Hot off the presses! Apr 15 Dev Biol

The Apr 15 issue of the Dev Biol is now up on Pubget (About Dev Biol): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Editorial Board
  - Dev Biol 328(2):i (2009)
  
• More constraint on ParaHox than Hox gene families in early metazoan evolution
  - Dev Biol 328(2):173-187 (2009)
  Hox and ParaHox (H/P) genes belong to evolutionary-sister clusters that arose through duplication of a ProtoHOX cluster early in animal evolution. In contrast to bilaterians, cnidarians express, beside PG1, PG2 and Gsx orthologs, numerous Hox-related genes with unclear origin. We characterized from marine hydrozoans three novel Hox-related genes expressed at medusa and polyp stages, which include a Pdx/Xlox ParaHox ortholog induced 1 day later than Gsx during embryonic development. To reconstruct H/P genes' early evolution, we performed multiple systematic comparative phylogenetic analyses, which identified derived sequences that blur the phylogenetic picture, recorded dramatically different evolutionary rates between ParaHox and Hox in cnidarians and showed the unexpected grouping of [Gsx–Pdx/Xlox–PG2–PG3] families in a single metagroup distinct from PG1. We propose a novel more parsimonious evolutionary scenario whereby H/P genes originated from a [Gsx–Pdx/Xl! ox–PG2–PG3]-related ProtoHox gene, the «posterior» and «anterior» H/P genes appearing secondarily. The ProtoHOX cluster would have contained the three Gsx/PG2, Pdx/PG3, Cdx/PG9 paralogs and produced through tandem duplication the primordial HOX and ParaHOX clusters in the Cnidaria–Bilateria ancestor. The stronger constraint on cnidarian ParaHox genes suggests that the primary function of pre-bilaterian H/P genes was to drive cellular evolutionary novelties such as neurogenesis rather than axis specification.
• The role of Wnt5a in prostate gland development
  - Dev Biol 328(2):188-199 (2009)
  The Wnt genes encode a large family of secreted glycoproteins that play important roles in controlling tissue patterning, cell fate and proliferation during development. Currently, little is known regarding the role(s) of Wnt genes during prostate gland development. The present study examines the role of the noncanonical Wnt5a during prostate gland development in rat and murine models. In the rat prostate, Wnt5a mRNA is expressed by distal mesenchyme during the budding stage and localizes to periductal mesenchymal cells with an increasing proximal-to-distal gradient during branching morphogenesis. Wnt5a protein is secreted and localizes to periductal stroma, extracellular matrix and epithelial cells in the distal ducts. While Wnt5a expression is high during active morphogenesis in all prostate lobes, ventral prostate (VP) expression declines rapidly following morphogenesis while dorsal (DP) and lateral lobe (LP) expression remains high into adulthood. Steroids modulate! prostatic Wnt5a expression during early development with testosterone suppressing Wnt5a and neonatal estrogen increasing expression. In vivo and ex vivo analyses of developing mouse and rat prostates were used to assess the functional roles of Wnt5a. Wnt5a−/− murine prostates rescued by organ culture exhibit disturbances in bud position and directed outgrowth leading to large bulbous sacs in place of elongating ducts. In contrast, epithelial cell proliferation, ductal elongation and branchpoint formation are suppressed in newborn rat prostates cultured with exogenous Wnt5a protein. While renal grafts of Wnt5a−/− murine prostates revealed that Wnt5a is not essential for cyto- and functional differentiation, a role in luminal cell polarity and lumenization of the ducts was indicated. Wnt5a suppresses prostatic Shh expression while Shh stimulates Wnt5a expression in a lobe-specific manner during early development indicating that Wnt5a participates in cross-talk with o! ther members of the gene regulatory network that control prost! ate development. Although Wnt5a does not influence prostatic expression of other Wnt morphogens, it suppresses Wif-1 expression and can thus indirectly modulate Wnt signaling. In summary, the present finds demonstrate that Wnt5a is essential for normal prostate development where it regulates bud outgrowth, ductal elongation, branching, cell polarity and lumenization. These findings contribute to the growing body of knowledge on regulatory mechanisms involved in prostate gland development which are key to understanding abnormal growth processes associated with aging.
• Functional equivalence of the zinc finger transcription factors Osr1 and Osr2 in mouse development
  - Dev Biol 328(2):200-209 (2009)
  Osr1 and Osr2 are the only mammalian homologs of the Drosophila odd-skipped family developmental regulators. The Osr1 protein contains three zinc-finger motifs whereas Osr2 exists in two isoforms, containing three and five zinc-finger motifs respectively, due to alternative splicing of the transcripts. Targeted null mutations in these genes in mice resulted in distinct phenotypes, with heart and urogenital developmental defects in Osr1−/− mice and with cleft palate and open eyelids at birth in Osr2−/− mice. To investigate whether these contrasting mutant phenotypes are due to differences in their protein structure or to differential expression patterns, we generated mice in which the endogenous Osr2 coding region was replaced by either Osr1 cDNA or Osr2A cDNA encoding the five-finger isoform. The knockin alleles recapitulated endogenous Osr2 mRNA expression patterns in most tissues and completely rescued cleft palate and cranial skeletal developmental defects o! f Osr2−/− mice. Mice hemizygous or homozygous for either knockin allele exhibited open-eyelids at birth, which correlated with differences in expression patterns between the knockin allele and the endogenous Osr2 gene during eyelid development. Molecular marker analyses in Osr2−/− and Osr2Osr1ki/Osr1ki mice revealed that Osr2 controls eyelid development through regulation of the Fgf10–Fgfr2 signaling pathway and that Osr1 rescued Osr2 function in maintaining Fgf10 expression during eyelid development in Osr2Osr1ki/Osr1ki mice. These results indicate that the distinct functions of Osr1 and Osr2 during mouse development result from evolutionary divergence of their cis regulatory sequences rather than distinct biochemical activities of their protein products.
• Regulation of Dictyostelium morphogenesis by RapGAP3
  - Dev Biol 328(2):210-220 (2009)
  Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell–cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior–posterior axis of the organism is formed, by controlling cell–cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3− cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3− cells exhibit some increased EDTA/EGTA sensitive cell–cell adhesion at the late mound stage. RapGAP3 transiently and ra! pidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3− cells may result in reduced directional movement of the mutant cells to the apex of the mound.
• Chordin is required for neural but not axial development in sea urchin embryos
  - Dev Biol 328(2):221-233 (2009)
  The oral–aboral (OA) axis in the sea urchin is specified by the TGFβ family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development ar! e both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.
• The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/β-catenin asymmetry pathway
  - Dev Biol 328(2):234-244 (2009)
  Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a β-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/β-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/β-catenin, WRM-1/β-catenin, or POP-1/Tcf show defects similar to dsh-2 muta! nts, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/β-catenin asymmetry pathway and regulating anterior–posterior fate decisions required for proper morphogenesis.
• CWN-1 functions with DSH-2 to regulate C. elegans asymmetric neuroblast division in a β-catenin independent Wnt pathway
  - Dev Biol 328(2):245-256 (2009)
  In Caenorhabditis elegans, Wnt signaling regulates many asymmetric cell divisions. During embryogenesis, the C. elegans Dishevelled (Dsh) homolog, DSH-2, regulates asymmetric neuroblast division of the ABpl/rpppa blast cell. Dsh is a key intracellular component of both β-catenin dependent and β-catenin independent Wnt pathways. In C. elegans, most of the well-characterized asymmetric cell divisions regulated by Wnts are dependent on β-catenin. In the ABpl/rpppa neuroblast division, however, we determined that DSH-2 regulates cell polarity through a β-catenin independent Wnt pathway. We also established that the C. elegans Wnt homolog, cwn-1, functions to regulate asymmetric division of the ABpl/rpppa blast cell. Our results indicated that cwn-1 does not act alone in this process, and it functions with another redundant ligand that appears not to be a Wnt. Finally, we show widespread requirements for DSH-2 during embryogenesis in the generation of many other neurons! . In particular, DSH-2 function is necessary for the correct production of the embryonic ventral cord motor neurons. This study demonstrates a role for DSH-2 and Wnt signaling in neuronal specification during C. elegans embryogenesis.
• Bmi-1 over-expression in neural stem/progenitor cells increases proliferation and neurogenesis in culture but has little effect on these functions in vivo
  - Dev Biol 328(2):257-272 (2009)
  The polycomb gene Bmi-1 is required for the self-renewal of stem cells from diverse tissues, including the central nervous system (CNS). Bmi-1 expression is elevated in most human gliomas, irrespective of grade, raising the question of whether Bmi-1 over-expression is sufficient to promote self-renewal or tumorigenesis by CNS stem/progenitor cells. To test this we generated Nestin-Bmi-1-GFP transgenic mice. Analysis of two independent lines with expression in the fetal and adult CNS demonstrated that transgenic neural stem cells formed larger colonies, more self-renewing divisions, and more neurons in culture. However, in vivo, Bmi-1 over-expression had little effect on CNS stem cell frequency, subventricular zone proliferation, olfactory bulb neurogenesis, or neurogenesis/gliogenesis during development. Bmi-1 transgenic mice were born with enlarged lateral ventricles and a minority developed idiopathic hydrocephalus as adults, but none of the transgenic mice formed de! tectable CNS tumors, even when aged. The more pronounced effects of Bmi-1 over-expression in culture were largely attributable to the attenuated induction of p16Ink4a and p19Arf in culture, proteins that are generally not expressed by neural stem/progenitor cells in young mice in vivo. Bmi-1 over-expression therefore has more pronounced effects in culture and does not appear to be sufficient to induce tumorigenesis in vivo.
• Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology
  - Dev Biol 328(2):273-284 (2009)
  Activating mutations of FGFRs1–3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast ! maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form.
• Catweasel mice: A novel role for Six1 in sensory patch development and a model for branchio-oto-renal syndrome
  - Dev Biol 328(2):285-296 (2009)
  Large-scale mouse mutagenesis initiatives have provided new mouse mutants that are useful models of human deafness and vestibular dysfunction. Catweasel is a novel N-ethyl-N-nitrosourea (ENU)-induced mutation. Heterozygous catweasel mutant mice exhibit mild headtossing associated with a posterior crista defect. We mapped the catweasel mutation to a critical region of 13 Mb on chromosome 12 containing the Six1, -4 and -6 genes. We identified a basepair substitution in exon 1 of the Six1 gene that changes a conserved glutamic acid (E) at position 121 to a glycine (G) in the Six1 homeodomain. Cwe/Cwe animals lack Preyer and righting reflexes, display severe headshaking and have severely truncated cochlea and semicircular canals. Cwe/Cwe animals had very few hair cells in the utricle, but their ampullae and cochlea were devoid of any hair cells. Bmp4, Jag1 and Sox2 expression were largely absent at early stages of sensory development and NeuroD expression was reduced in th! e developing vestibulo-acoustic ganglion. Lastly we show that Six1 genetically interacts with Jag1. We propose that the catweasel phenotype is due to a hypomorphic mutation in Six1 and that catweasel mice are a suitable model for branchio-oto-renal syndrome. In addition Six1 has a pivotal role in early sensory patch development and may act in the same genetic pathway as Jag1.
• The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis
  - Dev Biol 328(2):297-304 (2009)
  Tubulogenesis and lumen formation are critical to the development of most organs. We study Caenorhabditis elegans vulval and uterine development to probe the complex mechanisms that mediate these events. Development of the vulva and the ventral uterus is coordinated by the inductive cell-signaling activity of a gonadal cell called the anchor cell (AC). We demonstrate that in addition to its function in specifying fate, the AC directly promotes dorsal vulval tubulogenesis. Two types of mutants with defective anchor cell behavior reveal that anchor cell invasion of the vulva is important for forming the toroidal shape of the dorsal vulval cell, vulF. In fos-1 mutants, where the AC cannot breakdown the basement membranes between the gonad and the vulva, and in mutants in unc-6 netrin or its receptor unc-40, which cause AC migration defects, the AC fails to invade the vulva and no lumen is formed in vulF. By examining GFP markers of dorsal vulval cell fate, we demonstrate ! that fate specification defects do not account for the aberrant vulF shape. We propose that the presence of the AC in the center of the developing vulF toroid is required for dorsal vulval lumen formation to complete vulval tubulogenesis.
• Adult stem cell plasticity: Neoblast repopulation in non-lethally irradiated planarians
  - Dev Biol 328(2):305-314 (2009)
  Planarians are a model system for studying adult stem cells, as they possess the neoblasts, a population of pluripotent adult stem cells able to give rise to both somatic and germ cells. Although over the last years several efforts have been made to shed light on neoblast biology, only recent evidence indicate that this population of cells is heterogeneous. In this study we irradiated planarians with different non-lethal X-ray doses (1–5 Gy) and we identified subpopulations of neoblasts with diverse levels of tolerance to X-rays. We demonstrated that a dramatic reduction of neoblasts occurred soon after non-lethal irradiations and that de-novo proliferation of some radioresistant cells re-established the primary neoblast number. In particular, a strong proliferation activity occurred at the ventral side of irradiated animals close to the nervous system. The produced cells migrated towards the dorsal parenchyma and, together with some dorsal radioresistant cells, reco! nstituted the entire neoblast population demonstrating the extreme plasticity of this adult stem cell system.
• Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse
  - Dev Biol 328(2):315-327 (2009)
  The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated b! y previous RARE reporter mice and our direct biochemical measurements and thus, RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.
• Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice
  - Dev Biol 328(2):328-341 (2009)
  Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morpho! histogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.
• Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent
  - Dev Biol 328(2):342-354 (2009)
  Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregu! lating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell–cell adhesion for cell migration in vivo.
• Hedgehog does not guide migrating Drosophila germ cells
  - Dev Biol 328(2):355-362 (2009)
  In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and! consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.
• Phospholipase D1 is required for angiogenesis of intersegmental blood vessels in zebrafish
  - Dev Biol 328(2):363-376 (2009)
  Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline. Studies in cultured cells and Drosophila melanogaster have implicated PLD in the regulation of many cellular functions, including intracellular vesicle trafficking, cell proliferation and differentiation. However, the function of PLD in vertebrate development has not been explored. Here we report cloning and characterization of a zebrafish PLD1 (pld1) homolog. Like mammalian PLDs, zebrafish Pld1 contains two conservative HKD motifs. Maternally contributed pld1 transcripts are uniformly distributed in early embryo. Localized expression of pld1 is observed in the notochord during early segmentation, in the somites during later segmentation and in the liver at the larval stages. Studies in intact and cell-free preparations demonstrate evolutionary conservation of regulation. Inhibition of Pld1 expression using antisense morpholino oligonucleotides (MO) interfering with the tran! slation or splicing of pld1 impaired intersegmental vessel (ISV) development. Incubating embryos with 1-butanol, which diverts production of phosphatidic acid to a phosphatidylalcohol, caused similar ISV defects. To determine where Pld1 is required for ISV development we performed transplantation experiments. Analyses of the mosaic Pld1 deficient embryos showed partial suppression of ISV defects in the segments containing transplanted wild-type notochord cells but not in the ones containing wild-type somitic cells. These results provide the first evidence that function of Pld1 in the developing notochord is essential for vascular development in vertebrates.
• BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1Ter/Ter mice
  - Dev Biol 328(2):377-383 (2009)
  A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1Ter/Ter XX germ cells developed in! a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.
• Remodeling of insulin producing β-cells during Xenopus laevis metamorphosis
  - Dev Biol 328(2):384-391 (2009)
  Insulin-producing β-cells are present as single cells or in small clusters distributed throughout the pancreas of the Xenopus laevis tadpole. During metamorphic climax when the exocrine pancreas dedifferentiates to progenitor cells, the β-cells undergo two changes. Insulin mRNA is down regulated at the beginning of metamorphic climax (NF62) and reexpressed again near the end of climax. Secondly, the β-cells aggregate to form islets. During climax the increase in insulin cluster size is not caused by cell proliferation or by acinar-to-β-cell transdifferentiation, but rather is due to the aggregation of pre-existing β-cells. The total number of β-cells does not change during the 8 days of climax. Thyroid hormone (TH) induction of premetamorphic tadpoles causes an increase in islet size while prolonged treatment of tadpoles with the goitrogen methimazole inhibits this increase. Expression of a dominant negative form of the thyroid hormone receptor (TRDN) driven by t! he elastase promoter not only protects the exocrine pancreas of a transgenic tadpole from TH-induced dedifferentiation but also prevents aggregation of β-cells at climax. These transgenic tadpoles do however undergo normal loss and resynthesis of insulin mRNA at the same stage as controls. In contrast transgenic tadpoles with the same TRDN transgene driven by an insulin promoter do not undergo down regulation of insulin mRNA, but do aggregate β-cells to form islets like controls. These results demonstrate that TH controls the remodeling of β-cells through cell–cell interaction with dedifferentiating acinar cells and a cell autonomous program that temporarily shuts off the insulin gene.
• The Xenopus MEF2 gene family: Evidence of a role for XMEF2C in larval tendon development
  - Dev Biol 328(2):392-402 (2009)
  MEF2 transcription factors are well-established regulators of muscle development. In this report, we describe the cloning of multiple splicing isoforms of the XMEF2A and XMEF2C encoding genes, differentially expressed during Xenopus development. Using whole-mount in situ hybridization, we found that the accumulation of XMEF2C mRNA in the tadpole stages was restricted to intersomitic regions and to the peripheral edges of hypaxial and cranial muscle masses in contrast to XMEF2A and XMEF2D, characterized by a continuous muscle cell expression. The XMEF2C positive cells express the bHLH transcription factor, Xscleraxis, known as a specific marker for tendons. Gain of function experiments revealed that the use of a hormone-inducible XMEF2C construct is able to induce Xscleraxis expression. Furthermore, XMEF2C specifically cooperates with Xscleraxis to induce tenascin C and betaig-h3, two genes preferentially expressed in Xenopus larval tendons. These findings 1) highlight ! a previously unappreciated and specific role for XMEF2C in tendon development and 2) identify a novel gene transactivation pathway where MEF2C cooperates with the bHLH protein, Xscleraxis, to activate specific gene expression.
• Abd-B suppresses lepidopteran proleg development in posterior abdomen
  - Dev Biol 328(2):403-409 (2009)
  Pterygotes lack abdominal appendages except for pleuropods and prolegs. The larvae of some holometabolous insects develop prolegs, which are used for locomotion. We analyzed the role of the homeotic genes abd-A and Abd-B in lepidopteran proleg development using mutant analysis and embryonic RNAi in the silkworm Bombyx mori. The EMu mutant developed extra prolegs in its posterior abdomen and showed the misexpression of both genes, suggesting their involvement in proleg formation. The depletion of Abd-B by embryonic RNAi caused the development of extra prolegs on all segments posterior to A6, indicating the suppressive function of Abd-B. The abd-A RNAi animals failed to develop prolegs. These results indicate that abd-A and Abd-B are involved in proleg development in B. mori.
• Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal
  - Dev Biol 328(2):410-421 (2009)
  Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These res! ults suggest that Spoltud-1 is required for long term stem cell self renewal.
• Origins and control of the differentiation of inhibitory interneurons and glia in the cerebellum
  - Dev Biol 328(2):422-433 (2009)
  Cerebellar GABAergic interneurons and glia originate from progenitors that delaminate from the ventricular neuroepithelium and proliferate in the prospective white matter. Even though this population of progenitor cells is multipotent as a whole, clonal analysis indicates that different lineages are already separated during postnatal development and little is known about the mechanisms that regulate the specification and differentiation of these cerebellar types at earlier stages. Here, we investigate the role of Ascl1 in the development of inhibitory interneurons and glial cells in the cerebellum. This gene is expressed by maturing oligodendrocytes and GABAergic interneurons and is required for the production of appropriate quantities of these cells, which are severely reduced in Ascl1−/− mouse cerebella. Nevertheless, the two lineages are not related and the majority of oligodendrocytes populating the developing cerebellum actually derive from extracerebellar sou! rces. Targeted electroporation of Ascl1-expression vectors to ventricular neuroepithelium progenitors enhances the production of interneurons and completely suppresses astrocytic differentiation, whereas loss of Ascl1 function has opposite effects on both cell types. Our results indicate that Ascl1 directs ventricular neuroepithelium progenitors towards inhibitory interneuron fate and restricts their ability to differentiate along the astroglial lineage.
• Bicaudal C and trailer hitch have similar roles in gurken mRNA localization and cytoskeletal organization
  - Dev Biol 328(2):434-444 (2009)
  Bicaudal C and trailer hitch are both required for dorsoventral patterning of the Drosophila oocyte. Each mutant produces ventralized eggs, a phenotype typically associated with failure of the oocyte to provide a dorsalization signal – the Gurken protein – to the follicle cells. Bicaudal C and trailer hitch are both implicated in post-transcriptional gene regulation. Bicaudal C acts in recruiting a deadenylase to specific mRNAs, leading to translational repression. The role of trailer hitch is less well defined, but mutants have defects in protein secretion, and show aberrant distribution of an endoplasmic reticulum exit site marker whose mRNA is associated with Trailer hitch protein. We show that Bicaudal C and trailer hitch interact genetically. Mutants of these two genes have shared defects in localization of gurken and other anteriorly-localized mRNAs, as well as altered microtubule organization which may underlie the mRNA localization defects. Bicaudal C and t! railer hitch mutants also share a syndrome of actin-related abnormalities, including the formation of ectopic actin cages near the anterior of the oocyte. The cages sequester Gurken protein, blocking its secretion and thus interfering with signaling of the follicle cells to specify dorsal fate.
• Temporal and spatial windows delimit activation of the outer ring of wingless in the Drosophila wing
  - Dev Biol 328(2):445-455 (2009)
  Extracellular signalling molecules play many roles in the development of higher organisms. They are used reiteratively in different tissues and stages, but the response of the receiving cells is controlled in a context dependent manner. The pattern of expression of the signalling molecule Wingless/WNT in Drosophila is extraordinarily complex. We have studied the mechanism that controls its expression and function in the outer ring of the Drosophila wing hinge. Our findings indicate that wingless expression is controlled by a dual mechanism: its initial activation requires the product of zinc finger homeodomain 2 and is subsequently repressed by the product of the gene complex elbow/no ocelli. This tight regulation restricts the activation of wingless temporally and spatially. Later in development, wingless expression is maintained by an autoregulatory loop that involves the product of homothorax. We have analyzed the phenotype of a wingless allelic combination that spe! cifically removes the outer ring, and our results show that Wingless is required to promote local proliferation of the wing base cells. Thus, cell proliferation in the proximal–distal axis is controlled by the sequential activation of wingless in the inner ring and the outer ring at different stages of development.
• Regulation of mouse embryonic stem cell neural differentiation by retinoic acid
  - Dev Biol 328(2):456-471 (2009)
  Pluripotent mouse embryonic stem cells (ESCs) derived from the early blastocyst can differentiate in vitro into a variety of somatic cell types including lineages from all three embryonic germ layers. Protocols for ES cell neural differentiation typically involve induction by retinoic acid (RA), or by exposure to growth factors or medium conditioned by other cell types. A serum-free differentiation (SFD) medium completely lacking exogenous retinoids was devised that allows for efficient conversion of aggregated mouse ESCs into neural precursors and immature neurons. Neural cells produced in this medium express neuronal ion channels, establish polarity, and form functional excitatory and inhibitory synapses. Brief exposure to RA during the period of cell aggregation speeds neuronal maturation and suppresses cell proliferation. Differentiation without RA yields neurons and neural progenitors with apparent telencephalic identity, whereas cells differentiated with exposure! to RA express markers of hindbrain and spinal cord. Transcriptional profiling indicates a substantial representation of transit amplifying neuroblasts in SFD cultures not exposed to RA.
• Differential requirement for BMP signaling in atrial and ventricular lineages establishes cardiac chamber proportionality
  - Dev Biol 328(2):472-482 (2009)
  The function of an organ relies upon the proper relative proportions of its individual operational components. For example, effective embryonic circulation requires the appropriate relative sizes of each of the distinct pumps created by the atrial and ventricular cardiac chambers. Although the differences between atrial and ventricular cardiomyocytes are well established, little is known about the mechanisms regulating production of proportional numbers of each cell type. We find that mutation of the zebrafish type I BMP receptor gene alk8 causes reduction of atrial size without affecting the ventricle. Loss of atrial tissue is evident in the lateral mesoderm prior to heart tube formation and results from the inhibition of BMP signaling during cardiac progenitor specification stages. Comparison of the effects of decreased and increased BMP signaling further demonstrates that atrial cardiomyocyte production correlates with levels of BMP signaling while ventricular cardi! omyocyte production is less susceptible to manipulation of BMP signaling. Additionally, mosaic analysis provides evidence for a cell-autonomous requirement for BMP signaling during cardiomyocyte formation and chamber fate assignment. Together, our studies uncover a new role for BMP signaling in the regulation of chamber size, supporting a model in which differential reception of cardiac inductive signals establishes chamber proportion.
• Non-autonomous modulation of heart rhythm, contractility and morphology in adult fruit flies
  - Dev Biol 328(2):483-492 (2009)
  The outermost layer of the vertebrate heart originates from migratory mesothelial cells (epicardium) that give rise to coronary vascular smooth muscles and fibroblasts. The role of the epicardium in myocardial morphogenesis and establishment of normal heart function is still largely unknown. Here, we use Drosophila to investigate non-autonomous influences of epicardial-like tissue surrounding the heart tube on the structural and functional integrity of the myocardium. It has previously been shown that during Drosophila heart formation, mesodermal expression of the homeobox transcription factor even-skipped (eve) is required for specification of a subset of non-myocardial progenitors in the precardiac mesoderm. These progenitors may share some similarities with the vertebrate epicardium. To investigate a non-autonomous epicardial-like influence on myocardial physiology, we studied the consequences of reduced mesodermal Eve expression and epi/pericardial cell numbers on ! the maturation of the myocardial heart tube, its contractility, and acquisition of a normal heart rhythm in the Drosophila model. Targeting the eve repressor ladybird early (lbe) with the minimal eve mesodermal enhancer efficiently eliminates the mesodermal Eve lineages. These flies exhibit defects in heart structure, including a reduction in systolic and diastolic diameter (akin to 'restrictive cardiomyopathy'). They also exhibit an elevated incidence of arrhythmias and intermittent asystoles, as well as compromised performance under stress. These abnormalities are restored by eve reexpression or by lbe-RNAi co-overexpression. The data suggest that adult heart function in Drosophila is likely to be modulated non-autonomously, possibly by paracrine influences from neighboring cells, such as the epi/pericardium. Thus, Drosophila may serve as a model for finding genetic effectors of epicardial–myocardial interactions relevant to higher organisms.
• Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors
  - Dev Biol 328(2):493-505 (2009)
  TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1−/− mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1−/− mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, w! as either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.
• A SHH-independent regulation of Gli3 is a significant determinant of anteroposterior patterning of the limb bud
  - Dev Biol 328(2):506-516 (2009)
  The family of GLI proteins (GLI1–3) comprises the intracellular mediators of the hedgehog pathway, which regulates a myriad of developmental processes, one of which is limb development. Whereas GLI1 and GLI2 seem to be dispensable during limb development, GLI3 is especially crucial since all GLI3-associated human congenital diseases comprise limb malformations. Furthermore, Gli3−/− mouse embryos exhibit pronounced polydactyly in conjunction with a loss of digit identities. Here we examined how the quantity of GLI3 contributes to its function by using different Gli3 mutants in order to vary overall GLI3 levels. In addition, we made use of the Gli3Δ699 allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3Δ699 mutant made it feasible to analyze isoform-specific contributions of GLI3 within the context of anteroposterior patterning of the limb bud. We revealed a so far unappreciated variation in the quantitative demand for GLI3 within different phases and aspects of distal limb formation. In addition, our analyses provide evidence that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud. Instead, digit identities are most likely defined by GLI3 repressor activity alone. Furthermore, we present evidence that the anteroposterior grading of GLI3 activity by the action of SHH is supported by a prototype patterning, which regulates Gli3 indepe! ndently from SHH.
• Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization
  - Dev Biol 328(2):518-528 (2009)
  Insulators or chromatin boundary elements are defined by their ability to block transcriptional activation by an enhancer and to prevent the spread of active or silenced chromatin. Recent studies have increasingly suggested that insulator proteins play a role in large-scale genome organization. To better understand insulator function on the global scale, we conducted a genome-wide analysis of the binding sites for the insulator protein CTCF in Drosophila by Chromatin Immunoprecipitation (ChIP) followed by a tiling-array analysis. The analysis revealed CTCF binding to many known domain boundaries within the Abd-B gene of the BX-C including previously characterized Fab-8 and MCP insulators, and the Fab-6 region. Based on this finding, we characterized the Fab-6 insulator element. In genome-wide analysis, we found that dCTCF-binding sites are often situated between closely positioned gene promoters, consistent with the role of CTCF as an insulator protein. Importantly, CT! CF tends to bind gene promoters just upstream of transcription start sites, in contrast to the predicted binding sites of the insulator protein Su(Hw). These findings suggest that CTCF plays more active roles in regulating gene activity and it functions differently from other insulator proteins in organizing the Drosophila genome.
• Ascl1 and Neurog2 form novel complexes and regulate Delta-like3 (Dll3) expression in the neural tube
  - Dev Biol 328(2):529-540 (2009)
  Delta-like 3 (Dll3) is a Delta family member expressed broadly in the developing nervous system as neural progenitor cells initiate differentiation. A proximal promoter sequence for Dll3 is conserved across multiple species and is sufficient to direct GFP expression in a Dll3-like pattern in the neural tube of transgenic mice. This promoter contains multiple E-boxes, the consensus binding site for bHLH factors. Dll3 expression and the activity of the Dll3-promoter in the dorsal neural tube depends on the basic helix–loop–helix (bHLH) transcription factors Ascl1 (Mash1) and Neurog2 (Ngn2). Mutations in each E-box identified in the Dll3-promoter allowed distinct enhancer or repressor properties to be assigned to each site individually or in combination. In addition, each E-box has distinct characteristics relative to binding of bHLH factors Ascl1, Neurog1, and Neurog2. Surprisingly, novel Ascl1 containing DNA binding complexes are identified that interact with specif! ic E-box sites within the Dll3-promoter in vitro. These complexes include Ascl1/Ascl1 homodimers and Ascl1/Neurog2 heterodimers, complexes that in some cases require additional undefined factors for efficient DNA binding. Thus, a complex interplay of E-box binding proteins spatially and temporally regulate Dll3 levels during neural tube development.
• EcR-B1 and Usp nuclear hormone receptors regulate expression of the VM32E eggshell gene during Drosophila oogenesis
  - Dev Biol 328(2):541-551 (2009)
  Ecdysone signaling plays key roles in Drosophila oogenesis, as its activity is required at multiple steps during egg chamber maturation. Recently, its involvement has been reported on eggshell production by controlling chorion gene transcription and amplification. Here, we present evidence that ecdysone signaling also controls the expression of the eggshell gene VM32E, whose product is a component of vitelline membrane and endochorion layers. Specifically blocking the function of the different Ecdysone receptor (EcR) isoforms we demonstrate that EcR-B1 is responsible for ecdysone-mediated VM32E transcriptional regulation. Moreover, we show that the EcR partner Ultraspiracle (Usp) is also necessary for VM32E expression. By analyzing the activity of specific VM32E regulatory regions in usp2 clones we identify the promoter region mediating ecdysone-dependent VM32E expression. By in vitro binding assay and site-directed mutagenesis we demonstrate that this region contains ! a Usp binding site necessary for VM32E regulation. Our results further support the crucial role of ecdysone signaling in controlling transcription of eggshell structural genes and suggest that the heterodimeric complex EcR-B1/Usp mediates the ecdysone-dependent VM32E transcriptional activation in the main body follicle cells.
• Spatio-temporal intersection of Lhx3 and Tbx6 defines the cardiac field through synergistic activation of Mesp
  - Dev Biol 328(2):552-560 (2009)
  Mesp encodes a bHLH transcription factor required for specification of the cardiac mesoderm in Ciona embryos. The activities of Macho-1 and β-catenin, two essential maternal determinants, are required for Mesp expression in the B7.5 blastomeres, which constitute the heart field. The T-box transcription factor Tbx6 functions downstream of Macho-1 as a direct activator of Mesp expression. However, Tbx6 cannot account for the restricted expression of Mesp in the B7.5 lineage since it is expressed throughout the presumptive tail muscles. Here we present evidence that the LIM-homeobox gene Lhx3, a direct target of β-catenin, is essential for localized Mesp expression. Lhx3 is expressed throughout the presumptive endoderm and B7.5 blastomeres. Thus, the B7.5 blastomeres are the only cells to express sustained levels of the Tbx6 and Lhx3 activators. Like mammalian Lhx3 genes, Ci-Lhx3 encodes two isoforms with distinct N-terminal peptides. The Lhx3a isoform appears to be exp! ressed both maternally and zygotically, while the Lhx3b isoform is exclusively zygotic. Misexpression of Lhx3b is sufficient to induce ectopic Mesp activation in cells expressing Tbx6b. Injection of antisense morpholino oligonucleotides showed that the Lhx3b isoform is required for endogenous Mesp expression. Mutations in the Lhx3 half-site of Tbx6/Lhx3 composite elements strongly reduced the activity of a minimal Mesp enhancer. We discuss the delineation of the heart field by the synergistic action of muscle and gut determinants.
• The evolutionarily conserved leprecan gene: Its regulation by Brachyury and its role in the developing Ciona notochord
  - Dev Biol 328(2):561-574 (2009)
  In Ciona intestinalis, leprecan was identified as a target of the notochord-specific transcription factor Ciona Brachyury (Ci-Bra) (Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R.W., Levine, M., Satoh, N., 1999. Brachyury downstream notochord differentiation in the ascidian embryo. Genes Dev. 13, 1519–1523). By screening not, vert, similar 14 kb of the Ci-leprecan locus for cis-regulatory activity, we have identified a 581-bp minimal notochord-specific cis-regulatory module (CRM) whose activity depends upon T-box binding sites located at the 3′-end of its sequence. These sites are specifically bound in vitro by a GST-Ci-Bra fusion protein, and mutations that abolish binding in vitro result in loss or decrease of regulatory activity in vivo. Serial deletions of the 581-bp notochord CRM revealed that this sequence is also able to direct expression in muscle cells through the same T-box sites that are utilized by Ci-Bra in the notochord, which are al! so bound in vitro by the muscle-specific T-box activators Ci-Tbx6b and Ci-Tbx6c. Additionally, we created plasmids aimed to interfere with the function of Ci-leprecan and categorized the resulting phenotypes, which consist of variable dislocations of notochord cells along the anterior–posterior axis. Together, these observations provide mechanistic insights generally applicable to T-box transcription factors and their target sequences, as well as a first set of clues on the function of Leprecan in early chordate development.