Intestinal expression patterns of transcription factors and markers for interstitial cells in the larval zebrafish.

For the digestion of food, it is important for the gut to be differentiated regionally and to have proper motor control. However, the number of transcription factors that regulate its development is still limited. Meanwhile, the interstitial cells of the gastrointestinal (GI) tract are necessary for intestinal motility in addition to the enteric nervous system. There are anoctamine1 (Ano1)-positive and platelet-derived growth factor receptor α (Pdgfra)-positive interstitial cells in mammal, but Pdgfra-positive cells have not been reported in the zebrafish. To identify new transcription factors involved in GI tract development, we used RNA sequencing comparing between larval and adult gut. We isolated 40 transcription factors that were more highly expressed in the larval gut. We demonstrated expression patterns of the 13 genes, 7 of which were newly found to be expressed in the zebrafish larval gut. Six of the 13 genes encode nuclear receptors. The osr2 is expressed in the anterior part, while foxP4 in its distal part. Also, we reported the expression pattern of pdgfra for the first time in the larval zebrafish gut. Our data provide fundamental knowledge for studying vertebrate gut regionalization and motility by live imaging using zebrafish.

Ca2+-imaging and photo-manipulation of the simple gut of zebrafish larvae in vivo.

Zebrafish larval gut could be considered as an excellent model to study functions of vertebrate digestive organs, by virtue of its simplicity and transparency as well as the availability of mutants. However, there has been scant investigation of the detailed behavior of muscular and enteric nervous systems to convey bolus, an aggregate of digested food. Here we visualized peristalsis using transgenic lines expressing a genetically encoded Ca2+ sensor in the circular smooth muscles. An intermittent Ca2+ signal cycle was observed at the oral side of the bolus, with Ca2+ waves descending and ascending from there. We also identified a regular cycle of weaker movement that occurs regardless of the presence or absence of bolus, corresponding likely to slow waves. Direct photo-stimulation of circular smooth muscles expressing ChR2 could cause local constriction of the gut, while the stimulation of a single or a few neurons could cause the local induction or arrest of gut movements. These results indicate that the larval gut of zebrafish has basic features found in adult mammals despite the small number of enteric neurons, providing a foundation for the study, at the single-cell level in vivo, in controlling the gut behaviors in vertebrates.

The enteric nervous system in zebrafish larvae can regenerate via migration into the ablated area and proliferation of neural crest-derived cells.

The enteric nervous system (ENS), which is derived from neural crest, is essential for gut function, and its deficiency causes severe congenital diseases. Since the capacity for ENS regeneration in mammals is limited, additional complementary models would be useful. Here, we show that the ENS in zebrafish larvae at 10-15 days postfertilization is highly regenerative. After laser ablation, the number of enteric neurons recovered to ∼50% of the control by 10 days post-ablation (dpa). Using transgenic lines in which enteric neural crest-derived cells (ENCDCs) and enteric neurons are labeled with fluorescent proteins, we live imaged the regeneration process and found covering by neurites that extended from the unablated area and entry of ENCDCs into the ablated areas by 1-3 dpa. BrdU assays suggested that ∼80% of the enteric neurons and ∼90% of the Sox10-positive ENCDCs therein at 7 dpa were generated through proliferation. Thus, ENS regeneration involves proliferation, entrance and neurogenesis of ENCDCs. This is the first report regarding the regeneration process of the zebrafish ENS. Our findings provide a basis for further in vivo research at single-cell resolution in this vertebrate model.

Local heat-shock mediated multi-color labeling visualizing behaviors of enteric neural crest cells associated with division and neurogenesis in zebrafish gut.

BACKGROUND: The enteric nervous system (ENS) is derived from enteric neural crest cells (ENCCs) that migrate into the gut. The zebrafish larva is a good model to study ENCC development due to its simplicity and transparency. However, little is known how individual ENCCs divide and become neurons. RESULTS: Here, by applying our new method of local heat-shock mediated Cre-recombination around the dorsal vagal area of zebrafish embryos we produced multicolored clones of ENCCs, and performed in vivo time-lapse imaging from ca. 3.5 to 4 days post-fertilization after arrival of ENCCs in the gut. Individual ENCCs migrated in various directions and were highly intermingled. The cell divisions were not restricted to a specific position in the gut. Antibody staining after imaging with anti-HuC/D and anti-Sox10 showed that an ENCC produced two neurons, or formed a neuron and an additional ENCC that further divided. At division, the daughter cells immediately separated. Afterward, some made soma-soma contact with other ENCCs. CONCLUSIONS: We introduced a new method of visualizing individual ENCCs in the zebrafish gut, describing their behaviors associated with cell division, providing a foundation to study the mechanism of proliferation and neurogenesis in the ENS in vertebrates.

Early development of the enteric nervous system visualized by using a new transgenic zebrafish line harboring a regulatory region for choline acetyltransferase a (chata) gene.

The enteric nervous system (ENS) is the largest part of the peripheral nervous system in vertebrates. Toward the visualization of the development of the vertebrate ENS, we report our creation of a new transgenic line, Tg(chata:GGFF2) which has a 1.5-kb upstream region of the zebrafish choline acetyltransferase a (chata) gene followed by modified green fluorescent protein (gfp). During development, GFP + cells were detected in the gut by 60 h post-fertilization (hpf). In the gut of 6- and 12-days post-fertilization (dpf) larvae, an average of 92% of the GFP + cells were positive for the neuronal marker HuC/D, suggesting that GFP marks enteric neurons in this transgenic line. We also observed that 66% of the GFP + cells were choline acetyltransferase (ChAT)-immunopositive at 1.5 months. Thus, GFP is expressed at the larval stages at which ChAT protein expression is not yet detected by immunostaining. We studied the spatiotemporal pattern of neural differentiation in the ENS by live-imaging of this transgenic line. We observed that GFP + or gfp + cells initially formed a pair of bilateral rows at 60 hpf or 53 hpf, respectively, in the migrating enteric neural crest cells. Most of the GFP + cells did not migrate, and most of the new GFP + cells were added to fill the space among the previously formed GFP + cells. GFP expression reached the anus by 72 hpf. New GFP + cells then also appeared in the dorsal and ventral sides of the initial GFP + rows, resulting in their distribution on the entire gut by 4 dpf. A small number of new GFP + cells were found to move among older GFP + cells just before the cells stopped migration, suggesting that the moving GFP + cells may represent neural precursor cells searching for a place for the final differentiation. Our data suggest that the Tg(chata:GGFF2) line could serve as a useful tool for studies of enteric neural differentiation and cell behavior.

Retinoic acid is required and Fgf, Wnt, and Bmp signaling inhibit posterior lateral line placode induction in zebrafish.

The lateral line system is a mechanosensory systems present in aquatic animals. The anterior and posterior lateral lines develop from anterior and posterior lateral line placodes (aLLp and pLLp), respectively. Although signaling molecules required for the induction of other cranial placodes have been well studied, the molecular mechanisms underlying formation of the lateral line placodes are unknown. In this study we tested the requirement of multiple signaling pathways, such as Wnt, Bmp Fgf, and Retinoic Acid for aLLp and pLLp induction. We determined that aLLp specification requires Fgf signaling, whilst pLLp specification requires retinoic acid which inhibits Fgf signaling. pLLp induction is also independent of Wnt and Bmp activities, even though these pathways limit the boundaries of the pLLp. This is the first report that the aLLp and pLLp depend on different inductive mechanisms and that pLLp induction requires the inhibition of Fgf, Wnt and Bmp signaling.

Munch's SCREAM: A spontaneous movement by zebrafish larvae featuring strong abduction of both pectoral fins often associated with a sudden bend.

Stereotyped movement of paired pectoral fins in zebrafish larvae could be considered a simple model with which to investigate the neural basis of behavior. Using a high-speed camera, we explored the repertoire of pectoral fin movements by naturally behaving larvae at 5-6 days post-fertilization. Previously, two types of fin movements were characterized in association with locomotion: 'CRAWLing,' an alternating fin movement associated with slow swimming, and 'TUCKing,' the adduction of both fins associated with fast swimming. We here describe a third mode of fin movement, which we call 'Munch's SCREAM', in which both pectoral fins were flipped anteriorly so that they reached the skin on the sides of the head, thus covering the otic vesicles. This behavior occurred spontaneously and was often associated with a slight regression or a sudden bending and change in body orientation. It could be also induced effectively in the agarose-embedded larvae by tactile stimulation on the skin around the eye and nose, some of which are associated with struggling, in which waves of bending propagate from the tail to the head. Larvae can still CRAWL and perform the SCREAM even when their forebrain and midbrain have been removed, suggesting that the neural circuits involved in the SCREAM are present in the hindbrain and/or spinal cord.

High expression of organic anion transporter 2 and organic cation transporter 2 is an independent predictor of good outcomes in patients with metastatic colorectal cancer treated with FOLFOX-based chemotherapy.

Although metastatic colorectal cancer (mCRC) is commonly treated with 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX), their response to FOLFOX varies, and no biomarkers predictive of treatment outcome have been validated. Organic anion transporter 2 (OAT2) and organic cation transporter 2 (OCT2) are critical determinants in uptake of 5-FU and oxaliplatin, respectively. In this study, we evaluated whether OAT2 and OCT2 levels can predict effectiveness of FOLFOX-based therapy. We retrospectively assessed 90 patients with mCRC who were treated with first-line FOLFOX with or without bevacizumab. We immunohistochemically determined OAT2 and OCT2 expression levels at invasion fronts of their tumors and correlated the levels to clinicopathological parameters, including objective tumor response (OTR) and progression-free survival (PFS). High expression of OAT2 (OAT2(High)) and OCT2 (OCT2(High)) were detected in 36% and 60% of the tumors, respectively. OCT2(High) was significantly associated with invasion depth (P=0.03), whereas OAT2(High) was not associated with any clinicopathological parameters. In univariate analysis, OAT2(High) was significantly correlated with good OTR (P=0.02), and OCT2(High) with long PFS (P=0.03). Multivariate analyses showed that OAT2(High) and OCT2(High), respectively, were the sole independent predictors of good OTR (P=0.02) and long PFS (P=0.03). We found that patients with OAT2(High)/OCT2(High) showed the best treatment outcomes (good OTR and long PFS) with significantly higher frequency than patients with other expression patterns (P=0.003). OAT2(High)/OCT2(High) status was also the only independent predictive factor in multivariate analysis. This study suggests that OAT2(High) and OCT2(High) are important independent predictors of good outcomes in FOLFOX-treated mCRC.

Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.

The apparent irreversibility of the loss of complex traits in evolution (Dollo's Law) has been explained either by constraints on generating the lost traits or the complexity of selection required for their return. Distinguishing between these explanations is challenging, however, and little is known about the specific nature of potential constraints. We investigated the mechanisms underlying the irreversibility of trait loss using reduction of dentition in cypriniform fishes, a lineage that includes the zebrafish (Danio rerio) as a model. Teeth were lost from the mouth and upper pharynx in this group at least 50 million y ago and retained only in the lower pharynx. We identified regional loss of expression of the Ectodysplasin (Eda) signaling ligand as a likely cause of dentition reduction. In addition, we found that overexpression of this gene in the zebrafish is sufficient to restore teeth to the upper pharynx but not to the mouth. Because both regions are competent to respond to Eda signaling with transcriptional output, the likely constraint on the reappearance of oral teeth is the alteration of multiple genetic pathways required for tooth development. The upper pharyngeal teeth are fully formed, but do not exhibit the ancestral relationship to other pharyngeal structures, suggesting that they would not be favored by selection. Our results illustrate an underlying commonality between constraint and selection as explanations for the irreversibility of trait loss; multiple genetic changes would be required to restore teeth themselves to the oral region and optimally functioning ones to the upper pharynx.

Organic cation transporter 2 and tumor budding as independent prognostic factors in metastatic colorectal cancer patients treated with oxaliplatin-based chemotherapy.

Oxaliplatin is currently approved for patients with metastatic colorectal cancer (mCRC). Its uptake and consequent cytotoxicity is determined by the levels of organic cation transporter 2 (OCT2). In addition, tumor budding (TB) is associated with high malignant potential. However, the impact of the levels of OCT2 and TB on clinicopathological findings and the prognosis of mCRC patients treated with oxaliplatin-based chemotherapy remains unclear. Here, 80 mCRC patients were retrospectively assessed. Immunohistochemistry was performed to determine the levels of OCT2 and TB. High levels of OCT2 (47/80, 59%) were detected at the invasion front and were associated with depth of invasion (P=0.03), whereas high levels of TB (40/80, 50%) were associated with extensive lymphatic invasion (P=0.03). In univariate analysis, high OCT2 levels were significantly correlated with longer progression-free survival (PFS) (P=0.02) whereas high TB levels were associated with shorter PFS (P=0.01). In combined analysis, patients with 2 favorable factors (high OCT2/low TB) had longer PFS than those with 1 (P=0.03) or 0 (P<0.001) favorable factors. Multivariate analysis confirmed that the OCT2 level (P=0.007), TB level (P=0.004), and combined OCT2/TB status (P=0.001) were independent predictors for PFS. These results suggest that high levels of OCT2 indicate severe invasion, but also better prognosis in mCRC patients treated with oxaliplatin-based chemotherapy, possibly because of its role in oxaliplatin susceptibility. Combined analysis of OCT2 and TB status may guide the selection of patients for successful oxaliplatin-based chemotherapy.

Identification of initially appearing glycine-immunoreactive neurons in the embryonic zebrafish brain.

Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 h post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level.

Stochastic Ca(2+) waves that propagate through the neuroepithelium in limited distances of the brain and retina imaged with GCaMP3 in zebrafish embryos.

Ca(2+) plays important roles in animal development and behavior. Various Ca(2+) transients during development have been reported in non-neuronal tissues, mainly by using synthesized calcium indicators. Here we used GCaMP3, a genetically encoded calcium indicator, to monitor stochastic Ca(2+) waves, in zebrafish embryos. To express GCaMP3 systemically throughout the body, its mRNA was injected into fertilized eggs. In the neuroepithelium of developing anterior brain and retina at 12-20 hours post-fertilization, we found spontaneously occurring stochastic Ca(2+) waves. Each Ca(2+) wave typically appeared in a randomly distributed spot, spread for 5-60 sec to form an area whose position and size varied each time with a diameter ranging from 10 to 160 µm, and then shrank and decreased to 50% brightness in 4-67 sec. A precise examination of the cellular distribution using Nipkow disk multibeam confocal laser scanning indicated that the Ca(2+) waves spread cell by cell. 2-APB, IP3-receptor inhibitor, but not carbenoxolone, a gap junction blocker, inhibit these Ca(2+) waves. Stronger fluorescence was found in the cytoplasm compared to the nuclei in the resting cells, and localized fluorescence was observed at the spindle poles in dividing cells. Ca(2+) waves also spread through the dividing cells. Our results reveal a novel type of cell-to-cell communication through the neuroepithelium in the developing zebrafish brain and retina, distinct from communication through neuron-neuron circuits. Our findings also indicated that GCaMP3 was useful for monitoring both stochastic and behavior-related Ca(2+) waves in the nervous system and skeletal muscles in zebrafish embryos.

Brief hypo-osmotic shock causes test cell death, prevents neurula rotation, and disrupts left-right asymmetry in Ciona intestinalis.

Ascidian Ciona intestinalis tadpole larvae exhibit left-right asymmetry. The photoreceptors are situated on the right side of the sensory vesicle, and the tail curls along the left side of the trunk within the chorion. In tailbud embryos, the Ci-pitx gene is expressed in the left-side epidermis. It was previously reported that embryos generated from naked eggs, which lack the chorionic membrane and accessory cells (follicle cells attached to the outside of the chorion and test cells covering the inner surface of the chorion), show bilateral expression of Ci-pitx. This suggested that the chorion or accessory cells are needed for generation of asymmetry. Here, we show that a brief treatment with 60% artificial seawater (ASW) before, but not after, the neurula stage results in bilateral expression of Ci-pitx in the chorion of tailbud embryos, loss of follicle cells, and randomization of both the direction of tail curling and the locations of photoreceptors in larvae. This treatment also impaired the transient counterclockwise rotation within the chorion at the neurula stage. Nearly all test cells in the chorion died following 60% ASW treatment. These results suggest that dead test cells blocked the neural rotation and impaired left-right asymmetry. We also showed that tailbud embryos and larvae generated from defolliculated eggs produced by 80% ASW treatment, in which the test cells were alive, showed normal left-right asymmetry, suggesting that the follicle cells were not essential for asymmetric morphogenesis.

[Our experiences of anal squamous cell carcinoma treated by chemoradiotherapy].

We reviewed the clinical records of 6 cases with anal squamous cell carcinoma to evaluate the clinical effectiveness of chemoradiotherapy (CRT). The radiotherapy consisted of 40 Gy delivered to the pelvis and bilateral inguinal lesion, and a perianal booster dose of 20 Gy, in fractions of 2.0 Gy per day, 5 days per week. 5-FU and mitomycin C were administrated 3 times every 4 weeks as standard chemotherapy. On the first day of radiation therapy, 750 mg/m2 of 5-FU in the form of a continuous 24-hour infusion for 5 days was given. On the first day of chemotherapy, 10 mg/m2 of mitomycin C was also given as a single bolus infusion. One aged patient with a T3 tumor was administrated oral S-1 during radiotherapy. Four patients had a T2 tumor, 1 had a T1 tumor, and 1 had a T3 tumor. One patient had metastases in the Virchow lymph node that originated from synchronous vaginal cancer. No patient had hematogenous metastases. Grade 2 adverse effects occurred in 3 patients, and Grade 3 in 1 patient, during CRT, but the completion of CRT was achieved in all 6 patients. All patients had complete response (CR) in the anal lesion after CRT. Only the patient with a T3 tumor who was administrated S- 1 showed signs of recurrence in the anal lesion. CRT is expected to be a safe and effective treatment for improving the prognosis of anal squamous carcinoma.

Transgenic line with gal4 insertion useful to study morphogenesis of craniofacial perichondrium, vascular endothelium-associated cells, floor plate, and dorsal midline radial glia during zebrafish development.

Zebrafish is a good model for studying vertebrate development because of the availability of powerful genetic tools. We are interested in the study of the craniofacial skeletal structure of the zebrafish. For this purpose, we performed a gene trap screen and identified a Gal4 gene trap line, SAGFF(LF)134A. We then analyzed the expression pattern of SAGFF(LF)134A;Tg(UAS:GFP) and found that green fluorescent protein (GFP) was expressed not only in craniofacial skeletal elements but also in the vascular system, as well as in the nervous system. In craniofacial skeletal elements, strong GFP expression was detected not only in chondrocytes but also in the perichondrium. In the vascular system, GFP was expressed in endothelium-associated cells. In the spinal cord, strong GFP expression was found in the floor plate, and later in the dorsal radial glia located on the midline. Taking advantage of this transgenic line, which drives Gal4 expression in specific tissues, we crossed SAGFF(LF)134A with several UAS reporter lines. In particular, time-lapse imaging of photoconverted floor-plate cells of SAGFF(LF)134A;Tg(UAS:KikGR) revealed that the floor-plate cells changed their shape within 36 h from cuboidal/trapezoidal to wine glass shaped. Moreover, we identified a novel mode of association between axons and glia. The putative paths for the commissural axons, including pax8-positive CoBL interneurons, were identified as small openings in the basal endfoot of each floor plate. Our results indicate that the transgenic line would be useful for studying the morphogenesis of less-well-characterized tissues of interest, including the perichondrium, dorsal midline radial glia, late-stage floor plate, and vascular endothelium-associated cells.

Formation of the spinal network in zebrafish determined by domain-specific pax genes.

In the formation of the spinal network, various transcription factors interact to develop specific cell types. By using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) was inserted into the pax8 gene. RFP insertion marked putative pax8-lineage cells with fluorescence and inhibited pax8 expression in homozygous embryos. Pax8 homozygous embryos displayed defects in the otic vesicle, as previously reported in studies with morpholinos. The pax8 homozygous embryos survived to adulthood, in contrast to mammalian counterparts that die prematurely. RFP is expressed in the dorsal spinal cord. Examination of the axon morphology revealed that RFP(+) neurons include commissural bifurcating longitudinal (CoBL) interneurons, but other inhibitory neurons such as commissural local (CoLo) interneurons and circumferential ascending (CiA) interneurons do not express RFP. We examined the effect of inhibiting pax2a/pax8 expression on interneuron development. In pax8 homozygous fish, the RFP(+) cells underwent differentiation similar to that of pax8 heterozygous fish, and the swimming behavior remained intact. In contrast, the RFP(+) cells of pax2a/pax8 double mutants displayed altered cell fates. CoBLs were not observed. Instead, RFP(+) cells exhibited axons descending ipsilaterally, a morphology resembling that of V2a/V2b interneurons.

Early glycinergic axon contact with the Mauthner neuron during zebrafish development.

Glycinergic neurons are the major inhibitory neurons in the vertebrate central nervous system. In teleosts, they play important roles in the escape response by regulating the activity of the Mauthner (M-) cells. Here we studied the contact between glycinergic axons and the M-cells in early zebrafish embryos by double immunostaining with an anti-glycine antibody and the 3A10 antibody that labels M-cells. We also studied a transgenic line, Tg(GlyT2:GFP), in which GFP is expressed under the control of the promoter for the glycine transporter-2 gene. The initial contacts by ascending glycinergic axons on the M-soma were observed within 27h post-fertilization (hpf) on the lateral part of the ventral surface of the M-soma. Stochastic labeling of glycinergic neurons was then performed by injecting a GlyT2:GFP construct into early cleaving eggs. We identified the origin of the earliest glycinergic axons that contact the M-soma as commissural neurons, located in the anterior spinal cord, whose axons ascend along the lateral longitudinal fascicles with a short descending branch. We also found, in the fourth rhombomere, late-developed glycinergic commissural neurons whose axons contact anterior or posterior edge of both M-somas. This study provides the first example of the initial development of an inhibitory network on an identifiable neuron in vertebrates.

Retinoic acid-dependent establishment of positional information in the hindbrain was conserved during vertebrate evolution.

Zebrafish hoxb1b is expressed during epiboly in the posterior neural plate, with its anterior boundary at the prospective r4 region providing a positional cue for hindbrain formation. A similar function and expression is known for Hoxa1 in mice, suggesting a shared regulatory mechanism for hindbrain patterning in vertebrate embryos. To understand the evolution of the regulatory mechanisms of key genes in patterning of the central nervous system, we examined how hoxb1b transcription is regulated in zebrafish embryos and compared the regulatory mechanisms between mammals and teleosts that have undergone an additional genome duplication. By promoter analysis, we found that the expression of the reporter gene recapitulated hoxb1b expression when driven in transgenic embryos by a combination of the upstream 8.0-kb DNA and downstream 4.6-kb DNA. Furthermore, reporter expression expanded anteriorly when transgenic embryos were exposed to retinoic acid (RA) or LiCl, or injected with fgf3/8 mRNA, implicating the flanking DNA examined here in the responsiveness of hoxb1b to posteriorizing signals. We further identified at least two functional RA responsive elements in the downstream DNA that were shown to be major regulators of early hoxb1b expression during gastrulation, while the upstream DNA, which harbors repetitive sequences with apparent similarity to the autoregulatory sequence of mouse Hoxb1, contributed only to later hoxb1b expression, during somitogenesis. Possible implications in vertebrate evolution are discussed based on these findings.

Planar polarization of node cells determines the rotational axis of node cilia.

Rotational movement of the node cilia generates a leftward fluid flow in the mouse embryo because the cilia are posteriorly tilted. However, it is not known how anterior-posterior information is translated into the posterior tilt of the node cilia. Here, we show that the basal body of node cilia is initially positioned centrally but then gradually shifts toward the posterior side of the node cells. Positioning of the basal body and unidirectional flow were found to be impaired in compound mutant mice lacking Dvl genes. Whereas the basal body was normally positioned in the node cells of Wnt3a(-/-) embryos, inhibition of Rac1, a component of the noncanonical Wnt signalling pathway, impaired the polarized localization of the basal body in wild-type embryos. Dvl2 and Dvl3 proteins were found to be localized to the apical side of the node cells, and their location was polarized to the posterior side of the cells before the posterior positioning of the basal body. These results suggest that posterior positioning of the basal body, which provides the posterior tilt to node cilia, is determined by planar polarization mediated by noncanonical Wnt signalling.