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1.  Recruitment of 5′ Hoxa genes in the allantois is essential for proper extra-embryonic function in placental mammals 
Development (Cambridge, England)  2012;139(4):731-739.
The Hox gene family is well known for its functions in establishing morphological diversity along the anterior-posterior axis of developing embryos. In mammals, one of these genes, Hoxa13, is crucial for embryonic survival, as its function is required for the proper expansion of the fetal vasculature in the placenta. Thus, it appears that the developmental strategy specific to placental mammals is linked, at least in part, to the recruitment of Hoxa13 function in developing extra-embryonic tissues. Yet, the mechanism underlying this extra-embryonic recruitment is unknown. Here, we provide evidence that this functional novelty is not exclusive to Hoxa13 but is shared with its neighboring Hoxa11 and Hoxa10 genes. We show that the extra-embryonic function of these three Hoxa genes stems from their specific expression in the allantois, an extra-embryonic hallmark of amniote vertebrates. Interestingly, Hoxa10–13 expression in the allantois is conserved in chick embryos, which are non-placental amniotes, suggesting that the extra-embryonic recruitment of Hoxa10, Hoxa11 and Hoxa13 most likely arose in amniotes, i.e. prior to the emergence of placental mammals. Finally, using a series of targeted recombination and transgenic assays, we provide evidence that the regulatory mechanism underlying Hoxa expression in the allantois is extremely complex and relies on several cis-regulatory sequences.
PMCID: PMC4508127  PMID: 22219351 CAMSID: cams4757
Hox genes; Endothelial cells; Enhancer; Placenta; Transcriptional regulation; Vascular progenitors; Mouse; Chick
2.  A secreted factor represses cell proliferation in Dictyostelium 
Development (Cambridge, England)  2005;132(20):4553-4562.
Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that inhibits the proliferation of wild-type and aprA− cells; this activity is not secreted by aprA− cells. AprA purified by immunoprecipitation also slows the proliferation of wild-type and aprA− cells. Compared with wild type, there is a higher percentage of multinucleate cells in the aprA− population, and when starved, aprA− cells form abnormal structures that contain fewer spores. AprA may thus decrease the number of multinucleate cells and increase spore production. Together, the data suggest that AprA functions as part of a Dictyostelium chalone.
PMCID: PMC4484793  PMID: 16176950
Chalone; Proliferation; Growth; Size regulation
3.  Sall genes regulate region-specific morphogenesis in the mouse limb by modulating Hox activities 
Development (Cambridge, England)  2009;136(4):585-594.
The genetic mechanisms that regulate the complex morphogenesis of generating cartilage elements in correct positions with precise shapes during organogenesis, fundamental issues in developmental biology, are still not well understood. By focusing on the developing mouse limb, we confirm the importance of transcription factors encoded by the Sall gene family in proper limb morphogenesis, and further show that they have overlapping activities in regulating regional morphogenesis in the autopod. Sall1/Sall3 double null mutants exhibit a loss of digit1 as well as a loss or fusion of digit2 and digit3, metacarpals and carpals in the autopod. We show that Sall activity affects different pathways, including the Shh signaling pathway, as well as the Hox network. Shh signaling in the mesenchyme is partially impaired in the Sall mutant limbs. Additionally, our data suggest an antagonism between Sall1-Sall3 and Hoxa13-Hoxd13. We demonstrate that expression of Epha3 and Epha4 is downregulated in the Sall1/Sall3 double null mutants, and, conversely, is upregulated in Hoxa13 and Hoxd13 mutants. Moreover, the expression of Sall1 and Sall3 is upregulated in Hoxa13 and Hoxd13 mutants. Furthermore, by using DNA-binding assays, we show that Sall and Hox compete for a target sequence in the Epha4 upstream region. In conjunction with the Shh pathway, the antagonistic interaction between Hoxa13-Hoxd13 and Sall1-Sall3 in the developing limb may contribute to the fine-tuning of local Hox activity that leads to proper morphogenesis of each cartilage element of the vertebrate autopod.
PMCID: PMC3266482  PMID: 19168674 CAMSID: cams4756
Sall; Townes-Brocks syndrome; Hox; Limb development; Shh; Eph; Mouse
4.  Rostral and caudal pharyngeal arches share a common neural crest ground pattern 
Development (Cambridge, England)  2009;136(4):637-645.
In vertebrates, face and throat structures, such as jaw, hyoid and thyroid cartilages develop from a rostrocaudal metameric series of pharyngeal arches, colonized by cranial neural crest cells (NCCs). Colinear Hox gene expression patterns underlie arch specific morphologies, with the exception of the first (mandibular) arch, which is devoid of any Hox gene activity. We have previously shown that the first and second (hyoid) arches share a common, Hox-free, patterning program. However, whether or not more posterior pharyngeal arch neural crest derivatives are also patterned on the top of the same ground-state remained an unanswered question. Here, we show that the simultaneous inactivation of all Hoxa cluster genes in NCCs leads to multiple jaw and first arch-like structures, partially replacing second, third and fourth arch derivatives, suggesting that rostral and caudal arches share the same mandibular arch-like ground patterning program. The additional inactivation of the Hoxd cluster did not significantly enhance such a homeotic phenotype, thus indicating a preponderant role of Hoxa genes in patterning skeletogenic NCCs. Moreover, we found that Hoxa2 and Hoxa3 act synergistically to pattern third and fourth arch derivatives. These results provide insights into how facial and throat structures are assembled during development, and have implications for the evolution of the pharyngeal region of the vertebrate head.
PMCID: PMC4482666  PMID: 19168678 CAMSID: cams4754
Hox genes; Head morphogenesis; Hyoid bone; Jaw development; Mouse; Neural crest cells
5.  Kruppel-like factor 5 is required for perinatal lung morphogenesis and function 
Development (Cambridge, England)  2008;135(15):2563-2572.
The transition to air breathing after birth requires both anatomic and biochemical maturation of the lung. Lung morphogenesis is mediated by complex paracrine interactions between respiratory epithelial cells and mesenchymal cells that direct transcriptional programs guiding patterning and cytodifferentiation of the lung. In the present study, transgenic mice were generated in which the Kruppel-like factor 5 gene (Klf5) was conditionally deleted in respiratory epithelial cells in the fetal lung. Lack of KLF5 inhibited maturation of the lung during the saccular stage of development. Klf5Δ/Δ mice died of respiratory distress immediately after birth. Abnormalities in lung maturation and morphogenesis were observed in the respiratory epithelium, the bronchiolar smooth muscle, and the pulmonary vasculature. Respiratory epithelial cells of both the conducting and peripheral airways were immature. Surfactant phospholipids were decreased and lamellar bodies, the storage form of surfactant, were rarely found. mRNA microarray analysis demonstrated that KLF5 influenced the expression of genes regulating surfactant lipid and protein homeostasis, vasculogenesis, including Vegfa, and smooth muscle cell differentiation. KLF5 regulates genes controlling paracrine interactions during lung morphogenesis, as well as those regulating the maturation of the respiratory epithelium that is required for lung function after birth.
PMCID: PMC4459582  PMID: 18599506
Pulmonary; Transcription factor; Vasculogenesis; Paracrine signaling; VEGF; Mouse
6.  Lrp6 is required for convergent extension during Xenopus gastrulation 
Development (Cambridge, England)  2007;134(22):4095-4106.
Wnt signaling regulates β-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the β-catenin arm. We show that Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. We show that Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells and that Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, we identify the region of the Lrp6 protein with Wnt/PCP activity to a stretch of 36 amino acids, distinct from regions required for Wnt/β-catenin signaling. We propose a model in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/β-catenin signaling.
PMCID: PMC4428168  PMID: 17965054
Lrp6; Wnt; Planar cell polarity; Convergent extension; Gastrulation; Xenopus
7.  Metastasis-associated kinase modulates Wnt signaling to regulate brain patterning and morphogenesis 
Development (Cambridge, England)  2006;133(15):2845-2854.
Wnt signaling is a major pathway regulating cell fate determination, cell proliferation and cell movements in vertebrate embryos. Distinct branches of this pathway activate β-catenin/TCF target genes and modulate morphogenetic movements in embryonic tissues by reorganizing the cytoskeleton. The selection of different molecular targets in the pathway is driven by multiple phosphorylation events. Here, we report that metastasis-associated kinase (MAK) is a novel regulator of Wnt signaling during morphogenetic movements, and eye and brain development in Xenopus embryos. Injected MAK RNA suppressed Wnt transcriptional reporters and activated Jun N-terminal kinase. Furthermore, MAK was recruited to the cell membrane by Frizzled 3, formed a complex with Dishevelled and phosphorylated Dsh in vitro. The regional brain markers Otx2, En2 and Gbx2 were affected in embryos with modulated MAK activity in a manner consistent with a role for MAK in midbrainhindbrain boundary formation. Confirming the inhibitory role for this kinase in Wnt/β-catenin signaling, the midbrain patterning defects in embryos depleted of MAK were rescued by the simultaneous depletion of β-catenin. These findings indicate that MAK may function in different developmental processes as a switch between the canonical and non-canonical branches of Wnt signaling.
PMCID: PMC4428341  PMID: 16790480
Wnt; Xenopus; Dsh; Midbrain; Morphogenesis; Kinase; JNK; SNF-1
8.  ‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation 
Development (Cambridge, England)  2003;130(2):379-389.
Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously – although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.
PMCID: PMC4386654  PMID: 12466204
Alopecia; Hair cycle; Hair differentiation; Homeobox genes; Msx2; Foxn1; Ha3; Fgf5; Mouse
9.  Conditional root expansion mutants of Arabidopsis 
Development (Cambridge, England)  1995;121(4):1237-1252.
Regulation of cell expansion is essential to the formation of plant organs. We have characterized 21 mutations, representing six loci, that cause abnormal cell expansion in the root of Arabidopsis thaliana. The phenotype of these mutants is conditional upon the rate of root growth. Calculation of cell volumes indicated that the mutations resulted in defects in either the orientation or the extent of expansion or in both. Analysis of cortical microtubules in the mutants suggested that a shift in the orientation of cell expansion may not be dependent on a change in the orientation of the microtubules. Double mutant combinations resulted in loss of the conditional phenotype suggesting that the genes may act in a similar pathway or encode partially redundant functions.
PMCID: PMC4353850  PMID: 7743935
root development; cell expansion; cell polarity; microtubules; core mutants; Arabidopsis
10.  Hedgehog signaling to distinct cell types differentially regulates coronary artery and vein development 
Development (Cambridge, England)  2008;135(18):3161-3171.
Vascular development begins with formation of a primary capillary plexus that is later remodeled to give rise to the definitive vasculature. While the mechanism by which arterial and venous fates are acquired is well understood, little is known about when during vascular development arterial and venous vessels emerge and how their growth is regulated. Previously, we have demonstrated that a Hedgehog (HH)/ Vascular endothelial growth factor (VEGF) and Angiopoietin2 (ANG2) signaling pathway is essential for the development of the coronary vasculature. Here we use conditional gene targeting to identify the cell types that receive HH signaling and mediate coronary vascular development. We show that HH signaling to the cardiomyoblast is required for the development of coronary veins, while HH signaling to the perivascular cell (PVC) is necessary for coronary arterial growth. Moreover, the cardiomyoblast and PVC appear to be the exclusive cell types that receive HH signals, as ablation of HH signaling in both cell types leads to an arrest in coronary development. Finally, we present evidence suggesting that coronary arteries and veins may be derived from distinct lineages.
PMCID: PMC4306353  PMID: 18725519
Hedgehog (HH); Vascular endothelial growth factor (VEGF); Angiopoietin (Ang); heart development; coronary vascular development; myocardium; pericyte
11.  Vascular remodeling of the mouse yolk sac requires hemodynamic force 
Development (Cambridge, England)  2007;134(18):3317-3326.
The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a−/−). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.
PMCID: PMC4260474  PMID: 17720695
Blood flow; Viscosity; Shear stress; Cardiovascular; Angiogenesis; eNOS (Nos3); Mlc2a (Myl7); Mouse
12.  Dual requirement for Pax6 in retinal progenitor cells 
Development (Cambridge, England)  2008;135(24):4037-4047.
Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. Here, we performed a detailed study of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools.
PMCID: PMC4231183  PMID: 19004853
Pax6; Retinal progenitor cells; Retinogenesis; Crx; Cre/loxP
13.  Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential 
Development (Cambridge, England)  2008;135(22):3789-3799.
Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells.
PMCID: PMC4213142  PMID: 18842815
Hedgehog; Cardiac progenitor specification; Cyclopamine; Heart development; Smoothened; Zebrafish
14.  Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation 
Development (Cambridge, England)  2006;133(9):1767-1778.
The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.
PMCID: PMC4158842  PMID: 16571627
Mouse; Planar cell polarity; Convergent extension; Neurulation
15.  Chromatin silencing and the maintenance of a functional germline in Caenorhabditis elegans 
Development (Cambridge, England)  1998;125(13):2451-2456.
The germline of the nematode Caenorhabditis elegans exhibits a remarkable ability to specifically silence transgenic DNA. We have shown that this silencing mechanism is disrupted in animals mutant for the maternal effect sterile genes mes-2, mes-3, mes-4 and mes-6. The proteins encoded by mes-2 and mes-6 have been shown to be related to the Polycomb Group of transcriptional repressors (Holdeman, R., Nehrt, S. and Strome, S. (1998). Development 125, 2457-2467; Korf, I., Fan, F. and Strome, S. (1998). Development 125, 2469-2478). These results suggest that a genetic silencing process is essential for sustained germline function, and that this silencing is mediated, at least in part, by Polycomb Group proteins.
PMCID: PMC4084878  PMID: 9609828
Caenorhabditis elegans; mes genes; Polycomb group; Repeat dependent silencing; Germline
16.  Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads 
Development (Cambridge, England)  2003;130(24):5895-5902.
The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.
PMCID: PMC4073601  PMID: 14561636
Primordial Germ cells; Meiosis; Sex Determination; Gonad; Testis; Sry
17.  Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development 
Development (Cambridge, England)  2012;139(10):1831-1841.
Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C2H2 zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons observed in Bcl11a mutant mice interferes with their correct innervation by cutaneous sensory neurons. To understand the mechanism underlying the innervation deficit, we characterized changes in gene expression in the dorsal horn of Bcl11a mutants and identified dysregulated expression of the gene encoding secreted frizzled-related protein 3 (sFRP3, or Frzb). Frzb mutant mice show a deficit in the innervation of the spinal cord, suggesting that the dysregulated expression of Frzb can account in part for the phenotype of Bcl11a mutants. Thus, our genetic analysis of Bcl11a reveals essential functions of this transcription factor in neuronal morphogenesis and sensory wiring of the dorsal spinal cord and identifies Frzb, a component of the Wnt pathway, as a downstream acting molecule involved in this process.
PMCID: PMC4067532  PMID: 22491945
Spinal cord; Transcription factor; Neuronal differentiation; Bcl11a (CTIP1); Mouse
18.  X-chromosome silencing in the germline of C. elegans 
Development (Cambridge, England)  2002;129(2):479-492.
Germline maintenance in the nematode C. elegans requires global repressive mechanisms that involve chromatin organization. During meiosis, the X chromosome in both sexes exhibits a striking reduction of histone modifications that correlate with transcriptional activation when compared with the genome as a whole. The histone modification spectrum on the X chromosome corresponds with a lack of transcriptional competence, as measured by reporter transgene arrays. The X chromosome in XO males is structurally analogous to the sex body in mammals, contains a histone modification associated with heterochromatin in other species and is inactivated throughout meiosis. The synapsed X chromosomes in hermaphrodites also appear to be silenced in early meiosis, but genes on the X chromosome are detectably expressed at later stages of oocyte meiosis. Silencing of the sex chromosome during early meiosis is a conserved feature throughout the nematode phylum, and is not limited to hermaphroditic species.
PMCID: PMC4066729  PMID: 11807039
C. elegans; Germline; Silencing; X-inactivation; Histone modifications; Gametogenesis
19.  Normal myoblast fusion requires myoferlin 
Development (Cambridge, England)  2005;132(24):5565-5575.
Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.
PMCID: PMC4066872  PMID: 16280346
Myoblast; Fusion; Ferlin
20.  Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons 
Development (Cambridge, England)  1999;126(18):3969-3979.
In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.
PMCID: PMC4059008  PMID: 10457007
narrowminded; Neural crest; Sensory neurons; Cell signalling; Zebrafish
21.  Wt1 negatively regulates β-catenin signaling during testis development 
Development (Cambridge, England)  2008;135(10):1875-1885.
β-Catenin, as an important effector of the canonical Wnt signaling pathway and as a regulator of cell adhesion, has been demonstrated to be involved in multiple developmental processes and tumorigenesis. β-Catenin expression was found mainly on the Sertoli cell membrane starting from embryonic day 15.5 in the developing testes. However, its potential role in Sertoli cells during testis formation has not been examined. To determine the function of β-catenin in Sertoli cells during testis formation, we either deleted β-catenin or expressed a constitutively active form of β-catenin in Sertoli cells. We found that deletion caused no detectable abnormalities. However, stabilization caused severe phenotypes, including testicular cord disruption, germ cell depletion and inhibition of Müllerian duct regression. β-Catenin stabilization caused changes in Sertoli cell identity and misregulation of inter-Sertoli cell contacts. As Wt1 conditional knockout in Sertoli cells causes similar phenotypes to our stabilized β-catenin mutants, we then investigated the relationship of Wt1 and β-catenin in Sertoli cells and found Wt1 inhibits β-catenin signaling in these cells during testis development. Wt1 deletion resulted in upregulation of β-catenin expression in Sertoli cells both in vitro and in vivo. Our study indicates that Sertoli cell expression of β-catenin is dispensable for testis development. However, the suppression of β-catenin signaling in these cells is essential for proper testis formation and Wt1 is a negative regulator of β-catenin signaling during this developmental process.
PMCID: PMC4038296  PMID: 18403409
β-Catenin; Wt1; Testis; Sertoli cell; Mouse
22.  spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer 
Development (Cambridge, England)  2001;128(21):4165-4176.
The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and neuronal differentiation in the midbrain and anterior hindbrain. Formation of this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and maintenance of its regional identity and signaling activities. Juxtaposition of the Otx2 and Gbx2 expression domains positions the MHB. How the positional information is translated into activation of Pax2, Wnt1 and Fgf8 expression during MHB establishment remains unclear. In zebrafish spiel ohne grenzen (spg) mutants, the MHB is not established, neither isthmus nor cerebellum form, the midbrain is reduced in size and patterning abnormalities develop within the hindbrain. In spg mutants, despite apparently normal expression of otx2, gbx1 and fgf8 during late gastrula stages, the initial expression of pax2.1, wnt1 and eng2, as well as later expression of fgf8 in the MHB primordium are reduced. We show that spg mutants have lesions in pou2, which encodes a POU-domain transcription factor. Maternal pou2 transcripts are distributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection of pou2 mRNA can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 mutants without inducing ectopic expression. This indicates an essential but permissive role for pou2 during MHB establishment. pou2 is expressed normally in noi/pax2.1 and ace/fgf8 zebrafish mutants, which also form no MHB. Thus, expression of pou2 does not depend on fgf8 and pax2.1. Our data suggest that pou2 is required for the establishment of the normal expression domains of wnt1 and pax2.1 in the MHB primordium.
PMCID: PMC4027960  PMID: 11684654
Hindbrain; MHB; Midbrain; Isthmus; engrailed; fgf8; gbx2; otx2; pax2.1; spg; wnt1; POU domain; Danio rerio
23.  Dlx proteins position the neural plate border and determine adjacent cell fates 
Development (Cambridge, England)  2003;130(2):331-342.
The lateral border of the neural plate is a major source of signals that induce primary neurons, neural crest cells and cranial placodes as well as provide patterning cues to mesodermal structures such as somites and heart. Whereas secreted BMP, FGF and Wnt proteins influence the differentiation of neural and non-neural ectoderm, we show here that members of the Dlx family of transcription factors position the border between neural and non-neural ectoderm and are required for the specification of adjacent cell fates. Inhibition of endogenous Dlx activity in Xenopus embryos with an EnR-Dlx homeodomain fusion protein expands the neural plate into non-neural ectoderm tissue whereas ectopic activation of Dlx target genes inhibits neural plate differentiation. Importantly, the stereotypic pattern of border cell fates in the adjacent ectoderm is re-established only under conditions where the expanded neural plate abuts Dlx-positive non-neural ectoderm. Experiments in which presumptive neural plate was grafted to ventral ectoderm reiterate induction of neural crest and placodal lineages and also demonstrate that Dlx activity is required in non-neural ectoderm for the production of signals needed for induction of these cells. We propose that Dlx proteins regulate intercellular signaling across the interface between neural and non-neural ectoderm that is critical for inducing and patterning adjacent cell fates.
PMCID: PMC4018238  PMID: 12466200
Dlx; Neural crest; Neural induction; Xenopus; hairy2a; slug; snail; msx
24.  Drosophila Fragile X Mental Retardation Protein Developmentally Regulates Activity-Dependent Axon Pruning 
Development (Cambridge, England)  2008;135(8):1547-1557.
Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both; a distinction vital for engineering intervention strategies. To address this critical issue, we have used the Drosophila FraX model to investigate the developmental roles of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement in late brain development.
PMCID: PMC3988902  PMID: 18321984
Fragile X Syndrome; mental retardation; autism; neural development; translation; synaptogenesis; synaptic pruning
25.  Pdm and Castor close successive temporal identity windows in the NB3–1 lineage 
Development (Cambridge, England)  2008;135(21):3491-3499.
Neurogenesis in Drosophila and mammals requires the precise integration of spatial and temporal cues. In Drosophila, embryonic neural progenitors (neuroblasts) sequentially express the transcription factors Hunchback, Kruppel, Pdm1/Pdm2 (Pdm) and Castor as they generate a stereotyped sequence of neuronal and glial progeny. Hunchback and Kruppel specify early temporal identity in two posterior neuroblast lineages (NB7–1 and NB7–3), whereas Pdm and Castor specify late neuronal identity in the NB7–1 lineage. Because Pdm and Castor have only been assayed in one lineage, it is unknown whether their function is restricted to neuronal identity in the NB7–1 lineage, or whether they function more broadly as late temporal identity genes in all neuroblast lineages. Here, we identify neuronal birth-order and molecular markers within the NB3–1 cell lineage, and then use this lineage to assay Pdm and Castor function. We show that Hunchback and Kruppel specify first and second temporal identities, respectively. Surprisingly, Pdm does not specify the third temporal identity, but instead acts as a timing factor to close the second temporal identity window. Similarly, Castor closes the third temporal identity window. We conclude that Hunchback and Kruppel specify the first and second temporal identities, an unknown factor specifies the third temporal identity, and Pdm and Castor are timing factors that close the second and third temporal identity windows in the NB3–1 lineage. Our results provide a new neuroblast lineage for investigating temporal identity and reveal the importance of Pdm and Cas as timing factors that close temporal identity windows.
PMCID: PMC3989073  PMID: 18832394
Castor; Pdm (Nubbin); Cell fate; Lineage; Temporal identity; Timer; Hunchback; Kruppel

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