Showing papers in "Developmental Dynamics in 2007"

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

Abstract: Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies

Abstract: Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal- and spatial-specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross-linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein-NTR (CFP-NTR) under control of tissue-specific promoters allowed us to induce the death of cardiomyocytes, pancreatic beta-cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration.

Gateway compatible vectors for analysis of gene function in the zebrafish.

Abstract: The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos.

Hox patterning of the vertebrate axial skeleton.

Abstract: The axial skeleton in all vertebrates is composed of similar components that extend from anterior to posterior along the body axis: the occipital skull bones and cervical, thoracic, lumbar, sacral, and caudal vertebrae. Despite significant changes in the number and size of these elements during evolution, the basic character of these anatomical elements, as well as the order in which they appear in vertebrate skeletons, have remained remarkably similar. Through extensive expression analyses, classic morphological perturbation experiments in chicken and targeted loss-of-function analyses in mice, Hox genes have proven to be critical regulators in the establishment of axial skeleton morphology. The convergence of these studies to date allows an emerging understanding of Hox gene function in patterning the vertebrate axial skeleton. This review summarizes genetic and embryologic findings regarding the role of Hox genes in establishing axial morphology and how these combined results impact our current understanding of the vertebrate Hox code. Developmental Dynamics 236:2454 –2463, 2007. © 2007 Wiley-Liss, Inc.

Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene

Abstract: Defects in tendon patterning and differentiation are seldom assessed in mouse mutants due to the difficulty in visualizing connective tissue structures. To facilitate tendon analysis, we have generated mouse lines harboring two different transgene reporters, alkaline phosphatase (AP) and green fluorescent protein (GFP), each expressed using regulatory elements derived from the endogenous Scleraxis (Scx) locus. Scx encodes a transcription factor expressed in all developing tendons and ligaments as well as in their progenitors. Both the ScxGFP and ScxAP transgenes are expressed in patterns recapitulating almost entirely the endogenous developmental expression of Scx including very robust expression in the tendons and ligaments. These reporter lines will facilitate isolation of tendon cells and phenotypic analysis of these tissues in a variety of genetic backgrounds.

Epigenetics in development.

Abstract: It has become increasingly evident in recent years that development is under epigenetic control. Epigenetics is the study of heritable changes in gene function that occur independently of alterations to primary DNA sequence. The best-studied epigenetic modifications are DNA methylation, and changes in chromatin structure by histone modifications, and histone exchange. An exciting, new chapter in the field is the finding that long-distance chromosomal interactions also modify gene expression. Epigenetic modifications are key regulators of important developmental events, including X-inactivation, genomic imprinting, patterning by Hox genes and neuronal development. This primer covers these aspects of epigenetics in brief, and features an interview with two epigenetic scientists.

A web‐based interactive developmental table for the ascidian Ciona intestinalis, including 3D real‐image embryo reconstructions: I. From fertilized egg to hatching larva

Abstract: The ascidian chordate Ciona intestinalis is an established model organism frequently exploited to examine cellular development and a rapidly emerging model organism with a strong potential for developmental systems biology studies. However, there is no standardized developmental table for this organism. In this study, we made the standard web-based image resource called FABA: Four-dimensional Ascidian Body Atlas including ascidian's three-dimensional (3D) and cross-sectional images through the developmental time course. These images were reconstructed from more than 3,000 high-resolution real images collected by confocal laser scanning microscopy (CLSM) at newly defined 26 distinct developmental stages (stages 1-26) from fertilized egg to hatching larva, which were grouped into six periods named the zygote, cleavage, gastrula, neurula, tailbud, and larva periods. Our data set will be helpful in standardizing developmental stages for morphology comparison as well as for providing the guideline for several functional studies of a body plan in chordate.

Back to basics: Sox genes.

Abstract: Sox genes are indispensable for multiple aspects of development. This primer briefly describes shared properties of the Sox gene family, and five well-characterized examples of vertebrate developmental mechanisms governed by Sox gene subgroups: testis development, central nervous system neurogenesis, oligodendrocyte development, chondrogenesis, and neural crest cell development. Also featured is an interview about current issues in the field with experts Jonas Muhr, Ph.D. and Robert Kelsh, Ph.D.

Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency.

Abstract: Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenic mice that expressed dn-Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild-type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that Runx2 is not essential for maintenance of the expression of major bone matrix protein genes in postnatal bone development and maintenance.

Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss.

Abstract: Early differentiation in rainbow trout gonads was investigated by expression profiling and in situ hybridization (ISH). Expression of cyp19a1 and fst in females and sox9a1 in males were sexually dimorphic between 32 to 35 days post-fertilization (dpf). After 35 dpf, the differentiation proceeded with sexually dimorphic profiles for sox9a2, dmrt1, cyp11b2.1, amh in males and foxl2a, foxl2b, hsd3b1, inha in females. cyp17a1, cyp11a1, star, nr5a1b increased only after 40 dpf in both sexes with a slightly higher expression in females. cyp19a1 expression was localized in a cluster of somatic cells in the ventral side of female gonads, and sox9a2 and amh in somatic cells surrounding the germ cells, at 28 dpf and thereafter, both in male and female gonads. cyp11b2.1, cyp17a1, and cyp11a1 expressions were only detected in scattered somatic cells in males after 46 dpf. This confirms the early implication of cyp19a1 in trout ovarian differentiation and suggests that early testicular differentiation does not need androgen production.

Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan

Abstract: DNA methylation is an epigenetic mechanism involved in gene regulation and implicated in the functioning of the nervous system. The de novo DNA methyltransferase Dnmt3a is expressed in neurons, but its specific role has not been clarified. Dnmt3a is activated around embryonic day 10.5 in mouse neuronal precursor cells and remains active in postmitotic neurons in the adult. We assessed the role of neuronal Dnmt3a by conditional gene targeting. Mice lacking functional Dnmt3a in the nervous system were born healthy, but degenerated in adulthood and died prematurely. Mutant mice were hypoactive, walked abnormally, and underperformed on tests of neuromuscular function and motor coordination. Loss of Dnmt3a also led to fewer motor neurons in the hypoglossal nucleus and more fragmented endplates in neuromuscular junctions of the diaphragm muscle. Our results implicate a role for Dnmt3a in the neuromuscular control of motor movement.

Maternally imprinted microRNAs are differentially expressed during mouse and human lung development

Abstract: MicroRNAs (miRNAs) are a recently discovered class of noncoding genes that regulate the translation of target mRNA. More than 300 miRNAs have now been discovered in humans, although the function of most is still unknown. A highly sensitive, semiquantitative real-time polymerase chain reaction method was used to reveal the differential expression of several miRNAs during the development of both mouse and human lung. Of note was the up-regulation in neonatal mouse and fetal human lung of a maternally imprinted miRNA cluster located at human chromosome 14q32.31 (mouse chromosome 12F2), which includes the miR-154 and miR-335 families and is situated within the Gtl2-Dio3 domain. Conversely, several miRNAs were up-regulated in adult compared with neonatal/fetal lung, including miR-29a and miR-29b. Differences in the spatial expression patterns of miR-154, miR-29a, and miR-26a was demonstrated using in situ hybridization of mouse neonatal and adult tissue using miRNA-specific locked nucleic acid (LNA) probes. Of interest, miR-154 appeared to be localized to the stroma of fetal but not adult lungs. The overall expression profile was similar for mouse and human tissue, suggesting evolutionary conservation of miRNA expression during lung development and demonstrating the importance of maternally imprinted miRNAs in the developmental process.

Anti-Müllerian hormone and 11 β-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males

Abstract: During development all zebrafish males first develop a "juvenile ovary" that later degenerates and transforms into a testis. In this study, individuals undergoing gonadal transformation were identified from a vas::egfp transgenic line and used for gene expression analysis of anti-Mullerian hormone (amh), ovarian aromatase (cyp19a1a) and 11 beta-hydroxylase (cyp11b, also known as P450(11 beta)) by real-time polymerase chain reaction and in situ hybridization. In the "normal (i.e., nontransforming) juvenile ovary" cyp19a1a was expressed around the oocytes, but cyp11b and amh could not be detected. During gonadal transformation cyp19a1a was down-regulated and could not be detected anymore; in contrast amh was up-regulated and highly expressed at similar regions where cyp19a1a had been expressed earlier. Furthermore, the normalized transcript levels of cyp19a1a and amh showed a reciprocal picture, i.e., the higher was the level of amh, the lower was that of cyp19a1a. Expression of cyp11b was also up-regulated but later than amh, and its localization was not related to the position of degenerating oocytes. These data indicate that amh is a candidate gene down-regulating cyp19a1a, leading to "juvenile ovary-to-testis" transformation. Whereas, cyp11b or its product, 11-ketotestosterone, is unlikely to be the inducer of zebrafish gonad transformation, as proposed earlier for some protogynous hermaphroditic fish species.

Amniote somite derivatives

Abstract: Somites are segments of paraxial mesoderm that give rise to a multitude of tissues in the vertebrate embryo. Many decades of intensive research have provided a wealth of data on the complex molecular interactions leading to the formation of various somitic derivatives. In this review, we focus on the crucial role of the somites in building the body wall and limbs of amniote embryos. We give an overview on the current knowledge on the specification and differentiation of somitic cell lineages leading to the development of the vertebral column, skeletal muscle, connective tissue, meninges, and vessel endothelium, and highlight the importance of the somites in establishing the metameric pattern of the vertebrate body.

Nitric oxide-dependence of satellite stem cell activation and quiescence on normal skeletal muscle fibers

Abstract: Satellite cells (quiescent precursors in normal adult skeletal muscle) are activated for growth and regeneration. Signaling by nitric oxide (NO) and hepatocyte growth factor (HGF) during activation has not been examined in a model that can distinguish quiescent from activated satellite cells. We tested the hypothesis that NO and HGF are required to regulate activation using the single-fiber culture model. In normal fibers, HGF and inhibition of NO synthase (NOS) each increased activation without stretching, and NOS inhibition reduced stretchactivation. Activation in unstretched mdx and NOS-I(/ ) fibers was three- to fourfold higher than normal, and was reduced by stretching. Distinctions were not due to different pax7-expressing populations on normal and mdx fibers. The population of c-met– expressing satellite cells on normal fibers was increased by stretch, demonstrating functional heterogeneity among normal satellite cells. Cycloheximide did not prevent the stretch-related increase in c-met expression, suggesting c-met may be an immediate– early gene in satellite cell activation. Results have important implications for designing new therapies that target the role of exercise in health, aging, and disease. Developmental Dynamics 236:240 –250, 2007. © 2006 Wiley-Liss, Inc.

The commonly used marker ELAV is transiently expressed in neuroblasts and glial cells in the Drosophila embryonic CNS.

Abstract: Glial cells in the Drosophila embryonic nervous system can be monitored with the marker Reversed-polarity (Repo), whereas neurons lack Repo and express the RNA-binding protein ELAV (Embryonic Lethal, Abnormal Vision). Since the first description of the ELAV protein distribution in 1991 (Robinow and White), it is believed that ELAV is an exclusive neuronal and postmitotic marker. Looking at ELAV expression, we unexpectedly observed that, in addition to neurons, ELAV is transiently expressed in embryonic glial cells. Furthermore, it is transiently present in the proliferating longitudinal glioblast, and it is transcribed in embryonic neuroblasts. Likewise, elav-Gal4 lines, which are generally used as postmitotic neuronal driver lines, show expression in neural progenitor cells and nearly all embryonic glial cells. Thus, in the embryo, elav can no longer be considered an exclusive marker or driver for postmitotic neurons. elav loss-of-function mutants show no obvious effects on the number and pattern of embryonic glia.

Prox1 expression patterns in the developing and adult murine brain.

Abstract: Prox1, a homeobox gene related to the Drosophila gene prospero, is necessary for retina, lens, liver, pancreas, and lymphatics development. However, not much is yet known about Prox1 expression during central nervous system development. Here we provide a detailed analysis of Prox1 mRNA and protein expression during prenatal and postnatal murine brain development. Prenatally, Prox1 is expressed in the subventricular zone or in early differentiating regions of the brain. At these stages, Prox1 mRNA, but not Prox1 protein, was also detected in several regions of the prethalamus and hypothalamus. At an early postnatal stage, Prox1 expression is mainly detected in several nuclei of the thalamus, the cerebellum, and the hippocampus. In adulthood, Prox1 expression remains only in the hippocampus and cerebellum. These complex patterns of expression suggest that Prox1 activity is differentially required during brain development and adulthood.

Sharp developmental thresholds defined through bistability by antagonistic gradients of retinoic acid and FGF signaling.

Abstract: The establishment of thresholds along morphogen gradients in the embryo is poorly understood. Using mathematical modeling, we show that mutually inhibitory gradients can generate and position sharp morphogen thresholds in the embryonic space. Taking vertebrate segmentation as a paradigm, we demonstrate that the antagonistic gradients of retinoic acid (RA) and Fibroblast Growth Factor (FGF) along the presomitic mesoderm (PSM) may lead to the coexistence of two stable steady states. Here, we propose that this bistability is associated with abrupt switches in the levels of FGF and RA signaling, which permit the synchronized activation of segmentation genes, such as mesp2, in successive cohorts of PSM cells in response to the segmentation clock, thereby defining the future segments. Bistability resulting from mutual inhibition of RA and FGF provides a molecular mechanism for the all-or-none transitions assumed in the "clock and wavefront" somitogenesis model. Given that mutually antagonistic signaling gradients are common in development, such bistable switches could represent an important principle underlying embryonic patterning.

Sox6 is required for normal fiber type differentiation of fetal skeletal muscle in mice.

Abstract: Sox6, a member of the Sox family of transcription factors, is highly expressed in skeletal muscle. Despite its abundant expression, the role of Sox6 in muscle development is not well understood. We hypothesize that, in fetal muscle, Sox6 functions as a repressor of slow fiber type-specific genes. In the wild-type mouse, differentiation of fast and slow fibers becomes apparent during late fetal stages (after approximately embryonic day 16). However, in the Sox6 null-p(100H) mutant mouse, all fetal muscle fibers maintain slow fiber characteristics, as evidenced by expression of the slow myosin heavy chain MyHC-beta. Knockdown of Sox6 expression in wild-type myotubes results in a significant increase in MyHC-beta expression, supporting our hypothesis. Analysis of the MyHC-beta promoter revealed a Sox consensus sequence that likely functions as a negative cis-regulatory element. Together, our results suggest that Sox6 plays a critical role in the fiber type differentiation of fetal skeletal muscle.

Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration.

Abstract: In the avian inner ear, nonsensory supporting cells give rise to new sensory hair cells through two distinct processes: mitosis and direct transdifferentiation. Regulation of supporting cell behavior and cell fate specification during avian hair cell regeneration is poorly characterized. Expression of Atoh1, a proneural transcription factor necessary and sufficient for developmental hair cell specification, was examined using immunofluorescence in quiescent and regenerating hair cell epithelia of mature chickens. In untreated birds, Atoh1 protein was not detected in the auditory epithelium, which is quiescent. In contrast, numerous Atoh1-positive nuclei were seen in the utricular macula, which undergoes continual hair cell turnover. Atoh1-positive nuclei emerged in the auditory epithelium by 15 hr post-ototoxin administration, before overt hair cell damage and supporting cell re-entry into the cell cycle. Subsequently, Atoh1 labeling was seen in 15% of dividing supporting cells. During cell division, Atoh1 was distributed symmetrically to daughter cells, but Atoh1 levels were dramatically regulated shortly thereafter. After cellular differentiation, Atoh1 labeling was confined to hair cells regenerated through either mitosis or direct transdifferentiation. However, Atoh1 expression in dividing progenitors did not necessarily predict hair cell fate specification in daughter cells. Finally, predominant modes of hair cell regeneration varied significantly across the radial axis of the auditory epithelium, with mitosis most frequent neurally and direct transdifferentiation most frequent abneurally. These observations suggest a role for Atoh1 in re-specifying supporting cells and in biasing postmitotic cells toward the hair cell fate during hair cell regeneration in the mature chicken ear. Developmental Dynamics 236:156–170, 2007. © 2006 Wiley-Liss, Inc.

Reprogramming of genetic networks during initiation of the Fetal Alcohol Syndrome

Abstract: Fetal Alcohol Spectrum Disorders (FASD) are birth defects that result from maternal alcohol use. We used a non a priori approach to prioritize candidate pathways during alcohol-induced teratogenicity in early mouse embryos. Two C57BL/6 substrains (B6J, B6N) served as the basis for study. Dosing pregnant dams with alcohol (2× 2.9 g/kg ethanol spaced 4 hr on day 8) induced FASD in B6J at a higher incidence than B6N embryos. Counter-exposure to PK11195 (4 mg/kg) significantly protected B6J embryos but slightly promoted FASD in B6N embryos. Microarray transcript profiling was performed on the embryonic headfold 3 hr after the first maternal alcohol injection (GEO data series accession GSE1074). This analysis revealed metabolic and cellular reprogramming that was substrain-specific and/or PK11195-dependent. Mapping ethanol-responsive KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed down-regulation of ribosomal proteins and proteasome, and up-regulation of glycolysis and pentose phosphate pathway in B6N embryos; and significant up-regulation of tight junction, focal adhesion, adherens junction, and regulation of the actin cytoskeleton (and near-significant up-regulation of Wnt signaling and apoptosis) pathways in both substrains. Expression networks constructed computationally from these altered genes identified entry points for EtOH at several hubs (MAPK1, ALDH3A2, CD14, PFKM, TNFRSF1A, RPS6, IGF1, EGFR, PTEN) and for PK11195 at AKT1. Our findings are consistent with the growing view that developmental exposure to alcohol alters common signaling pathways linking receptor activation to cytoskeletal reorganization. The programmatic shift in cell motility and metabolic capacity further implies cell signals and responses that are integrated by the mitochondrial recognition site for PK11195. Developmental Dynamics 236:613–631, 2007. © 2007 Wiley-Liss, Inc.

Monitoring Notch1 activity in development: evidence for a feedback regulatory loop.

Abstract: Signaling through Notch receptors, which regulate cell fate decisions and embryonic patterning, requires ligand-induced receptor cleavage to generate the signaling active Notch intracellular domain (NICD). Here, we show an analysis at specific developmental stages of the distribution of active mouse Notch1. We use an antibody that recognizes N1ICD, and a highly sensitive staining technique. The earliest N1ICD expression was observed in the mesoderm and developing heart, where we detected expression in nascent endocardium, presumptive cardiac valves, and ventricular and atrial endocardium. During segmentation, N1ICD was restricted to the presomitic mesoderm. N1ICD expression was also evident in arterial endothelium, and in kidney and endodermal derivatives such as pancreas and thymus. Ectodermal N1ICD expression was found in central nervous system and sensory placodes. We found that Notch1 transcription and activity was severely reduced in zebrafish and mouse Notch pathway mutants, suggesting that vertebrate Notch1 expression is regulated by a positive feedback loop. Developmental Dynamics 236:2594–2614, 2007. © 2007 Wiley-Liss, Inc.

Evolutionary conservation and divergence of the segmentation process in arthropods

Abstract: A fundamental characteristic of the arthropod body plan is its organization in metameric units along the anterior-posterior axis The segmental organization is laid down during early embryogenesis Our view on arthropod segmentation is still strongly influenced by the huge amount of data available from the fruit fly Drosophila melanogaster (the Drosophila paradigm) However, the simultaneous formation of the segments in Drosophila is a derived mode of segmentation Successive terminal addition of segments from a posteriorly localized presegmental zone is the ancestral mode of arthropod segmentation This review focuses on the evolutionary conservation and divergence of the genetic mechanisms of segmentation within arthropods The more downstream levels of the segmentation gene network (eg, segment polarity genes) appear to be more conserved than the more upstream levels (gap genes, Notch/Delta signaling) Surprisingly, the basally branched arthropod groups also show similarities to mechanisms used in vertebrate somitogenesis Furthermore, it has become clear that the activation of pair rule gene orthologs is a key step in the segmentation of all arthropods Important findings of conserved and diverged aspects of segmentation from the last few years now allow us to draw an evolutionary scenario on how the mechanisms of segmentation could have evolved and led to the present mechanisms seen in various insect groups including dipterans like Drosophila

Emerging roles for zic genes in early development

Abstract: Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. They are involved in development of neural tissues and the neural crest, in left-right axis patterning, in somite development, and in formation of the cerebellum. In addition to their roles in cell-fate specification, zic genes also promote cell proliferation. Further, they are expressed in postmitotic cells of the cerebellum and in retinal ganglion cells. Efforts to determine the role of individual zic genes within an array of developmental and cellular processes are complicated by overlapping patterns of zic gene expression and strong sequence conservation within this gene family. Nevertheless, substantial progress has been made. This review summarizes our knowledge of the molecular events that govern the activities of zic family members, including emerging relationships between upstream signaling pathways and zic genes. In addition, advancements in our understanding of the molecular events downstream of Zic transcription factors are reviewed. Despite significant progress, however, much remains to be learned regarding the mechanisms through which zic genes exert their function in a variety of different contexts. Developmental Dynamics 236:922–940, 2007. © 2007 Wiley-Liss, Inc.

Expression of Sox9, Mis, and Dmrt1 in the gonad of a species with temperature‐dependent sex determination

Abstract: Sex determination in vertebrates, the process of forming an ovary or testis from a bipotential gonad, can be initiated by genetic or environmental factors. Elements of the downstream molecular pathways underlying these different sex-determining mechanisms have been evolutionarily conserved. We find the first evidence that Sox9 expression is preferentially organized in the testis early in the temperature-sensitive period in a species with temperature-dependent sex determination (Trachemys scripta). This pattern occurs before sexually dimorphic Mis expression and in a temporal hierarchy that is similar to mammals. Furthermore, we extend previous findings that Dmrt1 expression at early stages of sex determination has a dimorphic pattern consistent with a possible upstream role in determining the fate of the bipotential gonad.

Botryllus schlosseri: A model ascidian for the study of asexual reproduction

Abstract: Botryllus schlosseri, a cosmopolitan colonial ascidian reared in the laboratory for more than 50 years, reproduces both sexually and asexually and is used as a model organism for studying a variety of biological problems. Colonies are formed of numerous, genetically identical individuals (zooids) and undergo cyclical generation changes in which the adult zooids die and are replaced by their maturing buds. Because the progression of the colonial life cycle is intimately correlated with blastogenesis, a shared staging method of bud development is required to compare data coming from different laboratories. With the present review, we aim (1) to introduce B. schlosseri as a valuable chordate model to study various biological problems and, especially, sexual and asexual development; (2) to offer a detailed description of bud development up to adulthood and the attainment of sexual maturity; (3) to re-examine Sabbadin's (1955) staging method and re-propose it as a simple tool for in vivo recognition of the main morphogenetic events and recurrent changes in the blastogenetic cycle, as it refers to the developmental stages of buds and adults. Developmental Dynamics 236:335–352, 2007. © 2006 Wiley-Liss, Inc.

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling.

Abstract: The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild-type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh(-/-) embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh(-/-)/Gli3(-/-) mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3(R) in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild-type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin-expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components.

Abnormal vertebral segmentation and the notch signaling pathway in man

Abstract: Abnormal vertebral segmentation (AVS) in man is a relatively common congenital malformation but cannot be subjected to the scientific analysis that is applied in animal models. Nevertheless, some spectacular advances in the cell biology and molecular genetics of somitogenesis in animal models have proved to be directly relevant to human disease. Some advances in our understanding have come through DNA linkage analysis in families demonstrating a clustering of AVS cases, as well as adopting a candidate gene approach. Only rarely do AVS phenotypes follow clear Mendelian inheritance, but three genes-DLL3, MESP2, and LNFG-have now been identified for spondylocostal dysostosis (SCD). SCD is characterized by extensive hemivertebrae, trunkal shortening, and abnormally aligned ribs with points of fusion. In familial cases clearly following a Mendelian pattern, autosomal recessive inheritance is more common than autosomal dominant and the genes identified are functional within the Notch signaling pathway. Other genes within the pathway cause diverse phenotypes such as Alagille syndrome (AGS) and CADASIL, conditions that may have their origin in defective vasculogenesis. Here, we deal mainly with SCD and AGS, and present a new classification system for AVS phenotypes, for which, hitherto, the terminology has been inconsistent and confusing.

Adult bone marrow–derived stem cells for organ regeneration and repair

Abstract: Stem cells have been recognized as a potential tool for the development of innovative therapeutic strategies. There are in general two types of stem cells, embryonic and adult stem cells. While embryonic stem cell therapy has been riddled with problems of allogeneic rejection and ethical concerns, adult stem cells have long been used in the treatment of hematological malignancies. With the recognition of additional, potentially therapeutic characteristics, bone marrow-derived stem cells have become a tool in regenerative medicine. The bone marrow is an ideal source of stem cells because it is easily accessible and harbors two types of stem cells. Hematopoietic stem cells give rise to all blood cell types and have been shown to exhibit plasticity, while multipotent marrow stromal cells are the source of osteocytes, chondrocytes, and fat cells and have been shown to support and generate a large number of different cell types. This review describes the general characteristics of these stem cell populations and their current and potential future applications in regenerative medicine.

The genetics and embryology of zebrafish metamerism.

Abstract: Somites are the most obvious metameric structures in the vertebrate embryo. They are mesodermal segments that form in bilateral pairs flanking the notochord and are created sequentially in an anterior to posterior sequence concomitant with the posterior growth of the trunk and tail. Zebrafish somitogenesis is regulated by a clock that causes cells in the presomitic mesoderm (PSM) to undergo cyclical activation and repression of several notch pathway genes. Coordinated oscillation among neighboring cells manifests as stripes of gene expression that pass through the cells of the PSM in a posterior to anterior direction. As axial growth continually adds new cells to the posterior tail bud, cells of the PSM become relatively less posterior. This gradual assumption of a more anterior position occurs over developmental time and constitutes part of a maturation process that governs morphological segmentation in conjunction with the clock. Segment morphogenesis involves a mesenchymal to epithelial transition as prospective border cells at the anterior end of the mesenchymal PSM adopt a polarized, columnar morphology and surround a mesenchymal core of cells. The segmental pattern influences the development of the somite derivatives such as the myotome, and the myotome reciprocates to affect the formation of segment boundaries. While somites appear to be serially homologous, there may be variation in the segmentation mechanism along the body axis. Moreover, whereas the genetic architecture of the zebrafish, mouse, and chick segmentation clocks shares many common elements, there is evidence that the gene networks have undergone independent modification during evolution.