Normal skeletal pattern formation in chick limb bud with a mesenchymal hole is mediated by adjustment of cellular properties along the anterior-posterior axis in the limb bud.

The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 â€‹h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 â€‹h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.

TC10, a Rho family GTPase, is required for efficient axon regeneration in a neuron-autonomous manner.

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock-out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild-type and TC10 knock-out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron-intrinsic.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.

Chronically skipping breakfast impairs hippocampal memory-related gene expression and memory function accompanied by reduced wakefulness and body temperature in mice.

Acute or chronic effects of consuming or skipping breakfast on cognitive performance in humans are controversial. To evaluate the effects of chronically skipping breakfast (SB) on hippocampus-dependent long-term memory formation, we examined hippocampal gene expression and applied the novel object recognition test (NORT) after two weeks of repeated fasting for six hours from lights off to mimic SB in mice. We also examined the effects of SB on circadian rhythms of locomotor activity, food intake, core body temperature (CBT) and sleep-wake cycles. Skipping breakfast slightly but significantly decreased total daily food intake without affecting body weight gain. Locomotor activity and CBT significantly decreased during the fasting period under SB. The degree of fasting-dependent CBT reduction gradually increased and then became stabilized after four days of SB. Electroencephalographic data revealed that repeated SB significantly decreased the duration of wakefulness and increased that of rapid eye movement (REM) and of non-REM (NREM) sleep during the period of SB. Furthermore, total daily amounts of wakefulness and NREM sleep were significantly decreased and increased, respectively, under SB, suggesting that SB disrupts sleep homeostasis. Skipping breakfast significantly suppressed mRNA expression of the memory-related genes, Camk2a, Fkbp5, Gadd45b, Gria1, Sirt1 and Tet1 in the hippocampus. Recognition memory assessed by NORT was impaired by SB in accordance with the gene expression profiles. These findings suggested that chronic SB causes dysregulated CBT, sleep-wake cycles and hippocampal gene expression, which results in impaired long-term memory formation.

Tissue regeneration during lower jaw restoration in zebrafish shows some features of epimorphic regeneration.

Zebrafish have the ability to regenerate skeletal structures, including the fin, skull roof, and jaw. Although fin regeneration proceeds by epimorphic regeneration, it remains unclear whether this process is involved in other skeletal regeneration in zebrafish. Initially in epimorphic regeneration, the wound epidermis covers the wound surface. Subsequently, the blastema, an undifferentiated mesenchymal mass, forms beneath the epidermis. In the present study, we re-examined the regeneration of the zebrafish lower jaw in detail, and investigated whether epimorphic regeneration is involved in this process. We performed amputation of the lower jaw at two different positions; the proximal level (presence of Meckel's cartilage) and the distal level (absence of Meckel's cartilage). In both manipulations, a blastema-like cellular mass was initially formed. Subsequently, cartilaginous aggregates were formed in this mass. In the proximal amputation, the cartilaginous aggregates were then fused with Meckel's cartilage and remained as a skeletal component of the regenerated jaw, whereas in the distal amputation, the cartilaginous aggregates disappeared as regeneration progressed. Two molecules that were observed during epimorphic regeneration, Laminin and msxb, were expressed in the regenerating lower jaw, although the domain of msxb expression was out of the main plain of the aggregate formation. Administration of an inhibitor of Wnt/ß-catenin signaling, a pathway associated with epimorphic regeneration, showed few effects on lower jaw regeneration. Our finding suggests that skeletal regeneration of the lower jaw mainly progresses through tissue regeneration that is dependent on the position in the jaw, and epimorphic regeneration plays an adjunctive role in this regeneration.

Characterization of cis-regulatory elements for Fgf10 expression in the chick embryo.

BACKGROUND: Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. RESULTS: By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. CONCLUSIONS: The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018. © 2018 Wiley Periodicals, Inc.

Mon1-Ccz1 activates Rab7 only on late endosomes and dissociates from the lysosome in mammalian cells.

Rab GTPases act as molecular switches regulating various aspects of membrane trafficking. Among them, Rab5 and Rab7 play central roles in the endolysosomal network. Although many effectors downstream of Rab7 have been elucidated, our present understanding of the mechanism regulating Rab7 activity is extremely limited. It has only recently been accepted that the Mon1-Ccz1 complex is a Rab7 guanine nucleotide exchange factor, but it still remains unclear what the location where Mon1-Ccz1 works with Rab7 is. To address what kind of change or switch exists in the regulatory mechanism upstream of Rab7 during its transition from the late endosome to lysosome, we examined Rab7 activity in steady-state cells and during EGF-induced macropinocytosis using a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1-Ccz1 and is implicated in late-endosome-lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1-Ccz1 and active Rab7 played a role in perinuclear clustering of lysosomes.

cAMP-induced activation of protein kinase A and p190B RhoGAP mediates down-regulation of TC10 activity at the plasma membrane and neurite outgrowth.

Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth.

Determination of reference genes that are independent of feeding rhythms for circadian studies of mouse metabolic tissues.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis is a popular method for the measurement of mRNA expression level and is a critical tool for basic research. The identification of suitable reference genes that are stable and not affected by experimental conditions is a critical step in the accurate normalization of RT-PCR. On the other hand, the levels of numerous transcripts exhibit circadian oscillation in various peripheral tissues and it is thought to be regulated by feeding rhythms in addition to the molecular circadian clock. Here, we investigated the effects of feeding schedule on the temporal expression profiles of 13 common housekeeping genes in metabolic tissues of mice fed during either the sleep or the active phase. The expression of most of these genes fluctuated dependently on feeding rhythms in the liver and WAT, but not in skeletal muscle. Two-way analyses of variance (ANOVA) identified 18S ribosomal RNA (Rn18s) as the only gene that was stably expressed throughout the day independently of feeding schedules in the liver and WAT, although RefFinder software showed that peptidylprolyl isomerase A (Ppia) was the most stably expressed housekeeping gene. Both ANOVA and RefFinder software determined that Actb was the preferred reference gene for skeletal muscle. Furthermore, NormFinder proposed that the optimal pairs of reference genes were beta-2 microglobulin (B2m)-Ppia in the liver, Ppia-TATA box binding protein (Tbp) in WAT, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz)-glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in skeletal muscle, and that their stability value was better than that of a single stable gene. The appropriate reference gene pairs for normalizing genes of interest in mouse circadian studies are B2m-Ppia in the liver, Ppia-Tbp in WAT, and Ywhaz-Gapdh in skeletal muscle.

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the ß-catenin pathway.

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→ß-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton.

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

Longest neurite-specific activation of Rap1B in hippocampal neurons contributes to polarity formation through RalA and Nore1A in addition to PI3-kinase.

In a developing nervous system, axon-dendrite formation is instructed by extrinsic cues, and the mechanism whereby a developing neuron interprets these cues using intracellular signaling is particularly important. Studies using dissociated hippocampal neurons have identified many signaling pathways underlying neuronal polarization. Among the components of these pathways, Rap1B is essential for axon specification in hippocampal cultures. However, spatiotemporal regulation of Rap1B activity in polarizing neurons and how it affects neuronal polarization remain unclear. Herein, we investigated spatiotemporal activity-change of Rap1B and its target molecules in hippocampal neurons. FRET imaging showed that specific activation of Rap1B was observed at the tip of a future axon. To dissect downstream signaling, we used three effector mutants of Rap1B. Expression of Rap1B-G12V/E37G and G12V/Y40C mutants resulted in supernumerary axons. The targets of Rap1B-G12V/E37G were RalA and Nore1A, whereas Rap1B-G12V/Y40C activated PI3-kinase. RalA was activated in the tip of stage 3 axons, and RalA-S28N expression reduced the fraction of neurons with supernumerary axons induced by Rap1B-G12V/E37G. Furthermore, Nore1A depletion reduced the number of cells without axons. These results indicate that specific activation of Rap1B contributes to neuronal polarization via interaction with RalA and Nore1A in addition to PI3-kinase.

Lateral bone ridge expansion and internal tissue replacement for vertebral body growth in Pacific bluefin tuna Thunnus orientalis.

Vertebral growth is an essential developmental process to support the expansion of the vertebrate body. In teleosts, the lateral side of the vertebral bodies develops to form different structures among species in the late stages of vertebral growth, although lateral structures are not apparent in the early stages. Lateral structures are one of the structural features that determine the diversity of teleost vertebrae. However, explanations for the formation of lateral structures are conflicting because few reports have investigated the growth of teleost vertebral bodies. To clarify the growth process, we analyzed the morphological changes in the vertebral body of Pacific bluefin tuna Thunnus orientalis at different developmental stages using micro-computed tomography (CT) scans. The micro-CT scans showed that the vertebral centrum formed a plate-like ridge on the lateral side along the cranial-caudal direction and extended laterally with increasing thickness. Simultaneously, the proximal region of the lateral ridges became porous as the vertebrae grew to form bone marrow cavities. Furthermore, we used histological observations to describe the relationship between these morphological changes and osteoblast and osteoclast activities. Osteoblasts accumulated on the distal edges of the lateral ridges, whereas osteoclasts were distributed in the bone marrow cavities. These observations suggest that bone resorption occurs proximally to form bone marrow cavities in addition to bone synthesis at the edges of the lateral ridges. The bone marrow cavities were occupied by blood vessels, extracellular matrix, and adipocytes, and the internal tissue composition changed to increase the area of adipose tissue. Because the ratio of bone volume decreases in large vertebrae, bone formation and resorption are regulated to separate the external cortical and internal trabecular bones to support the vertebrae. This study is the first to report the formation of lateral structures and can be applied to similar lateral structures in the vertebrae of other teleost species.

Developmental and evolutionary significance of the mandibular arch and prechordal/premandibular cranium in vertebrates: revising the heterotopy scenario of gnathostome jaw evolution.

The cephalic neural crest produces streams of migrating cells that populate pharyngeal arches and a more rostral, premandibular domain, to give rise to an extensive ectomesenchyme in the embryonic vertebrate head. The crest cells forming the trigeminal stream are the major source of the craniofacial skeleton; however, there is no clear distinction between the mandibular arch and the premandibular domain in this ectomesenchyme. The question regarding the evolution of the gnathostome jaw is, in part, a question about the differentiation of the mandibular arch, the rostralmost component of the pharynx, and in part a question about the developmental fate of the premandibular domain. We address the developmental definition of the mandibular arch in connection with the developmental origin of the trabeculae, paired cartilaginous elements generally believed to develop in the premandibular domain, and also of enigmatic cartilaginous elements called polar cartilages. Based on comparative embryology, we propose that the mandibular arch ectomesenchyme in gnathostomes can be defined as a Dlx1-positive domain, and that the polar cartilages, which develop from the Dlx1-negative premandibular ectomesenchyme, would represent merely posterior parts of the trabeculae. We also show, in the lamprey embryo, early migration of mandibular arch mesenchyme into the premandibular domain, and propose an updated version of the heterotopy theory on the origin of the jaw.

Dual origins of the prechordal cranium in the chicken embryo.

The prechordal cranium, or the anterior half of the neurocranial base, is a key structure for understanding the development and evolution of the vertebrate cranium, but its embryonic configuration is not well understood. It arises initially as a pair of cartilaginous rods, the trabeculae, which have been thought to fuse later into a single central stem called the trabecula communis (TC). Involvement of another element, the intertrabecula, has also been suggested to occur rostral to the trabecular rods and form the medial region of the prechordal cranium. Here, we examined the origin of the avian prechordal cranium, especially the TC, by observing the craniogenic and precraniogenic stages of chicken embryos using molecular markers, and by focal labeling of the ectomesenchyme forming the prechordal cranium. Subsequent to formation of the paired trabeculae, a cartilaginous mass appeared at the midline to connect their anterior ends. During this midline cartilage formation, we did not observe any progressive medial expansion of the trabeculae. The cartilages consisted of premandibular ectomesenchyme derived from the cranial neural crest. This was further divided anteroposteriorly into two portions, derived from two neural crest cell streams rostral and caudal to the optic vesicle, called preoptic and postoptic neural crest cells, respectively. Fate-mapping analysis elucidated that the postoptic neural crest cells were distributed exclusively in the lateroposterior part of the prechordal cranium corresponding to the trabeculae, whereas the preoptic stream of cells occupied the middle anterior part, differentiating into a cartilage mass corresponding to the intertrabecula. These results suggest that the central stem of the prechordal cranium of gnathostomes is composed of two kinds of distinct cartilaginous modules: a pair of trabeculae and a median intertrabecula, each derived from neural crest cells populating distinct places of the craniofacial primordia through specific migratory pathways.

Spatiotemporal changes in cell adhesiveness during vertebrate limb morphogenesis.

During vertebrate limb development, various molecules are expressed in the presumptive limb field or the limb bud in a spatiotemporal-specific manner. The combination of these molecules regulates cellular properties that affect limb initiation and its morphogenesis, especially cartilage formation. Cell adhesiveness of the limb mesenchyme is a key factor in the regulation of cell distribution. Differential adhesiveness of mesenchymal cells is first observed between cells in the presumptive limb field and flank region, and the adhesiveness of the cells in the limb field is higher than that of cells in the flank region. In the limb bud, the adhesiveness of mesenchymal cells shows spatiotemporal difference, which reflects the positional identity of the cells. Position-dependent cell adhesiveness is also observed in blastema cells of the regenerating limb. Therefore, local changes in cell adhesiveness are observed during limb development and regeneration, suggesting significant roles for cell adhesiveness in limb morphogenesis.

The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud.

Little effort has been made to apply the insights gained from studies of amphibian limb regeneration to higher vertebrates. During amphibian limb regeneration, a functional epithelium called the apical ectodermal cap (AEC) triggers a regenerative response. As long as the AEC is induced, limb regeneration will take place. Interestingly, similar responses have been observed in chicken embryos. The AEC is an equivalent structure to the apical ectodermal ridge (AER) in higher vertebrates. When a limb bud is amputated it does not regenerate; however, if the AER is grafted onto the amputation surface, damage to the amputated limb bud can be repaired. Thus, the AER/AEC is able to induce regenerative responses in both amphibians and higher vertebrates. It is difficult, however, to induce limb regeneration in higher vertebrates. One reason for this is that re-induction of the AER after amputation in higher vertebrates is challenging. Here, we evaluated whether AER re-induction was possible in higher vertebrates. First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred. These results open up new possibilities for limb regeneration in higher vertebrates since AER re-induction, which is considered a key factor in limb regeneration, is now possible.

Regenerative Polarity of the Fin Ray in Zebrafish Caudal Fin and Related Tissue Formation on the Cut Surface.

Zebrafish caudal fin rays are used as a model system for regeneration because of their high regenerative ability, but studies on the regeneration polarity of the fin ray are limited. To investigate this regeneration polarity, we made a hole to excise part of the fin ray and analyzed the regeneration process. We confirmed that the fin rays always regenerated from the proximal margin toward the distal margin, as previously reported; however, regeneration-related genes were expressed at both the proximal and distal edges of the hole in the early stage of regeneration, suggesting that the regenerative response also occurs at the distal edge. One difference between the proximal and distal margins is a sheet-like tissue that is formed on the apical side of the regenerated tissue at the proximal margin. This sheet-like tissue was not observed at the distal edge. To investigate whether the distal margin was also capable of forming this sheet-like tissue and subsequent regeneration, we kept the distal margin separated from the proximal margin by manipulation. Consequently, the sheet-like tissue was formed at the distal margin and regeneration of the fin ray was also induced. The regenerated fin rays from the distal margin protruded laterally from the caudal fin and then bent distally, and their ends showed the same characteristics as those of the normal fin rays. These results suggest that fin rays have an ability to regenerate in both directions; however, under normal conditions, regeneration is restricted to the proximal margin because the sheet-like tissue is preferentially formed on the apical side of the regenerating tissue from the proximal margin.

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development.

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.

Rab5 activation on macropinosomes requires ALS2, and subsequent Rab5 inactivation through ALS2 detachment requires active Rab7.

Macropinocytosis is a nonspecific bulk uptake of extracellular fluid. During endosome maturation, the Rab5-to-Rab7 switch machinery executes the conversion from early to late endosomes. However, how the Rab switch works during macropinosome maturation remains unclear. Here, we elucidate the Rab switch machinery in macropinosome maturation using Förster resonance energy transfer imaging. Rab5 is activated and concurrently recruited to macropinosomes during ruffle closure. ALS2 depletion abolishes transient Rab5 activation on macropinosomes, while ALS2 is recruited to macropinosomes simultaneously with Rab5 activation. Thus, we conclude ALS2 activates Rab5 on macropinosomes. The absence of active Rab7 prolongs ALS2 presence and Rab5 activation on macropinosomes, indicating that active Rab7 is necessary for Rab5 inactivation through ALS2 dissociation and plays key roles in the Rab switch on macropinosomes.