regulation of glycolysis and pentose phosphate pathways. These data suggest that TSPO plays a role in susceptibility to FAS.

Mosaic versus regulation development in avian blastoderms depends on the spatial distribution of Rauber's sickle material

We describe how to prepare unincubated avian eggs to obtain a greater number of clearly visible Rauber's sickles for experimental embryology. After hemi-sectioning of unincubated chicken (Gallus domesticus) blastoderms and cultivating both halves in vitro, two kinds of development can be discerned: (1) when the unincubated blastoderms were hemi-sectioned according to the plane of bilateral symmetry, going through the middle region of Rauber's sickle, we obtained two hemi-embryos (a left and a right one). Each contained a half primitive streak, localized at the cut edge (starting from the most median part of Rauber's sickle) giving rise to a half mesoblast mantle and half area vasculosa, thus indicating mosaic development (each part of the whole fertilized egg would be able to form independently on its own). (2) When the unincubated blastoderm is hemi-sectioned more obliquely, going through a more lateral part of Rauber's sickle (sickle horn), two complete bilaterally symmetrically miniature embryos will form, indicating the so-called regulation phenomena. We demonstrate that these two types of development are in reality due to the different spreading and concentration of Rauber's sickle tissue (containing gamma ooplasm) around the area centralis. Embryonic regulation thus must not be considered as a kind of totipotent regeneration capacity of isolated parts of the unincubated avian blastoderm, but depends on the spatial distribution of a kind of extraembryonic tissue (Rauber's sickle) built up by the oblique uptake of gamma ooplasm (ooplasmic mosaicism) at the moment of bilateral symmetrization (Callebaut [1994] Eur Arch Biol 105:111-123; Callebaut [2005] Dev Dyn 233:1194-1216).

Highlights in DD

“Highlights” is a feature that 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 will spotlight 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. Do the Strand (Dev Dyn 2007;236:84–92) In this Special Focus Research Article on the enteric nervous system (ENS), Druckenbrod and Epstein describe behaviors of migratory enteric neural crest-derived cells (ENCCs) as they populate the ENS. ENCCs follow a wavefront where connected chains of cells, or strands, form and extend to the gut mesenchyme. Using time-lapse imaging in chick and mice, the authors document how three cell types, distal, rostral, and anterior cells (DCs, RCs, ACs), influence overall strand dynamics. DCs, the leading cells of strands, are exploratory, extending filopodia and changing trajectories during their rostral to caudal route. RC dynamics vary with respect to distance from the wavefront; they chiefly elongate and maintain the integrity of strands. ACs play a prominent role in colonization of the cecum and proximal colon. Like RCs, they elongate strands, and also influence DC trajectories. Potential mechanisms that guide the intricate strand cell dance are discussed. Time Is of the Essence (Dev Dyn 2007;236:142–155) Most are familiar with the concept that circadian rhythms dictate when you are prone to feeling sleepy or wakeful. But did you know that the cell cycle in proliferating bone marrow is also synchronized to day–night cycles? These facts and much more are brought to light in this compelling review on circadian rhythm. The article not only covers the molecular bases and physiological outputs of circadian rhythm, but also development of circadian timing and the impact of development on the circadian clock. Is early embryonic development also under the spell of day–night rhythms? Read the review to find out. Going Gray (Dev Dyn 2007;236:282–289) It is tempting to blame newfound gray hairs on mounting stresses at work. This paper highlights another possible cause, malfunctioning Notch receptors. During hair growth, melanocytes deliver pigment to the hair matrix. As the melanocyte stem cell population is depleted, hair pigmentation decreases, producing gray hair. The authors show that mice with a conditional deletion of Notch 1 or 2 from melanocytes undergo premature graying; eye pigmentation is unaffected. Moreover, graying is dose-dependent; it is accelerated upon deletion of additional Notch1/2 alleles. The underlying defect is precocious depletion of Dct-expressing cells, which labels melanoblasts and melanocytes. This work suggests that Notch 1 and 2 have overlapping functions in regulating self-renewal of hair follicle melanocyte stem cells.

Highlights in DD

“Highlights” is a feature that 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 will spotlight 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. Macrophages and morphology (Dev Dyn 235:3222–3229). If you think that macrophages are just gluttons that gobble up cellular debris and foreign material, think again. According to Pollard's group, macrophages also shape mammary gland branching morphology. During female puberty, rudimentary ducts in mammary glands start to grow. At the leading edge of growing ducts are epithelial structures called terminal end buds (TEBs), whose surrounding stromal layer is collagen-rich. Here, the authors show that mice deficient in macrophages have abnormal TEB morphology, but its total amount of collagen I is unchanged. This finding demonstrates that macrophages regulate TEB morphology, but not by producing monomeric collagen. The fascinating observation that macrophages travel along the TEB collagen matrix in vivo agrees with their conclusion that macrophages instead promote the organization of collagen into fibrillar bundles. It takes guts (Dev Dyn 235:3259–3267). Sea cucumbers (Holothuria glaberrima) eject their intestines to distract or repel predators. Before you get any ideas, be advised that while they have the remarkable capacity to regenerate their internal organs, humans do not. In documenting intestine regeneration, this study focuses on a population of sphere-shaped, vesicle-filled cells called spherulocytes. With the help of spherulocyte-specific antibodies, the authors track the cells during the regeneration process. Found in connective tissue throughout the organism, spherulocytes increase in number, change morphology, appear to fuse with intestinal extracellular matrix, and dispel their vesicular contents. These data are highly suggestive that spherulocytes participate in regeneration and/or wound healing. Identifying the vesicular contents may be key to understanding exactly what these mysterious cells do. Sugar coated fish (Dev Dyn 235:3423–3431, 3432–3437). In back-to-back papers, Cadwallader and Yost present compelling evidence that sugar does not deserve such a bad rap. Heparin sulfate (HS) proteoglycans bind to extracellular molecules, modulating several biological processes, including cell adhesion and cell signaling. HS ligand binding specificity is influenced by O-sulfotransferases (OSTs), which position sulfates on HS sugar chains. Here, the authors detail the zebrafish developmental expression patterns of two OST families, 3- and 6-OSTs, most members of which are conserved in humans. Interestingly, they find that the enzymes show unique tissue distribution patterns, suggesting that each plays a specialized role in development.

Building the Right Ventricle

See related article, pages 1000–1007 Abnormal development of the arterial pole of the heart underlies a significant fraction of congenital heart defects. Critical steps in arterial pole development are formation of the myocardial outflow tract (or conotruncal region) and its subsequent division into separate left and right ventricular outlets. Division of the cylindrical outflow tract is a complex morphogenetic process driven by cardiac neural crest cell influx and associated with rotation of the myocardial wall and cell death, ensuring alignment of the ascending aorta and pulmonary trunk with the left and right ventricles.1–3 The transient nature of the embryonic outflow tract raises existential but also clinically relevant questions as to the origin of this structure and its fate. In an article in this issue, Rana et al have addressed the latter in the developing chick heart with important inferences for the origin of the right ventricle.4 Using scanning confocal microscopy, Rana et al monitored the rise and fall of the myocardial outflow tract. After a 4-fold increase in length to reach a maximum extension the myocardial outflow tract shortens 5-fold. At the same time a nonmyocardial component appears, giving rise to the ascending aorta and pulmonary trunk. Rana et al focused on the retraction phase by following the fate of clusters of DiI labeled cells in ovo at 2 developmental timepoints and concluded that the proximal outflow tract gives rise to a large part of the right ventricular free wall. Although the concept of ventricularisation of the proximal outflow tract (or conal absorption) to form the right ventricular outlet is not new,5–7 the extent to which myocardium initially in the outflow tract contributes to the trabeculated part of the free ventricular wall …

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. Shapely models (Dev Dyn 2006;235:2907–2919) In classic experiments, Spemann and Mangold transplanted dorsal “organizer” cells from one newt to another, creating the unforgettable two-headed embryo. This and later works led to the hypothesis that the Spemann organizer specifies the primary body axes. In this review, Meinhardt eschews convention and proposes that the Spemann organizer plays a secondary role in axis formation. His model states that the head anterior/posterior (AP) axis is specified by the blastopore, and the trunk's by oscillating expression of sequentially posteriorizing genes. The role of the Spemann organizer is to trigger formation of head and trunk midline organizers (prechordal plate and notochord), which specify the dorsal/ventral (DV) axis. Unlike other models, Meinhardt's predicts the correct AP and DV orientation of an induced second axis. This provocative model will surely shape the interpretation of results from both new and old experiments in the field. Creating boundaries (Dev Dyn 2006;235:3051–3058) Like a border fence between countries, cells with distinct fates need boundaries to keep one population from invading the other. Major and Irvine show for the first time that, in the Drosophila wing disc, Myosin II is a necessary boundary component downstream of Notch signaling. Of interest, Myosin II is required for DV, but not AP compartmentalization, showing that not all boundaries are alike. Building on their previous work, the authors propose that Myosin II creates a contractile force along an F-actin cable, making a physical barrier separating dorsal and ventral wing disc cells. Navigating the Xenopus embryo (Dev Dyn 2006;235:3059–3062). A frustration shared among Xenopus biologists is their inability to visualize individual cells in the depths of the murky, yolk-filled embryo. Finally, Jacobs' group has found a way to navigate through uncharted territory. They report that two dimensional microscopic magnetic resonance imaging (mMRI) successfully images the orientation, timing, and lineage of cell divisions. Ironically, the often-despised yolk benefits this technique. Its uneven distribution in a cell provides imaging contrast, enabling identification of animal and vegetal poles, blastocele, cell nuclei, and membranes without contrast enhancing labeling. This tool will make the already proven Xenopus model system even more powerful.

Highlights in DD

“Highlights” is a new feature that 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 will spotlight 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. May the (gravitational) force be with you (Dev Dyn 235:2686–2694). We are a product of the world in which we live. Nothing drives this point home better than the remarkable finding that gravity can affect gene expression. In their most recent work on the subject, Shimada and Moorman show that altered gravity changes expression of two lens-specific members of the zebrafish heat shock protein gene family, hsp70 and αA-crystallin. Environmental stresses like heat shock and oxidative stress can induce hsp70 expression in several cell types. Of interest, altered gravity only affects hsp70 and αA-crystallin expression in the lens during the period of lens development. This work suggests that, compared with other stressors, there is a specific biological response to altered gravity. Beauty secrets (Dev Dyn 235:2710–2721). The beauty of a leaf is due in part to the delicate network of veins within it. Venation patterns are markedly diverse between species, a consequence of varied leaf shape and size. Fujita and Mochizuki have devised a mathematical model that predicts venation patterns in different leaf types. Auxin is a plant hormone that is central to vein development, and also maintains cell polarity. PIN1, which is localized to the basal side of the cell, directionally transports auxin. The predictions of venation patterns made by the authors' model, which is based on this information, are enviably accurate. Their secret is to take into account the positive feedback regulation between auxin and PIN1. Putting it together (Dev Dyn 235:2795–2801). In the debut of the peer-reviewed section, Disease Connections, Oottamasathien et al., report on a method using tissue recombination to induce mature bladder tissue in recombination grafts. They combine mouse embryonic bladder mesenchyme with primary cultures of adult rat urothelium, the lining of the bladder, and graft them into a host mouse. Through a process that is believed to recapitulate normal bladder development, the mesenchyme induces the urothelium to differentiate as mature bladder tissue, complete with well-differentiated smooth muscle and connective tissue. When performed with genetically engineered tissue, the technique will be a powerful tool to understand the molecular mechanisms that govern bladder development and disease.

Regulation of chloride ion conductance during skeletal muscle development and in disease. Focus on “Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1”

productive investigations of disease mechanisms that also reveal new information about normal regulation elicit a particularly satisfying sense of a two-for-one deal. In the case of Lueck et al. (Ref. [13][1]; see page 1291 of this issue), a detailed study of the molecular basis for the myotonia (

Highlights inDD

Similar yet different (Dev Dyn 236:2493–2501) A mouse could never be mistaken for a chicken—unless their neural crests are being compared. Neural crest cells migrate from the neural tube to various tissues, where they differentiate into several cell types. Chick neural crest migration is under the control of bone morphogenetic proteins (BMPs). Seemingly in agreement, Bmp2 null mice lack migratory crest. But, is the mouse phenotype due to defects in migration or induction of crest fate? Correia et al. put this question to rest. Consistent with a role in migration, they show that early crest markers are induced in Bmp2 null embryos, but the cells remain associated with the neural tube. However, when they dig deeper, they find direct comparisons between mouse and chick neural crest falter. Whereas chick Cad6B is only expressed in premigratory crest, it is also in migratory crest in mouse. Additionally, unlike in chick, mouse neural crest Cad6B, Id2, and Wnt1 expression is not regulated by BMPs. The data illustrate that, although the top of the neural crest migration hierarchy is the same, downstream mechanisms differ. Not-so-“bad” cholesterol (Dev Dyn 236:2708–2712) Ever get a craving for fried food? You are not the only one. Work by Zhao and colleagues suggests that, at times, oligodendrocytes cannot get enough “bad” cholesterol either. Differentiated oligodendrocytes form a cholesterol-prich myelin sheath that enhances conduction of electrical signals along axons. One way that central nervous system glia procure cholesterol is by synthesizing their own. Here, the authors find evidence that the cells also uptake cholesterol from the surrounding environment. Mature oligodendrocytes express very low-density lipoprotein receptors (VLDLRs) and LDLRs in the spinal cord and brain, with some regional differences between the two. Highest expression of both receptors coincides with the peak of myelination in mouse spinal cords. These findings suggest that oligodendrocytes endocytose LDL and VLDL, and use them to build the myelin sheath. Apparently oligodendrocytes cannot get too much of a bad, er good, thing. Feast or famine? (Dev Dyn 236:2792–2799) In the face of an unpredictable food supply, the body's ability to adjust energy usage and storage is integral to survival. Energy homeostasis is a product of crosstalk between specific neurons in the hypothalamus and brainstem, and cues from the periphery, such as adipose tissue. Simon et al. show that, in the absence of the homeobox gene Sax2, these signals get crossed. Despite sufficient intake of food, energy stores in 2-week-old Sax2 null pups are depleted. They fail to incorporate lipids into white and brown adipose tissues, and liver glycogen stores and blood glucose levels are decreased. Consistent with a state of fasting, mRNA levels of neuropeptide Y in the hindbrain are low, whereas pro-opiomelanocortin levels are high. By contrast, serotonin mRNA levels are high, indicative of satiation. Because Sax2 is expressed in the brainstem close to serotonergic neurons, and not in affected peripheral tissues, the authors propose that the imbalance is due to dysregulation of central nervous system energy homeostasis signals by Sax2. Future studies promise to determine specifically where Sax2 fits into the energy homeostasis pathway.

Chronically Increased Transforming Growth Factor-β1 Strongly Inhibits Hippocampal Neurogenesis in Aged Mice

There is increasing evidence that hippocampal learning correlates strongly with neurogenesis in the adult brain. Increases in neurogenesis after brain injury also correlate with improved outcomes. With aging the capacity to generate new neurons decreases dramatically, both under normal conditions and after injury. How this decrease occurs is not fully understood, but we hypothesized that transforming growth factor (TGF)-beta1, a cell cycle regulator that rapidly increases after injury and with age, might play a role. We found that chronic overproduction of TGF-beta1 from astrocytes almost completely blocked the generation of new neurons in aged transgenic mice. Even young adult TGF-beta1 mice had 60% fewer immature, doublecortin-positive, hippocampal neurons than wild-type littermate controls. Bromodeoxyuridine labeling of dividing cells in 2-month-old TGF-beta1 mice confirmed this decrease in neuro-genesis and revealed a similar decrease in astrogenesis. Treatment of early neural progenitor cells with TGF-beta1 inhibited their proliferation. This strongly suggests that TGF-beta1 directly affects these cells before their differentiation into neurons and astrocytes. Together, these data show that TGF-beta1 is a potent inhibitor of hippocampal neural progenitor cell proliferation in adult mice and suggest that it plays a key role in limiting injury and age-related neurogenesis.

Is the Fetal Heart a Hematopoietic Organ?

To the Editor: The article by Tomanek et al1 presents well the in vivo data on the requirement of VEGF family members for tubulogenesis and coronary artery formation. The authors observed that VEGF-Trap (a chimera of R1 and R2), apart from precluding formation of coronary arteries, causes a massive accumulation of erythrocytes in the subepicardium and in the interventricular septum. The important finding of this study was that erythrocytes that are components of blood islands derive from epicardial-derived precursors. The authors assume that proepicardial cells include a hemangioblast population. From this observation a conclusion can be drawn that either the proepicardium or the fetal heart exhibits a hematopoietic activity. Considering the topic of blood island derivation in the embryonic heart I would like to comment that although the derivation of the endothelial cell component in these structures has been proven, the origin of erythrocytes is …