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Background: The two-pore domain potassium (K2P) channels are a major type of potassium channels that maintain the cell membrane potential by conducting passive potassium leak currents independent of voltage change. They play prominent roles in multiple physiological processes, including neuromodulation, perception of pain, breathing and mood control, and response to volatile anesthetics. Mutations in K2P channels have been linked to many human diseases, such as neuronal and cardiovascular disorders and cancers. Significant progress has been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and function during embryonic development remain largely unknown., Results: We employed the zebrafish model and identified 23 k2p genes using BLAST search and gene cloning. We first analyzed vertebrate K2P channel evolution by phylogenetic and syntenic analyses. Our data revealed that the six subtypes of the K2P genes have already evolved in invertebrates long before the emergence of vertebrates. Moreover, the vertebrate K2P gene number increased, most likely due to two whole-genome duplications. Furthermore, we examined zebrafish k2p gene expression during early embryogenesis by in situ hybridization. Each subgroup's genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues consistent with their known function of neural excitability regulation. However, a few k2p genes were expressed temporarily in specific tissues or organs, suggesting that these K2P channels may be needed for embryonic development., Conclusions: Our phylogenetic and developmental analyses of K2P channels shed light on their evolutionary history and potential roles during embryogenesis related to their physiological functions and human channelopathies., (© 2024 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Some marine invertebrate organisms are considered not to develop tumors due to unknown mechanisms. To gain an initial insight into how tumor-related genes may be expressed and function during marine invertebrate development, we here leverage sea urchin embryos as a model system and characterize the expressions of Myc and p53/p63/p73 which are reported to function synergistically in mammalian models as an oncogene and tumor suppressor, respectively., Results: During sea urchin embryogenesis, a combo gene of p53/p63/p73 is found to be maternally loaded and decrease after fertilization both in transcript and protein, while Myc transcript and protein are zygotically expressed. p53/p63/p73 and Myc proteins are observed in the cytoplasm and nucleus of every blastomere, respectively, throughout embryogenesis. Both p53/p63/p73 and Myc overexpression results in compromised development with increased DNA damage after the blastula stage. p53/p63/p73 increases the expression of parp1, a DNA repair/cell death marker gene, and suppresses endomesoderm gene expressions. In contrast, Myc does not alter the expression of specification genes or oncogenes yet induces disorganized morphology., Conclusions: p53/p63/p73 appears to be important for controlling cell differentiation, while Myc induces disorganized morphology yet not through conventional oncogene regulations or apoptotic pathways during embryogenesis of the sea urchin., (© 2023 American Association for Anatomy.)

Background: Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish., Results: We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization., Conclusion: The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results., (© 2023 American Association for Anatomy.)

Background: Members of the sulfotransferase superfamily (SULT) influence the activity of a wide range of hormones, neurotransmitters, metabolites and xenobiotics. However, their roles in developmental processes are not well characterized even though they are expressed during embryogenesis. We previously found in a microarray screen that Six1 up-regulates LOC100037047, which encodes XB5850668.L, an uncharacterized sulfotransferase., Results: Since Six1 is required for patterning the embryonic ectoderm into its neural plate, neural crest, preplacodal and epidermal domains, we used loss- and gain-of function assays to characterize the role of XB5850668.L during this process. Knockdown of endogenous XB5850668.L resulted in the reduction of epidermal, neural crest, cranial placode and otic vesicle gene expression domains, concomitant with neural plate expansion. Increased levels had minimal effects, but infrequently expanded neural plate and neural crest gene domains, and infrequently reduced cranial placode and otic vesicle gene domains. Mutation of two key amino acids in the sulfotransferase catalytic domain required for PAPS binding and enzymatic activity tended to reduce the effects of overexpressing the wild-type protein., Conclusions: Our analyses indicates that XB5850668.L is a member of the SULT2 family that plays important roles in patterning the embryonic ectoderm. Some aspects of its influence likely depend on sulfotransferase activity., (© 2023 American Association for Anatomy.)

Background: MLLT3 (AF9) is a nuclear transcription factor crucial for hematopoietic stem cell and progenitor cell maintenance, but its role during embryonic hematopoiesis remains uncertain. Here, we examine the role of mllt3 in developmental hematopoiesis during embryogenesis using zebrafish., Results: Cloning, sequencing, phylogenetic, and synteny analyses showed high evolutionary conservation between important functional domains of the zebrafish orthologue of mllt3 and MLLT3 in humans. Quantitative reverse transcription-PCR and in situ hybridization analyses revealed that mllt3 is maternally supplied and zygotically expressed throughout embryonic development, and that expression is highest between 10 and 24 hours post-fertilization (hpf) coincident with enrichment in the intermediate cell mass (ICM) and posterior blood island, which are the sites of the primitive and transient definitive hematopoiesis in zebrafish, respectively. Further, we found co-expression of mllt3 with the early hematopoietic progenitor markers tal1, gata2, and gata1a in the posterior ICM. By investigating zebrafish hematopoietic mutants, we discovered that mllt3 is involved in erythroid precursor formation. By 48-72 hpf, mllt3 expression proved to be restricted to non-hematopoietic tissues including head structures, pronephric tubules, and liver primordium., Conclusions: These findings establish a link between mllt3 and primitive erythropoiesis and provide the basis for future functional investigations., (© 2022 American Association for Anatomy.)

Background: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis., Results: To overcome these challenges, we adapted the chromatin profiling methods Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and CUT&Tag for use in zebrafish and applied these methods to generate high-resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z using tissue isolated from whole, mid-gastrula stage embryos. Using this data, we identify a subset of genes that may be bivalently regulated during both zebrafish and mouse gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the effectiveness of CUT&RUN for detecting H3K9me3 enrichment at repetitive sequences., Conclusions: Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis., (© 2021 American Association for Anatomy.)

Background: Inwardly rectifying potassium channels are essential for normal potassium homeostasis, maintaining the cellular resting membrane potential, and regulating electrolyte transportation. Mutations in Kir channels have been known to cause debilitating diseases ranging from neurological abnormalities to renal and cardiac failures. Many efforts have been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and functions during embryonic development remain largely unknown., Results: Using zebrafish as a model, we identified and renamed 31 kir genes. We also analyzed Kir gene evolution by phylogenetic and syntenic analyses. Our data indicated that the four subtypes of the Kir genes might have already evolved out in chordates. These vertebrate Kir genes most likely resulted from both whole-genome duplications and tandem duplications. In addition, we examined zebrafish kir gene expression during early embryogenesis. Each subgroup's genes showed similar but distinct gene expression domains. The gene expression of ohnologous genes from teleost-specific whole-genome duplication indicated subfunctionalization. Varied temporal gene expression domains suggest that Kir channels may be needed for embryonic patterning or regulation., Conclusions: Our phylogenetic and developmental analyses of Kir channels shed light on their evolutionary history and potential functions during embryogenesis related to congenital diseases and human channelopathies., (© 2021 American Association for Anatomy.)

Background: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression., Results: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation., Conclusions: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified., (© 2021 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus., Results: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap., Conclusions: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes., (© 2021 American Association for Anatomy.)

Background: Eya2 expression during mouse development has been studied by in situ hybridization and it has been shown to be involved skeletal muscle development and limb formation. Here, we generated Eya2 knockout (Eya2 - ) and a lacZ knockin reporter (Eya2 lacZ ) mice and performed a detailed expression analysis for Eya2 lacZ at different developmental stages to trace Eya2 lacZ -positive cells in Eya2-null mice. We describe that Eya2 is not only expressed in cranial sensory and dorsal root ganglia, retina and olfactory epithelium, and somites as previously reported, but also Eya2 is specifically detected in other organs during mouse development., Results: We found that Eya2 is expressed in ocular and trochlear motor neurons. In the inner ear, Eya2 lacZ is specifically expressed in differentiating hair cells in both vestibular and cochlear sensory epithelia of the inner ear and Eya2 -/- or Eya2 lacZ/lacZ mice displayed mild hearing loss. Furthermore, we detected Eya2 expression during both salivary gland and thymus development and Eya2-null mice had a smaller thymus., Conclusions: As Eya2 is coexpressed with other members of the Eya family genes, these results together highlight that Eya2 as a potential regulator may act synergistically with other Eya genes to regulate the differentiation of the inner ear sensory hair cells and the formation of the salivary gland and thymus., (© 2021 American Association of Anatomists.)

Background: The male-abnormal 21 like (MAB21L) genes are important in human ocular development. Homozygous loss of MAB21L1 leads to corneal dystrophy in all affected individuals along with cataracts and buphthalmos in some. The molecular function and downstream pathways of MAB21L factors are largely undefined., Results: We generated the first mab21l1 zebrafish mutant carrying a putative loss-of-function allele, c.107delA p.(Lys36Argfs*7). At the final stages of embryonic development, homozygous mab21l1 c.107delA fish displayed enlarged anterior chambers and corneal thinning which progressed with age. Additional studies revealed increased cell death in the mutant corneas, transformation of the cornea into a skin-like epithelium, and progressive lens degeneration with development of fibrous masses in the anterior chamber. RNA-seq of wild-type and mutant ocular transcriptomes revealed significant changes in expression of several genes, including irf1a and b, stat1, elf3, krt17, tlr9, and loxa associated with immunity and/or corneal function. Abnormal expression of lysyl oxidases have been previously linked with corneal thinning, fibrosis, and lens defects in mammals, suggesting a role for loxa misexpression in the progressive mab21l1 c.107delA eye phenotype., Conclusions: Zebrafish mab21l1 is essential for normal corneal development, similar to human MAB21L1. The identified molecular changes in mab21l1 c.107delA mutants provide the first clues about possible affected pathways., (© 2021 American Association of Anatomists.)

Background: Distinct boundaries between the proximal conducting airways and more peripheral-bronchial regions of the lung are established early in foregut embryogenesis, demarcated in part by the distribution of SOX family and NKX2-1 transcription factors along the cephalo-caudal axis of the lung. We used blastocyst complementation to identify the role of NKX2-1 in the formation of the proximal-peripheral boundary of the airways in mouse chimeric embryos., Results: While Nkx2-1 -/- mouse embryos form primordial tracheal cysts, peripheral pulmonary structures are entirely lacking in Nkx2-1 -/- mice. Complementation of Nkx2-1 -/- embryos with NKX2-1-sufficient embryonic stem cells (ESCs) enabled the formation of all tissue components of the peripheral lung but did not enhance ESC colonization of the most proximal regions of the airways. In chimeric mice, a precise boundary was formed between NKX2-1-deficient basal cells co-expressing SOX2 and SOX9 in large airways and ESC-derived NKX2-1 + SOX9 + epithelial cells of smaller airways. NKX2-1-sufficient ESCs were able to selectively complement peripheral, rather than most proximal regions of the airways. ESC complementation did not prevent ectopic expression of SOX9 but restored β-catenin signaling in Nkx2-1 -/- basal cells of large airways., Conclusions: NKX2-1 and β-catenin function in an epithelial cell-autonomous manner to establish the proximal-peripheral boundary along developing airways., (© 2021 American Association of Anatomists.)

Background: Genetic programs underlying preimplantation development and early lineage segregation are highly conserved across mammals. It has been suggested that nonhuman primates would be better model organisms for human embryogenesis, but a limited number of studies have investigated the monkey preimplantation development. In this study, we collect single cells from cynomolgus monkey preimplantation embryos for transcriptome profiling and compare with single-cell RNA-seq data derived from human and mouse embryos., Results: By weighted gene-coexpression network analysis, we found that cynomolgus gene networks have greater conservation with human embryos including a greater number of conserved hub genes than that of mouse embryos. Consistently, we found that early ICM/TE lineage-segregating genes in monkeys exhibit greater similarity with human when compared to mouse, so are the genes in signaling pathways such as LRP1 and TCF7 involving in WNT pathway. Last, we tested the role of one conserved pre-EGA hub gene, SIN3A, using a morpholino knockdown of maternal RNA transcripts in monkey embryos followed by single-cell RNA-seq. We found that SIN3A knockdown disrupts the gene-silencing program during the embryonic genome activation transition and results in developmental delay of cynomolgus embryos., Conclusion: Taken together, our study provided new insight into evolutionarily conserved and divergent transcriptome dynamics during mammalian preimplantation development., (© 2021 American Association of Anatomists.)

Background: Vertebrate eye formation requires coordinated inductive interactions between different embryonic tissue layers, first described in amphibians. A network of transcription factors and signaling molecules controls these steps, with mutations causing severe ocular, neuronal, and craniofacial defects. In eyeless mutant axolotls, eye morphogenesis arrests at the optic vesicle stage, before lens induction, and development of ventral forebrain structures is disrupted., Results: We identified a 5-bp deletion in the rax (retina and anterior neural fold homeobox) gene, which was tightly linked to the recessive eyeless (e) axolotl locus in an F2 cross. This frameshift mutation, in exon 2, truncates RAX protein within the homeodomain (P154fs35X). Quantitative RNA analysis shows that mutant and wild-type rax transcripts are equally abundant in E/e embryos. Translation appears to initiate from dual start codons, via leaky ribosome scanning, a conserved feature among gnathostome RAX proteins. Previous data show rax is expressed in the optic vesicle and diencephalon, deeply conserved among metazoans, and required for eye formation in other species., Conclusion: The eyeless axolotl mutation is a null allele in the rax homeobox gene, with primary defects in neural ectoderm, including the retinal and hypothalamic primordia., (© 2020 Wiley Periodicals LLC.)

Background: Embryonic cells and cancer cells share various cellular characteristics important for their functions. It has been thus proposed that similar mechanisms of regulation may be present in these otherwise disparate cell types., Results: To explore how regulative embryonic cells are fundamentally different from cancerous cells, we report here that a fine balance of a tumor suppressor protein Retinoblastoma1 (Rb1) and a germline factor Vasa are important for proper cell proliferation and differentiation of the somatic cells during embryogenesis of the sea urchin. Rb1 knockdown blocked embryonic development and induced Vasa accumulation in the entire embryo, while its overexpression resulted in a smaller-sized embryo with differentiated body structures. These results suggest that a titrated level of Rb1 protein may be essential for a proper balance of cell proliferation and differentiation during development. Vasa knockdown or overexpression, on the other hand, reduced or increased Rb1 protein expression, respectively., Conclusions: Taken together, it appears that Vasa protein positively regulates Rb1 protein while Rb1 protein negatively regulates Vasa protein, balancing the act of these two antagonistic molecules in somatic cells. This mechanism may provide a fine control of cell proliferation and differentiation, which is essential for regulative embryonic development., (© 2019 Wiley Periodicals, Inc.)

Background: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process., Results: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression., Conclusion: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development., (© 2019 Wiley Periodicals, Inc.)

Background: Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis., Results: We identified two novel tetrapod-teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue-specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression., Conclusion: This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression., (© 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

Purpose: This study analyzes Prx1-specific conditional knockout of Acvr1 aiming to elucidate the endogenous role of Acvr1 during limb formation in early embryonic development. ACVR1 can exhibit activating and inhibiting function in BMP signaling. ACVR1 gain-of-function mutations can cause the rare disease fibrodysplasia ossificans progressiva (FOP), where patients develop ectopic bone replacing soft tissue, tendons and ligaments., Methods: Whole-mount in situ hybridization and skeletal preparations revealed that following limb-specific conditional knockout of Acvr1, metacarpals and proximal phalanges were shortened and additional cartilage and bone elements were formed., Results: The analysis of a set of marker genes including ligands and receptors of BMP signaling as well as genes involved in patterning and tendon and cartilage formation, revealed temporal disturbances with distinct spatial patterns. The most striking result was that in the absence of Acvr1 in mesoderm precursor cells, first digits were drastically malformed., Conclusion: In FOP, malformation of big toes can serve as a first soft marker in diagnostics. The surprising similarities in phenotype between the described conditional knockout of Acvr1 and the FOP mouse model, indicates a natural inhibitory function of ACVR1. This represents a further step towards better understanding the role of Acvr1 and developing treatment options for FOP., (© 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

The first stages of mammalian development, before implantation of the embryo in the maternal uterus, result in the establishment of three cell populations in the blastocyst: trophectoderm, epiblast, and primitive endoderm. These events involve only a small number of cells, and are initiated by morphological differences among them related to cell adhesion and polarity. Much attention has been paid to the master transcription factors that are critical for establishing and maintaining early lineage choices. Nevertheless, a large body of work also reveals that additional molecular mechanisms are involved. Here, we provide an updated view of the role of different signaling pathways in the first stages of mouse development, and how their cross-talk and interplay determine the initial lineage decisions occurring in the blastocyst. We will also discuss how these pathways are critical for translating cellular phenotypes, the product of the morphogenetic events occurring at these stages, into transcriptional responses and expression of lineage-specifying transcription factors. Developmental Dynamics 246:245-261, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Environmental temperature influences rates of embryonic development, but a detailed staging series for vertebrate embryos developing in the subzero cold of Antarctic waters is not yet available from fertilization to hatching. Given projected warming of the Southern Ocean, it is imperative to establish a baseline to evaluate potential effects of changing climate on fish developmental dynamics., Results: We studied the Bullhead notothen (Notothenia coriiceps), a notothenioid fish inhabiting waters between -1.9 and +2 °C. In vitro fertilization produced embryos that progressed through cleavage, epiboly, gastrulation, segmentation, organogenesis, and hatching. We compared morphogenesis spatially and temporally to Zebrafish and medaka. Experimental animals hatched after about 6 months to early larval stages. To help understand skeletogenesis, we analyzed late embryos for expression of sox9 and runx2, which regulate chondrogenesis, osteogenesis, and eye development. Results revealed that, despite their prolonged developmental time course, N. coriiceps embryos developed similarly to those of other teleosts with large yolk cells., Conclusions: Our studies set the stage for future molecular analyses of development in these extremophile fish. Results provide a foundation for understanding the impact of ocean warming on embryonic development and larval recruitment of notothenioid fish, which are key factors in the marine trophic system. Developmental Dynamics 245:1066-1080, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo in a process termed the endothelial to hematopoietic transition (EHT). EHT is most extensively studied in the yolk sac and dorsal aorta. Recently new sites of hematopoiesis have been described, including the heart, somites, head, and venous plexus of the yolk sac., Results: We examined sites of HSPC formation in well-studied and in less well-known sites by mapping the expression of the key EHT factor Runx1 along with several other markers by means of confocal microscopy. We identified sites of HSPC formation in the head, heart and somites. We also identified sites of HSPC formation in both the arterial and venous plexuses of the yolk sac, and show that progenitors with lymphoid potential are enriched in hematopoietic clusters in close proximity to arteries. Furthermore, we demonstrate that many of the cells in hematopoietic clusters resemble monocytes or granulocytes based on nuclear shape., Conclusions: We identified sites of HSPC formation in the head, heart, and somites, confirming that embryonic hematopoiesis is less spatially restricted than previously thought. Furthermore, we show that HSPCs in the yolk sac with lymphoid potential are located in closer proximity to arteries than to veins. Developmental Dynamics 245:1011-1028, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Cell polarity is essential for directed migration of mesenchymal cells and morphogenesis of epithelial tissues. Studies in cultured cells indicate that a condensed Golgi Complex (GC) is essential for directed protein trafficking to establish cell polarity underlying directed cell migration. Dynamic changes of the GC intracellular organization during early vertebrate development remain to be investigated., Results: We used antibody labeling and fusion proteins in vivo to study the organization and intracellular placement of the GC during early zebrafish embryogenesis. We found that the GC was dispersed into several puncta containing cis- and trans-Golgi Complex proteins, presumably ministacks, until the end of the gastrula period. By early segmentation stages, the GC condensed in cells of the notochord, adaxial mesoderm, and neural plate, and its intracellular position became markedly polarized away from borders between these tissues., Conclusions: We find that GC is dispersed in early zebrafish cells, even when cells are engaged in massive gastrulation movements. The GC accumulates into patches in a stage and cell-type specific manner, and becomes polarized away from borders between the embryonic tissues. With respect to tissue borders, intracellular GC polarity in notochord is independent of mature apical/basal polarity, Wnt/PCP, or signals from adaxial mesoderm. Developmental Dynamics 245:678-691, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Lin28 proteins are post-transcriptional regulators of gene expression with multiple roles in development and the regulation of pluripotency in stem cells. Much attention has focussed on Lin28 proteins as negative regulators of let-7 miRNA biogenesis; a function that is conserved in several animal groups and in multiple processes. However, there is increasing evidence that Lin28 proteins have additional roles, distinct from regulation of let-7 abundance. We have previously demonstrated that lin28 proteins have functions associated with the regulation of early cell lineage specification in Xenopus embryos, independent of a lin28/let-7 regulatory axis. However, the nature of lin28 targets in Xenopus development remains obscure., Results: Here, we show that mir-17∼92 and mir-106∼363 cluster miRNAs are down-regulated in response to lin28 knockdown, and RNAs from these clusters are co-expressed with lin28 genes during germ layer specification. Mature miRNAs derived from pre-mir-363 are most sensitive to lin28 inhibition. We demonstrate that lin28a binds to the terminal loop of pre-mir-363 with an affinity similar to that of let-7, and that this high affinity interaction requires to conserved a GGAG motif., Conclusions: Our data suggest a novel function for amphibian lin28 proteins as positive regulators of mir-17∼92 family miRNAs., (© 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.)

Background: Syncytial nuclei in Drosophila embryos undergo their first 13 divisions nearly synchronously. In the last several cell cycles, these division events travel across the anterior-posterior axis of the syncytial blastoderm in a wave. The phenomenon is well documented but the underlying mechanisms are not yet understood., Results: We study timing and positional data obtained from in vivo imaging of Drosophila embryos. We determine the statistical properties of the distribution of division times within and across generations with the null hypothesis that timing of division events is an independent random variable for each nucleus. We also compare timing data with a model of Drosophila cell cycle regulation that does not include internuclear signaling, and to a universal model of phase-dependent signaling to determine the probable form of internuclear signaling in the syncytial embryo., Conclusions: The statistical variance of division times is lower than one would expect from uncoordinated activity. In fact, the variance decreases between the 10th and 11th divisions, which demonstrates a contribution of internuclear signaling to the observed synchrony and division waves. Our comparison with a coupled oscillator model leads us to conclude that internuclear signaling must be of Response/Signaling type with a positive impulse., (© 2015 Wiley Periodicals, Inc.)

Background: The sphingosine 1-phosphate (S1P) signaling pathway regulates zebrafish cardiogenesis, and provides a paradigm for how signaling gradients coordinate collective cell migration across tissue layers. It is known that the S1P transporter (Spns2) functions in extra-embryonic YSL to activate G protein-coupled receptor (S1pr2) signaling in endoderm for deposition of positional cues (integrin, fibronectin, etc.). Such cues are recognized by overlying lateral precardiac mesoderm that migrates to the midline and fuses to form the primordial heart tube. However, the source of bio-active S1P is not known. There are multiple receptors and it is not known if there are earlier or even receptor-independent functions for S1P., Results: Because S1P can only be generated by sphingosine kinases, we targeted a mutation to the single kinase gene expressed during early embryogenesis (sphk2). Zygotic mutants survive to adulthood and appear normal, but maternal-zygotic mutant embryos phenocopy null zygotic mutants of spns2 or s1pr2., Conclusions: The data show that maternally derived sphk2 RNA is fully sufficient to generate an S1P signaling gradient in the YSL that ultimately controls precardiac mesoderm migration during embryogenesis. Furthermore, despite maternal expression of sphk2, there are no obvious developmental functions requiring its activity prior to stimulation of S1pr2 in endoderm., (© 2015 Wiley Periodicals, Inc.)

Background: For the delivery of oxygen, the correct size/number of erythrocytes is required for proper blood flow., Results: By combined analyses of wild-type (WT) medaka and the kyoho (kyo) mutant, we found proliferation-mediated adaptation for size/number of erythrocytes in the blood flow during medaka development. Before the start of heart beating in the WT medaka, the karyotype of erythrocytes was 2N-4N. After the start of blood flow, the karyotype changed to 4N-8N with tetraploidization, and the cell size became larger. After the start of intersegmental and pharyngeal blood flow, the erythrocytes became smaller. The medaka mutant kyo showed erythrocytes of large size, and positional cloning of kyo demonstrated the candidate gene TFDP1, indicating higher polyploidization due to arrest in S-phase in erythrocytes of the kyo mutant., Conclusions: From our findings, we uncovered a previously unrecognized system for the regulation of the size/number in the blood flow:proliferation of erythrocytes following tetraploidization during embryonic development., (© 2015 Wiley Periodicals, Inc.)

Background: The family of AP-1 complexes mediates protein sorting in the late secretory pathway and it is essential for the development of mammals. The ubiquitously expressed AP-1A complex consists of four adaptins γ1, β1, μ1A, and σ1A. AP-1A mediates protein transport between the trans-Golgi network and early endosomes. The polarized epithelia AP-1B complex contains the μ1B-adaptin. AP-1B mediates specific transport of proteins from basolateral recycling endosomes to the basolateral plasma membrane of polarized epithelial cells., Results: Analysis of the zebrafish genome revealed the existence of three μ1-adaptin genes, encoding μ1A, μ1B, and the novel isoform μ1C, which is not found in mammals. μ1C shows 80% sequence identity with μ1A and μ1B. The μ1C expression pattern largely overlaps with that of μ1A, while μ1B is expressed in epithelial cells. By knocking-down the synthesis of μ1A, μ1B and μ1C with antisense morpholino techniques we demonstrate that each of these μ1 adaptins is essential for zebrafish development, with μ1A and μ1C being involved in central nervous system development and μ1B in kidney, gut and liver formation., Conclusions: Zebrafish is unique in expressing three AP-1 complexes: AP-1A, AP-1B, and AP-1C. Our results demonstrate that they are not redundant and that each of them has specific functions, which cannot be fulfilled by one of the other isoforms. Each of the μ1 adaptins appears to mediate specific molecular mechanisms essential for early developmental processes, which depends on specific intracellular vesicular protein sorting pathways., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The vertebrate body axis extends sequentially from the posterior tip of the embryo, fueled by the gastrulation process at the primitive streak and its continuation within the tailbud. Anterior structures are generated early, and subsequent nascent tissues emerge from the posterior growth zone and continue to elongate the axis until its completion. The underlying processes have been shown to be disrupted in mouse mutants, some of which were described more than half a century ago., Results: Important progress in elucidating the cellular and genetic events involved in body axis elongation has recently been made on several fronts. Evidence for the residence of self-renewing progenitors, some of which are bipotential for neurectoderm and mesoderm, has been obtained by embryo-grafting techniques and by clonal analyses in the mouse embryo. Transcription factors of several families including homeodomain proteins have proven instrumental for regulating the axial progenitor niche in the growth zone. A complex genetic network linking these transcription factors and signaling molecules is being unraveled that underlies the phenomenon of tissue lengthening from the axial stem cells. The concomitant events of cell fate decision among descendants of these progenitors begin to be better understood at the levels of molecular genetics and cell behavior., Conclusions: The emerging picture indicates that the ontogenesis of the successive body regions is regulated according to different rules. In addition, parameters controlling vertebrate axial length during evolution have emerged from comparative experimental studies. It is on these issues that this review will focus, mainly addressing the study of axial extension in the mouse embryo with some comparison with studies in chick and zebrafish, aiming at unveiling the recent progress, and pointing at still unanswered questions for a thorough understanding of the process of embryonic axis elongation., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Hox proteins are key developmental regulators involved in almost every embryonic tissue for specifying cell fates along longitudinal axes or during organ formation. It is thought that the panoply of Hox activities relies on interactions with tissue-, stage-, and/or cell-specific transcription factors. High-throughput approaches in yeast or cell culture systems have shown that Hox proteins bind to various types of nuclear and cytoplasmic components, illustrating their remarkable potential to influence many different cell regulatory processes. However, these approaches failed to identify a relevant number of context-specific transcriptional partners, suggesting that these interactions are hard to uncover in non-physiological conditions. Here we discuss this problematic., Results: In this review, we present intrinsic Hox molecular signatures that are probably involved in multiple (yet specific) interactions with transcriptional partners. We also recapitulate the current knowledge on Hox cofactors, highlighting the difficulty to tracking context-specific cofactors through traditional large-scale approaches., Conclusion: We propose experimental approaches that will allow a better characterisation of interaction networks underlying Hox contextual activities in the next future., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Highly divergent morphology among the different goldfish strains (Carassius auratus) may make it a suitable model for investigating how artificial selection has altered developmental mechanisms. Here we describe the embryological development of the common goldfish (the single fin Wakin), which retains the ancestral morphology of this species., Results: We divided goldfish embryonic development into seven periods consisting of 34 stages, using previously reported developmental indices of zebrafish and goldfish. Although several differences were identified in terms of their yolk size, epiboly process, pigmentation patterns, and development rate, our results indicate that the embryonic features of these two teleost species are highly similar in their overall morphology from the zygote to hatching stage., Conclusions: These results provide an opportunity for further study of the evolutionary relationship between domestication and development, through applying well-established zebrafish molecular biological resources to goldfish embryos., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Inner ear development involves signaling from surrounding tissues, including the adjacent hindbrain, periotic mesenchyme, and notochord. These signals include SHH, FGFs, BMPs, and WNTs from the hindbrain and SHH from the notochord. Zic genes, which are expressed in the dorsal neural tube and act during neural development, have been implicated as effectors of these pathways. This report examines whether Zic genes' involvement in inner ear development is a tenable hypothesis based on their expression patterns., Results: In the developing inner ear of both the chick and mouse, all of the Zic genes were expressed in the dorsal neural tube and variably in the periotic mesenchyme, but expression of the Zic genes in the otic epithelium was not found. The onset of expression differed among the Zic genes; within any given region surrounding the otic epithelium, multiple Zic genes were expressed in the same place at the same time., Conclusions: Zic gene expression in the region of the developing inner ear is similar between mouse and chick. Zic expression domains overlap with sites of WNT and SHH signaling during otocyst patterning, suggesting a role for Zic genes in modulating signaling from these pathways., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The axial skeleton develops from the sclerotome, a mesenchymal cell population derived from somites. Sclerotomal cells migrate from somites to the perinotochordal and perineural space where they differentiate into chondrocytes to form cartilage and bone. In anurans, little is known about the way how the sclerotome changes as development proceeds and how these events are regulated at the molecular level. Pax1, Pax9, and Uncx4.1 genes play a central role in the morphogenesis of the axial skeleton in vertebrates, regulating cell proliferation and chondrogenic specification of the sclerotome., Results: In this work, we cloned and examined through whole-mount in situ hybridization and reverse transcriptase-polymerase chain reaction the expression patterns of pax1, pax9, and uncx transcription factors in the anuran Xenopus laevis., Conclusions: We found that these genes are similarly expressed in the sclerotome and in the pharyngeal pouch. A detailed analysis of the location of these transcripts showed that they are expressed in different subdomains of the sclerotomal compartment and differ from that observed in other vertebrates., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Unexpected phenotypes resulting from morpholino-mediated translational knockdown of Pitx3 in Xenopus laevis required further investigation regarding the genetic networks in which the gene might play a role. Microarray analysis was, therefore, used to assess global transcriptional changes downstream of Pitx3., Results: From the large data set generated, selected candidate genes were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization., Conclusions: We have identified four genes as likely direct targets of Pitx3 action: Pax6, β Crystallin-b1 (Crybb1), Hes7.1, and Hes4. Four others show equivocal promise worthy of consideration: Vent2, and Ripply2 (aka Ledgerline or Stripy), eFGF and RXRα. We also describe the expression pattern of additional and novel genes that are Pitx3-sensitive but that are unlikely to be direct targets., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Smad ubiquitination regulatory factor (Smurf) 1 and 2 are E3 ubiquitin ligases originally identified as inhibitors of transforming growth factor beta signaling and are shown to modulate multiple cellular activities. The roles of Smurfs in vertebrate embryogenesis, however, are not completely understood., Results: Here we investigate the function of Smurf2 during early Xenopus development. We show that distinctly from Smurf1, overexpression of Smurf2 in presumptive mesoderm interfered with mesoderm induction and caused axial defects, whereas knockdown of Smurf2 with antisense morpholino oligonucleotides resulted in expansion of the mesoderm. These results imply that Smurf2 may modulate nodal-mediated mesodermal induction. Consistently, ventral expression of Smurf2 induced a partial secondary axis with head structures. In the ectoderm, Smurf2 resembled Smurf1 in controlling neural and epidermal marker expression and influencing head formation. Smurf1, but not Smurf2, additionally affected neural tube closure. Interestingly, both Smurfs could enhance as well as repress neural crest markers, implying that they modulate their targets dynamically during neural plate border specification., Conclusion: Our data demonstrate that Smurf1 and Smurf2 have overlapping and distinct functionalities during early frog embryogenesis; collectively, they regulate ectodermal and mesodermal induction and patterning to ensure normal development of Xenopus embryos., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: DDX11 is a DNA helicase of the conserved FANCJ/RAD3/XPD family involved in maintaining genome stability. Studies in yeast and humans have shown requirements for DDX11 in sister chromatid cohesion and DNA repair. In mouse, loss of Ddx11 results in embryonic lethality. However, the developmental defects of Ddx11 mutants are poorly understood., Results: We describe the characterization and positional cloning of cetus, a mouse ENU-induced mutation in Ddx11. We demonstrate that cetus causes a nonconservative amino acid change in DDX11 motif V and that this mutation is a null allele of Ddx11. cetus mutant embryos failed to thrive beyond embryonic day 8.5 and displayed placental defects similar to those described in Ddx11 null embryos. Additionally, our characterization of Ddx11(cetus) mutants identified embryonic phenotypes that had not been previously reported in Ddx11(KO) embryos, including loss of somitic mesoderm, an open kinked neural tube and abnormal heart looping. We show that loss of Ddx11 causes widespread apoptosis from early embryonic stages and that loss of Ddx11 disrupts somitic mesoderm more dramatically than other embryonic cells., Conclusions: Our results identify novel roles of Ddx11 during embryo morphogenesis and demonstrate that the activity of its motif V is essential for DDX11 function., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: A challenge in age research is the absence of short-lived vertebrate model organisms. The turquoise killifish Nothobranchius furzeri has an exceptionally short lifespan of 4-10 months depending on the strain. Thus, it possesses the shortest known maximum lifespan of a vertebrate species that can be bred in captivity., Results: Here we show the successful introduction of DNA and RNA molecules into the one-cell embryo of N. furzeri. For this purpose, we adapted existing microinjection protocols to inject through the remarkably thick and robust chorion of N. furzeri's eggs. The injected DNA transgene was integrated into the genome and transmitted to subsequent generations as indicated by the expression of the fluorophore enhanced green fluorescent protein (EGFP). Furthermore, we could confirm a special phase during embryonic development in which embryogenesis occurs within a re-aggregated mass of previously dispersed cells as it has been described for other related cyprinodont fish species., Conclusions: The transgenesis protocol described here provides a basis for a variety of genetic manipulations including overexpression of genes and determining their effects on lifespan and longevity. The feasibility to perform transgenesis is an important step to establish N. furzeri as a new model in age research., (Copyright © 2012 Wiley Periodicals, Inc.)

Early placental development in mice involves patterning of the chorion into distinct layers, though little is understood regarding the interactions that regulate its organization. Here we demonstrate that keratin aggregates found in Mrj(-/-) chorionic trophoblast cells are associated with abnormal cell morphology, collapse of the actin cytoskeleton, E-cadherin and β-catenin misexpression and extracellular matrix (ECM) disorganization. Accordingly, Mrj(-/-) trophoblast cells in vitro are nonadherent and display erratic migratory behavior. These cells also fail to differentiate into syncytiotrophoblast cells since Rhox4b expression, a marker of syncytiotrophoblast progenitors, was maintained and Gcm1, Synb, and Syna expression failed to increase. This differentiation defect was not solely attributable to E-cadherin misexpression or ECM disorganization. However, plating Mrj-deficient cells on exogenous laminin-511 normalized their cell behavior. Lastly, we show that Mrj(-/-) chorions at embryonic day 8.5 have expanded Rhox4b expression domains and do not form normal layers of gene expression suggesting that chorion patterning requires Mrj., (Copyright © 2011 Wiley Periodicals, Inc.)

Yeast Periodic tryptophan protein 2 gene (Pwp2) is involved in ribosome biogenesis and has been implicated in regulation of the cell cycle in yeast. Here, we report a zebrafish protein-trap line that produces fluorescently tagged Periodic tryptophan protein 2 gene homologue (Pwp2h) protein, which can be dynamically tracked in living fish at subcellular resolution. We identified both full-length zebrafish Pwp2h and a short variant. The expression results show that Pwp2h is present in numerous sites in the early developing embryo, but later is restricted to highly proliferative regions, including the forebrain ventricular zone and endoderm-derived organs in the early larval stage. At the subcellular level, Pwp2h protein appears to be localized to the region of the nucleolus consistent with its presumed function in ribosomal RNA synthesis. This Pwp2h protein trap line offers a powerful tool to study the link between ribosome biogenesis and cell cycle progression during vertebrate development., (Copyright © 2011 Wiley Periodicals, Inc.)

Hepatocyte growth factor-regulated tyrosine kinase substrate that is encoded by Hgs promotes degradation of ubiquitinated signaling molecule in the early endosome. We previously reported that a targeted mutation in Hgs results in embryonic lethality soon after gastrulation in the mouse. Here, we report that downstream target genes for BMP signaling were highly down-regulated in the Hgs mutant embryos. We also showed that Hgs is required for phosphorylation of SMAD1/5/8 and TAK1/p38 to transduce BMP signaling. Furthermore, we found that HGS functions to localize TAK1 in early endosome for its activation. These results suggest that HGS is critical to localize TAK1 to early endosome for transducing BMP signaling for proper development. Our data revealed a new mechanism to modify BMP signaling by Hgs during early mouse development., (Copyright © 2011 Wiley-Liss, Inc.)

Mutated in Colorectal Cancer (MCC) encodes a multiple PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein implicated, as its name suggests, in the pathogenesis of human colon cancer. To date, however, what role, if any, MCC plays in normal tissue homeostasis and development remains unclear. In an effort to expand our understanding of MCC function and distribution, we examined the expression of the evolutionarily conserved mouse Mcc homolog between embryonic days (E) 6.5 and 12.5 using conventional whole-mount in situ hybridization and two independent Mcc reporter alleles. Mcc is expressed in the posterior primitive streak during gastrulation and in diverse tissues of both mesodermal and endodermal origin. In addition, Mcc transcripts localize to the posterior neural tube and identify discrete neuronal subtypes and ganglia within the developing central nervous system. Genetically, however, Mcc is entirely dispensable, as mice homozygous for the Mcc(Gt(D062B07)) gene trap allele, which generates a loss-of-function mutation, are viable and fertile, with no ostensible phenotype., (Copyright © 2011 Wiley-Liss, Inc.)

S100a8 is a cytosolic protein expressed in myeloid cells where it forms a stable heterodimer with another S100 protein family member, S100a9. The S100a9(-/-) mouse is viable and phenotypically normal, whereas the S100a8(-/-) condition is embryonic lethal. We present evidence that S100a8, without S100a9, has a previously unrecognized role in embryo development between fertilization and the 8-cell stage at embryonic day (E) 2.5. S100a8 also has a second role in the maternal deciduum, where expression is associated with the vasculature from the E8.5 stage to the formation of mature placenta. Uterine natural killer cells that have a role in vascular remodelling colocalise with the S100a8 vascular expression in the metrial triangle. In inflammatory responses in peripheral tissues, S100a8 is a potent chemoattractant and also an anti-oxidant. Both roles may be important in the developing placenta. Thus we highlight two new S100a9-independent roles for S100a8 in early embryo development., (Copyright © 2011 Wiley-Liss, Inc.)

Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm., (Copyright © 2011 Wiley-Liss, Inc.)

The fibroblast growth factor receptor 3 (Fgfr3) is expressed in a rostral(low) to caudal(high) gradient in the developing cerebral cortex. Therefore, we hypothesized that Fgfr3 contributes to the correct morphology and connectivity of the caudal cortex. Overall, the forebrain structures appeared normal in Fgfr3(-/-) mice. However, cortical and hippocampal volumes were reduced by 26.7% and 16.3%, respectively. Hypoplasia was particularly evident in the caudo-ventral region of the telencephalon where proliferation was mildly decreased at embryonic day 18.5. Dysplasia of GABAergic neurons in the amygdala and piriform cortex was seen following GAD67 immunohistochemistry. Dye-tracing studies and diffusion magnetic resonance imaging and tractography detected a subtle thalamocortical tract deficit, and significant decreases in the stria terminalis and lateral arms of the anterior commissure. These results indicate the subtle role of Fgfr3 in formation of caudal regions of the telencephalon affecting some brain projections., (Copyright © 2011 Wiley-Liss, Inc.)

Members of the Meis family of TALE homeobox transcription factors are involved in many processes of vertebrate development and morphogenesis, showing extremely complex transcriptional and spatiotemporal expression patterns. In this work, we performed a comprehensive study of chicken Meis genes using multiple approaches. First, we assessed whether the chicken genome contains a Meis3 ortholog or harbors only two Meis genes; we gathered several lines of evidence pointing to a specific loss of the Meis3 ortholog in an early ancestor of birds. Next, we studied the transcriptional diversity generated from chicken Meis genes through alternative splicing during development. Finally, we performed a detailed analysis of chick Meis1/2 expression patterns during early embryogenesis and organogenesis. We show that the expression of both Meis genes begins at the gastrulation stage in the three embryonic layers, presenting highly dynamic patterns with overlapping as well as distinct expression domains throughout development., (Copyright © 2011 Wiley-Liss, Inc.)

Members of the ProSAP/Shank family are important scaffolding proteins of the postsynaptic density (PSD). We investigated for the first time the expression of the three family members named Shank1, ProSAP1/Shank2, and ProSAP2/Shank3 during Xenopus laevis development. Shank1 is expressed in the neural tube, the retina, and the cranial ganglions. In contrast, ProSAP1/Shank2 transcripts could be visualized in the otic vesicle, the pronephros, the liver, the neural tube, and the retina. ProSAP2/Shank3 could be detected in the cardiovascular network, the neural tube, the pronephros, and the retina. Furthermore, we showed that LAPSER1 interacts with all three ProSAP/Shank family members in Xenopus embryos and co-localizes with ProSAP/Shank in a cell-based assay. In Xenopus, LAPSER1 is expressed in somites, brain, proctodeum, pronephros, and in some cranial ganglions. Thus, we suggest that members of the ProSAP/Shank family and LAPSER1 not only play a role in PSD formation and plasticity, but also during embryonic development., (Copyright © 2011 Wiley-Liss, Inc.)

Melanoblasts are derived from neural crest cells (NCCs) and are the only NCCs that migrate through the dorsolateral pathway. However, how melanoblasts evolved to migrate through a pathway different from other NCCs is still unclear, because little is known about common molecular mechanisms of melanoblast migration that are conserved between species. Endothelin receptor B2 (Ednrb2) is required for avian melanoblasts to enter the dorsolateral pathway. Here, we show that Endothelin-3 (ET3)/Ednrb2 signaling is also required for melanoblast migration in Xenopus laevis, although they migrate through the ventral pathway. In Xenopus, Ednrb2 is expressed by melanoblasts from pre-migration stages and ET3 is expressed around their destinations, suggesting that ET3/Ednrb2 signaling may determine melanophore localization. Furthermore, melanoblast migration is interrupted by aberrant ET3/Ednrb2 signaling in vivo and their invasive ability is enhanced by ET3 in vitro. Our results suggest that ET3/Ednrb2 signaling is required for melanoblast migration in Ednrb2 gene-conserved animals., (Copyright © 2011 Wiley-Liss, Inc.)

We have developed "b" and "c" isoform-specific chicken fibroblast growth factor (FGF) receptor 1-3 probes for in situ hybridization. We rigorously demonstrate the specificity of these probes by using both dot blot hybridization and whole-mount in situ hybridization during neurulation and early postneurulation stages, and we compare expression patterns of each of the three isoform-specific probes to one another and to generic probes to each of the three (non-isoform-specific) FGF receptors. We show that the expression pattern of each receptor is represented by the collective expression of each of its two isoforms, with the expression of each FGF receptor being most similar to that of its "c" isoform at two of the three stages studied, and that tissue and stage differences exist in the patterns of expression of the six isoforms. We demonstrate the usefulness of these probes for defining the differential tissue expression of FGF receptor 1-3 isoforms., (Copyright © 2011 Wiley-Liss, Inc.)

The amino acid sequence across the DNA-binding homeodomain of Gbx2 is highly conserved across multiple species. In mice, Gbx2 is essential for establishment of the midbrain-hindbrain boundary (MHB), and in development of anterior hindbrain structures, rhombomeres (r) 1-r3, and the r2/r3-derived cranial nerve V. In contrast, studies in zebrafish have implicated gbx1 in establishment of the MHB. Therefore, we tested potential roles for gbx2 in anterior hindbrain development in zebrafish. gbx2 knockdown with antisense morpholino results in increased cell death in r2, r3, and r5 and a truncation of the anterior hindbrain, similar to the defect in Gbx2(-/-) mice. Moreover, there is abnormal clustering of cranial nerve V cell bodies in r2 and r3 indicative of defects in aspects of anterior hindbrain patterning. These phenotypes can be rescued by expression of the mouse GBX2 protein. These results suggest that gbx2/Gbx2 has an evolutionarily conserved role in anterior hindbrain development., (Copyright © 2011 Wiley-Liss, Inc.)

In zebrafish, ovulated oocytes contain both maternal cortisol and the mRNA for the glucocorticoid receptor (gr), which is spread as granular structures throughout the ooplasm. At 0.2 hpf, this transcript is relocated in the blastodisc area and partitioned among blastomeres. At 6-8 hpf, it is replaced by zygotic transcript. We used morpholinos to block translation of both maternal and zygotic gr transcripts, and a missplicing morpholino to block post-transcriptionally the zygotic transcript alone. Only knockdown of translation produced an increase of apoptosis and subsequent craniofacial and caudal deformities with severe malformations of neural, vascular, and visceral organs in embryos and 5-dpf larvae. Such defects were rescued with trout gr2 mRNA. Microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal Gr protein deficiency in 5-hpf embryos. These results indicate that the maternal gr transcript and protein participate in the maternal programming of zebrafish development., (Copyright © 2011 Wiley-Liss, Inc.)