Hyperactive and impulsive behaviors of LMTK1 knockout mice.

Lemur tail kinase 1 (LMTK1), previously called Apoptosis-Associated Tyrosine Kinase (AATYK), remains an uncharacterized Ser/Thr protein kinase that is predominantly expressed in the brain. It is recently reported that LMTK1A, an isoform of LMTK1, binds to recycling endosomes through its palmitoylation and regulates endosomal trafficking by suppressing the activity of Rab11 small GTPase. In neurons, knockdown or knockout of LMTK1 results in longer axons, greater branching of dendrites and increased number of spines, suggesting that LMTK1 plays a role in neuronal circuit formation. However, its in vivo function remained to be investigated. Here, we examined the brain structures and behaviors of LMTK1 knockout (KO) mice. LMTK1 was expressed in most neurons throughout the brain. The overall brain structure appeared to be normal in LMTK1 KO mice, but the numbers of synapses were increased. LMTK1 KO mice had a slight impairment in memory formation and exhibited distinct psychiatric behaviors such as hyperactivity, impulsiveness and high motor coordination without social interaction deficits. Some of these abnormal behaviors represent core features of attention deficit hyperactive disorder (ADHD), suggesting the possible involvement of LMTK1 in the pathogenesis of ADHD.

CHARGE syndrome modeling using patient-iPSCs reveals defective migration of neural crest cells harboring CHD7 mutations.

CHARGE syndrome is caused by heterozygous mutations in the chromatin remodeler, CHD7, and is characterized by a set of malformations that, on clinical grounds, were historically postulated to arise from defects in neural crest formation during embryogenesis. To better delineate neural crest defects in CHARGE syndrome, we generated induced pluripotent stem cells (iPSCs) from two patients with typical syndrome manifestations, and characterized neural crest cells differentiated in vitro from these iPSCs (iPSC-NCCs). We found that expression of genes associated with cell migration was altered in CHARGE iPSC-NCCs compared to control iPSC-NCCs. Consistently, CHARGE iPSC-NCCs showed defective delamination, migration and motility in vitro, and their transplantation in ovo revealed overall defective migratory activity in the chick embryo. These results support the historical inference that CHARGE syndrome patients exhibit defects in neural crest migration, and provide the first successful application of patient-derived iPSCs in modeling craniofacial disorders.

Amniogenic somatopleure: a novel origin of multiple cell lineages contributing to the cardiovascular system.

The somatopleure is the amniotic primordium in amniote development, but its boundary to the embryonic body at early embryonic stages and the fate of cells constituting this structure are not well characterized. It also remains unclear how cells behave during the demarcation between intra- and extra-embryonic tissues. Here we identify cellular alignments, which indicate two streams towards the sites of dorsal amniotic closure and ventral thoracic wall formation. A subpopulation of mesodermal cells moving ventrally from the somatopleural region adjacent to the base of the head fold enter the body of the embryo and distribute to the thoracic wall, pharyngeal arches and heart. These cells are induced to differentiate into vascular endothelial cells and cardiomyocytes possibly by FGF and BMP signaling, respectively. These results indicate that the somatopleure acting as the amniotic primordium also serves as a source of embryonic cells, which may contribute to cardiovascular development.

LNGFR+THY-1+ human pluripotent stem cell-derived neural crest-like cells have the potential to develop into mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are defined as non-hematopoietic, plastic-adherent, self-renewing cells that are capable of tri-lineage differentiation into bone, cartilage or fat in vitro. Thus, MSCs are promising candidates for cell-based medicine. However, classifications of MSCs have been defined retrospectively; moreover, this conventional criterion may be inaccurate due to contamination with other hematopoietic lineage cells. Human MSCs can be enriched by selection for LNGFR and THY-1, and this population may be analogous to murine PDGFRα+Sca-1+ cells, which are developmentally derived from neural crest cells (NCCs). Murine NCCs were labeled by fluorescence, which provided definitive proof of neural crest lineage, however, technical considerations prevent the use of a similar approach to determine the origin of human LNGFR+THY-1+ MSCs. To further clarify the origin of human MSCs, human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) were used in this study. Under culture conditions required for the induction of neural crest cells, human ESCs and iPSCs-derived cells highly expressed LNGFR and THY-1. These LNGFR+THY-1+ neural crest-like cells, designated as LT-NCLCs, showed a strong potential to differentiate into both mesenchymal and neural crest lineages. LT-NCLCs proliferated to form colonies and actively migrated in response to serum concentration. Furthermore, we transplanted LT-NCLCs into chick embryos, and traced their potential for survival, migration and differentiation in the host environment. These results suggest that LNGFR+THY-1+ cells identified following NCLC induction from ESCs/iPSCs shared similar potentials with multipotent MSCs.

HINTW, a W-chromosome HINT gene in chick, is expressed ubiquitously and is a robust female cell marker applicable in intraspecific chimera studies.

Grafting and transplantation experiments in embryology require proper distinction between host and donor tissues. For the avian model this has traditionally been achieved by using two closely related species (e.g., chick and quail) followed by species-specific antibody staining. Here, we show that an in situ hybridization probe against the HINTW gene is a robust and reliable marker for female-derived chicken cells. At all pre-circulation stages tested, all cells in female embryos, independently confirmed by PCR analysis, were strongly positive for HINTW, whereas all male embryos were negative. This probe is broadly applicable in intra-specific chick/chick chimera studies, and as a proof of principle, we utilized this probe to detect female cells in three experimental settings: (1) to mark female donor cells in a node transplantation assay; (2) to distinguish female cells in male/female twins generated by the Cornish pasty culture; and (3) to detect female half of the embryo in artificially generated bilateral gynandromorphs. A rapid, PCR based pre-screening step increases the efficiency of obtaining desired donor/host sex combination from 25% to 100%. For most avian chimera studies, this female-specific in situ probe is a low cost alternative to the commonly used QCPN antibody and to ubiquitous-GFP chicken strains which are not widely available to the research community.

The dual origin of the peripheral olfactory system: placode and neural crest.

BACKGROUND: The olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos. RESULTS: Examination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa. CONCLUSION: Our data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration.

Identification of region-specific genes in the early chicken endoderm.

In vertebrates, the endoderm gives rise to the epithelial lining of the digestive tract, respiratory system and endocrine organs. After gastrulation, the newly formed endoderm gradually becomes regionalized and differentiates into specific organs. To understand the molecular basis of early endoderm regionalization, which is largely unknown, it is necessary to identify novel region-specific genes as candidates potentially involved in this process. Applying an Affymetrix Array based approach we aimed for the identification of genes specifically upregulated in the foregut or mid-/hindgut endoderm at the onset of regionalization. Several genes exhibiting spatial and temporal restricted expression patterns in the developing early endoderm were identified and their expression was validated via RT-PCR and whole mount in situ hybridization. We report here the detailed gene expression patterns of two novel genes specifically associated with foregut endoderm and of eight novel genes specifically expressed in the mid-/hindgut endoderm at HH stages 10-11. Future functional analysis of these genes may help to elucidate the mechanisms involved in endoderm development and regionalization.

Conserved origin of the ventral pancreas in chicken.

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.

Origin of pancreatic precursors in the chick embryo and the mechanism of endoderm regionalization.

To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.

Regional specification of the endoderm in the early chick embryo.

In the avian embryo, the endoderm, which forms a simple flat-sheet structure after gastrulation, is regionally specified in a gradual manner along the antero-posterior and dorso-ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA, a posterior endoderm marker, but not cSox2, an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid-hindgut endoderm expressed cSox2 but not CdxA. At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2, but anteriorly grafted presumptive mid-hindgut endoderm showed no changes in its posterior-specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid-hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid-hindgut.

Fate and plasticity of the endoderm in the early chick embryo.

In vertebrates, the endoderm is established during gastrulation and gradually becomes regionalized into domains destined for different organs. Here, we present precise fate maps of the gastrulation stage chick endoderm, using a method designed to label cells specifically in the lower layer. We show that the first population of endodermal cells to enter the lower layer contributes only to the midgut and hindgut; the next cells to ingress contribute to the dorsal foregut and followed finally by the presumptive ventral foregut endoderm. Grafting experiments show that some migrating endodermal cells, including the presumptive ventral foregut, ingress from Hensen's node, not directly into the lower layer but rather after migrating some distance within the middle layer. Cell transplantation reveals that cells in the middle layer are already committed to mesoderm or endoderm, whereas cells in the primitive streak are plastic. Based on these results, we present a revised fate map of the locations and movements of prospective definitive endoderm cells during gastrulation.

Expression patterns of the chicken peroxisome proliferator-activated receptors (PPARs) during the development of the digestive organs.

Peroxisome proliferator-activated receptors (PPARs) play very important roles in various biological phenomena such as regulation of lipid metabolism, homeostasis, cell differentiation and proliferation, in a variety of organs and tissues. However, their functions in the development of the digestive organs have not been studied yet, although it has been supposed that they are involved in the tumor development and regression of digestive organs. To provide fundamental data to analyze functions of PPARs in the developing digestive organs in the chicken embryos, we performed thorough analysis of expression of PPARalpha, beta (delta) and gamma in the esophagus, proventriculus (glandular stomach), gizzard (muscular stomach), small and large intestines from early developmental stages to post hatch stages. The results showed that each PPAR is expressed in spatio-temporally regulated manner. In general, PPARbeta is widely expressed among digestive organs whereas PPARalpha and gamma showed restricted expression. In the intestine, all PPARs are expressed after hatch, indicating that they play important roles in the physiology of the adult intestine.

Endoderm development in vertebrates: fate mapping, induction and regional specification.

The formation of the vertebrate body plan begins with the differentiation of cells into three germ layers: ectoderm, mesoderm and endoderm. Cells in the endoderm give rise to the epithelial lining of the digestive tract, associated glands and respiratory system. One of the fundamental problems in developmental biology is to elucidate how these three primary germ layers are established from the homologous population of cells in the early blastomere. To address this question, ectoderm and mesoderm development have been extensively analyzed, but study of endoderm development has only begun relatively recently. In this review, we focus on the 'where', 'when' and 'how' of endoderm development in four vertebrate model organisms: the zebrafish, Xenopus, chick and mouse. We discuss the classical fate mapping of the endoderm and the more recent progress in characterizing its induction, segregation and regional specification.

The molecular mechanisms of stomach development in vertebrates.

The tissue interactions between endodermal epithelium and mesenchyme originated from splanchnic mesoderm are essential during the formation of digestive tract. In this review, we introduce a series of works to elucidate the molecular mechanisms of the epithelial-mesenchymal interaction of stomach development in mainly the chicken embryo. We also describe some molecular studies in mouse stomach development.

Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development.

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. We found that Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) resulted in a luminal differentiation, while forced activation of Notch signaling promoted the specification of immature glandular cells, but prevented the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus.

Selected single blastocyst transfers maintained pregnancy outcome and eliminated multiple pregnancies.

Background: Transfer of more than one embryo following in vitro fertilization/intracytoplasmic sperm injection cycles have increased pregnancy rate at the cost of increasing the incidence of triplets and twins. It has been proposed that prolonged culture to the blastocyst stage would automatically result in the selection of good quality embryos for transfer and minimize the incidence of triplets and twins. Methods and Results: The objectives of the present retrospective analysis were to examine the pregnancy outcome, multiple pregnancy and related data following: (i) single blastocyst transfer (BT) and double BT; (ii) single BT in patients belonging to different age groups; and (iii) good, fair or poor quality of BT. A total of 260 BT were carried out between August 1998 and July 2002 and they are included in the current study. Sixty of the 260 BT patients received a single BT, and 41 of them received selected single good quality BT (SSBT). The implantation rate has no significant difference between following single BT (53.3%) and double BT (42.8%). No multiple pregnancy occurred following single BT, while significantly higher (P < 0.05) multiple pregnancy rate was observed following a double BT (45.8%). The clinical pregnancy and implantation rates following a single BT were similar (P > 0.05) in patients belonging to <30 years (62.5%), 30-34 years (57.9%) and 35-39 years old (35.8%). Conclusion: Selected single good quality BT maintained pregnancy and avoided multiple pregnancies. It is recommended for patients with a risk for high-order multiple pregnancy. (Reprod Med Biol 2004; 3: 13-18).

Down-regulation of endodermal Shh is required for gland formation in chicken stomach.

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.

Expression of DDSG1, a novel gene encoding a putative DNA-binding protein in the embryonic chicken nervous system.

In an attempt to clone genes expressed in the gizzard of the chicken embryo by differential display, we obtained a cDNA of a gene encoding a protein with a putative nuclear localization signal and a DNA-binding motif and designated it DDSG1 (differential display-screened gene expressed in the gizzard-1). Besides its expression in the gizzard, the gene is expressed in central and peripheral nervous systems such as brain, spinal cord and dorsal root ganglia in specific patterns.